Methods and vaccine compositions for LYME disease

Abstract

Described are compositions and methods for prevention of Lyme disease. Also described are methods for production of an RNA molecule comprising an open reading frame encoding a structural domain polypeptide and an open reading frame encoding an outer surface protein A (OspA) lipoprotein polypeptide, including structural domain polypeptides from an influenza neuraminidase structural domain polypeptide, a PIV5 structural domain polypeptide and a RSV- G structural domain polypeptide.

Description

[0001] METHODS AND VACCINE COMPOSITIONS FOR LYME DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 733,366, filed December 12, 2024, and U.S. Provisional Patent Application Serial No. 63 / 614,170, filed December 22, 2023, the disclosures of which are hereby incorporated by reference in their entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The instant application contains a sequence listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml file, named “PC073013A.xml]”, was created on December 19, 2024, and is 106 KB in size.

[0006] FIELD

[0007] The methods and compositions disclosed herein relate to design and development of mRNA vaccine constructs encoding Borrelia OspA.

[0008] BACKGROUND

[0009] Lyme disease is caused by spirochete bacterial members of the Borrelia burgdorferi sensu lato genospecies, that are vectored by Ixodes hard ticks. It is the most common vector borne disease in Northern latitudes. In North America, B. burgdorferi sensu stricto (B. burgdorferi) is the dominant genospecies. In Europe, a broader range of genospecies circulate, that include principally B. afzelii, B. garinii, and B. bavariensis, as well as B. burgdorferi. Symptoms of Lyme disease include rash, fever, headaches, neurological symptoms, tiredness, joint pain, memory problems, and heart palpitations. Thus, in view of the high prevalence and public health burden of Lyme disease, there remains a need for improved vaccines for the prevention of Lyme disease.

[0010] SUMMARY OF THE INVENTION

[0011] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the RNA molecules further comprise a linker sequence located between the open reading frame encoding a structural domain polypeptide and the open reading frame encoding an OspA polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is a full-length polypeptide. In some embodiments, the OspA polypeptide is a C-terminal fragment. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the open reading frame encodes at least one of ST1 , ST2, ST3, ST4, ST5, ST6, ST7, ST8, ST9, ST10, ST11 , ST12, ST13, ST14, ST15, ST16, ST17, ST18, ST19, ST20, ST21 , ST22, ST23, ST24 and ST25. In some embodiments, the open reading from encodes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more of ST1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types. In some embodiments, the OspA polypeptide has at leaast 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 , or 54. In some embodiments, the structural domain polypeptide comprises a cytoplasmic domain, a transmembrane domain and an extracellular stalk. In some embodiments, the structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:14-19 or 59-62. In some embodiments, the RNA molecules encode an influenza neuraminidase (NA) structural domain polypeptide and linker having at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:70- 75. In some aspects, the RNA molecules encode a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52. In some embodiments, the RNA molecule has at least 90% identity to SEQ ID NQ:20 or SEQ ID NO:21. In some embodiments, the RNA molecule has at least 90% identity to any one of SEQ ID NOs:22-27, 53, or 55.

[0012] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the RNA molecules further comprise a linker sequence located between the open reading frame encoding an influenza NA structural domain polypeptide and the open reading frame encoding an OspA polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID N0s:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 , or 54. In some embodiments, the NA structural domain polypeptide comprises a cytoplasmic domain, a transmembrane domain and an extracellular stalk. In some embodiments, the NA structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:14-19 or 62. In some embodiments, the RNA molecules encode an influenza NA structural domain polypeptide and linker having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:70-75. In some aspects, the RNA molecules encode a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52. In some embodiments, the RNA molecule has at least 90% identity to SEQ ID NQ:20 or SEQ ID NO:21. In some embodiments, the RNA molecule has at least 90% identity to any one of SEQ ID NOs:22-27, 53, or 55.

[0013] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the RNA molecules further comprise a linker sequence located between the open reading frame encoding a PIV5 structural domain polypeptide and the open reading frame encoding an OspA polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 or 54. In some embodiments, the PIV5 structural domain polypeptide comprises a cytoplasmic domain, a transmembrane domain and an extracellular stalk. In some embodiments, the PIV5 structural domain polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NOs:59. In some embodiments, the RNA molecule has at least 90% identity to any one of SEQ ID NO:76.

[0014] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the RNA molecules further comprise a linker sequence located between the open reading frame encoding an RSV-G structural domain polypeptide and the open reading frame encoding an OspA polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 , or 54. In some embodiments, the RSV-G structural domain polypeptide comprises a cytoplasmic domain, a transmembrane domain and an extracellular stalk. In some embodiments, the RSV-G structural domain polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NOs:60 or 61. In some embodiments, the RNA molecule has at least 90% identity to any one of SEQ ID NOs:77-78.

[0015] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-7, 50-51 , 54. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33- 49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types.

[0016] In some embodiments, the RNA molecules disclosed herein further comprise a 5' untranslated region (5' UTR). In some embodiments, the 5' UTR comprises SEQ ID NO:28. In some embodiments, the RNA molecules further comprise a 3' untranslated region (3' UTR). In some embodiments, the 3' UTR comprises SEQ ID NO:29. In some embodiments, the RNA molecules disclosed herein comprise a linker, wherein the linker sequence comprises SEQ ID NO:30. In some embodiments, the RNA molecules disclosed herein further comprise a 3' poly-A tail. In some embodiments, the poly-A tail comprises SEQ ID NO:31. In some embodiments, the RNA molecules disclosed herein further comprise a 5’ cap moiety. In some embodiments, the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%. In some embodiments, the RNA molecules disclosed herein comprise at least one modified nucleotide. In some embodiments, the RNA molecules disclosed herein comprise at least one uridine replaced by N1- methylpseudouridine (^P). In some embodiments, each uridine is replaced by N1- methylpseudouridine (^P). In some embodiments, the RNA molecules disclosed herein are selfamplifying RNA (saRNA).

[0017] In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog. In some embodiments, the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315). In some embodiments, the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycollipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3'- di(tetradecanoyloxy)propyl-1-O-((o- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>- methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the neutral lipid is 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl- oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE). In some embodiments, the steroid or steroid analog is cholesterol.

[0018] In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the infection, disease, or condition is Lyme disease. In some embodiments, the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. In some embodiments, the RNA molecule is administered as a vaccine. In some embodiments, the subject is administered a single dose, two doses, three doses, or more, and optionally, a booster dose, for example an annual booster dose, of the RNA molecule.

[0019] In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0020] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 , or 54. In some embodiments, the NA structural domain polypeptide, PIV5 structural domain or RSV-G structural domain has at least 90% identity to any one of SEQ ID NOs:14-19 or 59-62. In some embodiments, the RNA molecules encode an influenza NA structural domain polypeptide and linker having at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:70-75. In some aspects, the RNA molecules encode a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52. In some embodiments, the RNA molecule has at least 90% identity to SEQ ID NQ:20 or SEQ ID NO:21. In some embodiments, the 5' UTR comprises SEQ ID NO:28. In some embodiments, the 3' UTR comprises SEQ ID NO:29. In some embodiments, the linker sequence comprises SEQ ID NQ:30. In some embodiments, the poly-A tail comprises SEQ ID NO:31. In some embodiments, the RNA molecule further comprises a 5’ cap moiety. In some embodiments, the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%. In some embodiments, the RNA molecule comprises at least one modified nucleotide. In some embodiments, the RNA molecules disclosed herein comprise at least one uridine replaced by N1-methylpseudouridine (^P). In some embodiments, each uridine is replaced by N1 -methylpseudouridine (^P).

[0021] In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog. In some embodiments, the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315). In some embodiments, the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycollipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3'- di(tetradecanoyloxy)propyl-1-O-((o- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>- methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the neutral lipid is 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl- oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE). In some embodiments, the steroid or steroid analog is cholesterol.

[0022] In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide;

[0023] (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide;

[0024] (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail.

[0025] In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV- G) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail. In some embodiments, the infection, disease, or condition is Lyme disease. In some embodiments, the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. In some embodiments, the RNA molecule is administered as a vaccine. In some embodiments, the subject is administered a single dose, two doses, three doses, four doses or more, and optionally, a booster dose, for example an annual booster dose, of the RNA molecule.

[0026] In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutininneuraminidase (PIV5) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding a respiratory syncytial virus (RSV) protein G (RSV- G) structural domain polypeptide; (c) a linker sequence; (d) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (e) a 3’ untranslated region (3’ UTR); and (f) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types and / or serotypes. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4- 7, 50-51 , or 54. In some embodiments, the NA structural domain, PIV5 structural domain or RSV- G structural domain polypeptide has at least 90% identity to any one of SEQ ID NOs:14-19 or 59- 62. In some embodiments, the RNA molecules encode an influenza NA structural domain polypeptide and linker having at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:70-75. In some aspects, the RNA molecules encode a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52. In some embodiments, the RNA molecule has at least 90% identity to SEQ ID NQ:20 or SEQ ID NO:21. In some embodiments, the 5' UTR comprises SEQ ID NO:28. In some embodiments, the 3' UTR comprises SEQ ID NO:29. In some embodiments, the linker sequence comprises SEQ ID NQ:30. In some embodiments, the poly-A tail comprises SEQ ID NO:31 . In some embodiments, the RNA molecule further comprises a 5’ cap moiety. In some embodiments, the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%. In some embodiments, the RNA comprises at least one modified nucleotide. In some embodiments, at least one uridine is replaced by N1- methylpseudouridine (^P). In some embodiments, each uridine is replaced by N1- methylpseudouridine (^P).

[0027] In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog. In some embodiments, the cationic lipid is (4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). In some embodiments, the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycol-lipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- O-(2’,3'- di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(u>- methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE). In some embodiments, the steroid or steroid analog is cholesterol.

[0028] In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail. In some embodiments, the infection, disease, or condition is Lyme disease. In some embodiments, the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. In some embodiments, the RNA molecule is administered as a vaccine. In some embodiments, the subject is administered a single dose, two doses, three doses, four doses or more, and optionally, a booster dose, for example an annual booster dose, of the RNA molecule. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) a 5' untranslated region (5’ UTR); (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide; (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide; (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide; (e) a linker sequence; (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide; (g) a 3’ untranslated region (3’ UTR); and (h) a 3’ poly-A tail, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0029] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a signal peptide derived from an immunoglobulin kappa (IgGK) light chain; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the signal peptide comprises SEQ ID NO:32. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33- 49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types and / or serotypes. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 , or 54. In some embodiments, the RNA molecule has an mRNA sequence having at least 90% identity to SEQ ID NO:20 or SEQ ID NO:21 . In some embodiments, the RNA molecules disclosed herein further comprise a 5' untranslated region (5' UTR). In some embodiments, the 5' UTR comprises SEQ ID NO:28. In some embodiments, the RNA molecules disclosed herein further comprise a 3' untranslated region (3' UTR). In some embodiments, the 3' UTR comprises SEQ ID NO:29. In some embodiments, the RNA molecules disclosed herein further comprise a 3' poly-A tail. In some embodiments, the poly-A tail comprises SEQ ID NO:31. In some embodiments, the RNA molecules disclosed herein further comprise a 5’ cap moiety. In some embodiments, the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%. In some embodiments, the RNA molecule comprises at least one modified nucleotide. In some embodiments, at least one uridine is replaced by N1 -methylpseudouridine (^P). In some embodiments, each uridine is replaced by N1-methylpseudouridine (^P).

[0030] In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a signal peptide derived from an immunoglobulin kappa (IgGK) light chain; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog. In some embodiments, the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315). In some embodiments, the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycollipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3'- di(tetradecanoyloxy)propyl-1-O-((o- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>- methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the neutral lipid is 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl- oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE). In some embodiments, the steroid or steroid analog is cholesterol.

[0031] In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a signal peptide derived from an immunoglobulin kappa (IgGK) light chain; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a signal peptide derived from an immunoglobulin kappa (IgGK) light chain; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the infection, disease, or condition is Lyme disease. In some embodiments, the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. In some embodiments, the RNA molecule is administered as a vaccine. In some embodiments, the subject is administered a single dose, two doses, three doses, four doses or more, and optionally, a booster dose, for example an annual booster dose, of the RNA molecule. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a signal peptide derived from an immunoglobulin kappa (IgGK) light chain; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a signal peptide derived from an immunoglobulin kappa (IgGK) light chain; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0032] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding a membrane anchoring moiety polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the RNA molecules further comprise a linker sequence located between the open reading frame encoding a membrane anchoring moiety polypeptide and the open reading frame encoding an OspA polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types and / or serotypes. In some embodiments, the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25 and / or serotypes (ST) 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:63-69. In some embodiments, the open reading frame encoding an OspA polypeptide comprises one or more linkers. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides. In some embodiments, the at least two OspA fragment polypeptides are different in silico types and / or serotypes. In some embodiments, the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4- 7, 50-51 , or 54. In some embodiments, the membrane anchoring moiety polypeptide comprises an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide or a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide. In some embodiments, the NA structural domain polypeptide, PIV5 structural domain polypeptide or RSV-G structural domain polypeptide comprises a cytoplasmic domain, a transmembrane domain and an extracellular stalk. In some embodiments, the NA structural domain polypeptide, PI 5 structural domain polypeptide or RSV-G structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:14-19 or 59-62. In some embodiments, the RNA molecules encode an influenza NA structural domain polypeptide and linker having at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:70-75. In some aspects, the RNA molecules encode a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52. In some embodiments, the RNA molecule has at least 90% identity to SEQ ID NO:20 or SEQ ID NO:21. In some embodiments, the RNA molecule has at least 90% identity to any one of SEQ ID NOs:22-27, 53, 55 or 76-78. In some embodiments, the RNA molecules disclosed herein further comprise a 5' untranslated region (5' UTR). In some embodiments, the 5' UTR comprises SEQ ID NO:28. In some embodiments, the RNA molecules further comprise a 3' untranslated region (3' UTR). In some embodiments, the 3' UTR comprises SEQ ID NO:29. In some embodiments, the RNA molecules disclosed herein comprise a linker, wherein the linker sequence comprises SEQ ID NQ:30. In some embodiments, the RNA molecules disclosed herein further comprise a 3' poly-A tail. In some embodiments, the poly-A tail comprises SEQ ID NO:31. In some embodiments, the RNA molecules disclosed herein further comprise a 5’ cap moiety. In some embodiments, the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%. In some embodiments, the RNA molecules disclosed herein comprise at least one modified nucleotide. In some embodiments, the RNA molecules disclosed herein comprise at least one uridine replaced by N1-methylpseudouridine (^P). In some embodiments, each uridine is replaced by N1-methylpseudouridine (^P).

[0033] In some embodiments, disclosed herein are ribonucleic acid (RNA) molecules comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide and / or a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more of in silico types 1-25. In some embodiments, the OspA polypeptide comprises an OspA C-terminal fragment from one or more of in silico types 1- 25. In some embodiments, the OspA polypeptide comprises an aglycosylated OspA C-terminal fragment from one or more of in silico types 1-25. In some embodiments, the OspA polypeptide has at least 90% identity to an OspA C-terminal fragment of any one of the amino acid sequences of SEQ ID NOs:33-49 or 79-86. In some embodiments, the OspA C-terminal fragment of any one of the amino acid sequences of SEQ ID Nos:33-49 or 79-86 comprises about 100, 110, 120, 130, 140, or 150 C-terminal amino acids of SEQ ID Nos:33-49 or 79-86. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA C-terminal fragment polypeptides. In some embodiments, the open reading frame encoding an OspA polypeptide comprises at least two OspA C-terminal fragment polypeptides of different in silico types. In some embodiments, the OspA polypeptide has at least 90% identity to the OspA polypeptide portion of any one of the amino acid sequences of SEQ ID NOs: 1-13, 50-52, 54, or 63-69. In some embodiments, the influenza NA structural domain polypeptide has at least 90% identity to any one of SEQ ID NOs:14-19 or 62, the influenza NA structural domain polypeptide and a linker have at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:70- 75, the PIV5 structural domain polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:59 and / or the RSV-G structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:60-61. In some embodiments, the RNA molecule encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:8-13 or 52. In some embodiments, the RNA molecule has at least 90% identity to any one of SEQ ID NQs:20-27, 53, 55 or 76-78. In some embodiments, the RNA molecule further comprises a linker between the influenza NA, PIV5 and / or RSV-G structural domain polypeptide and the OspA polypeptide. In some embodiments, the RNA molecule further comprises a 5' cap moiety, a 5’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR) and 3’ poly-A tail. In some embodiments, the RNA comprises at least one modified nucleotide. In some embodiments, the RNA comprises at least one modified nucleotide wherein at least one uridine is replaced by N1-methylpseudouridine (^P). In some embodiments, disclosed herein are compositions comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide and / or a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog. In some embodiments, disclosed herein are methods of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of an RNA molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide and / or a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide. In some embodiments, the infection, disease, or condition is Lyme disease. In some embodiments, the RNA molecule is administered as a vaccine. In some embodiments, disclosed herein are methods of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of a composition comprising a ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction: (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide and / or a respiratory syncytial virus (RSV) protein G (RSV- G) structural domain polypeptide; and (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1A-C depicts expression of OspA and C-terminal fragment OspA in ExpiCHO cells. To remove N-glycosylation sites, sequences of 77 natural OspA variants (across in silico types 1- 10) were aligned and ‘N’ residues mutated to consensus amino acids across all loci (FIG. 1A). X-axis groupings refer to asparagines targeted for substitution based on their high probability of being N-glycosylated in mammalian cells (as predicted by ‘NetNGIyc’ software). Amino acid substitutions were chosen based on highest frequency (Y-axis) of natural occurrence variation among aligned sequences. FIG. 1B depicts locations of amino acid substitutions to remove N- glycosylation. FIG. 1C depicts an anti-His tag Western blot of OspA / C-terminal fragment OspA ST1-2 protein expression in ExpiCHO cell supernatants (S / N) and cells. When preparing cell culture samples for Western Blot analysis, cell pellets were resuspended in an equivalent volume of 1X PBS as the original sample, 200 pL.

[0036] FIG. 2 depicts an overview of the C-terminal fragment OspA ST1-2 protein, showing predicted disulfide bond formations and the 8x N-terminal His tag used in nickel chromatography purification.

[0037] FIG. 3 depicts an exemplary design for mRNA constructs encoding secreted C-terminal fragment OspA ST1-2. mRNA constructs were designed to encode OspA antigens with an N-terminal secretion signal sequence.

[0038] FIG. 4A-B depicts expression of secreted C-terminal fragment OspA ST1-2 from modRNA transcripts. Expi293 cells were transfected with BMD576 modRNA transcripts encoding secreted aglycosylated C-terminal fragment OspA ST1-2. FIG. 4A shows quantification of C-terminal fragment OspA ST1-2 in Expi293 culture supernatant over a 12-point titration, using lipidated C- terminal fragment OspA ST1-2 protein as standard. Detected using mAb 2-30. Measured by Octet. FIG. 4B shows anti-OspA Western blot with mAb 2-30, used at 1 pg / mL, on transfected Expi293 cells and cell culture supernatant. “Cell” = cell pellet, “S / N” = supernatant, “R” = reduced with 10 mM DTT, “NR” = non-reduced. Expected molecular weight = 34.3 kDa. When preparing cell culture samples for Western Blot analysis, cell pellets were resuspended in an equivalent volume of 1X PBS as the original sample, 200 pL.

[0039] FIG. 5 depicts an exemplary design for mRNA constructs encoding membrane-anchored OspA antigens. mRNA constructs were designed to encode C-terminal fragment OspA antigens with an N-terminal membrane targeting sequence.

[0040] FIG. 6 depicts the percentage of Expi293 cells expressing C-terminal fragment OspA ST1-2 on the surface. Expression of C-terminal fragment OspAST1-2 on the surface of Expi293 suspension cells was determined after overnight transfection with modRNA diluted five-fold over a range from 50.0 ng to 0.005 pg for membrane targeted C-terminal fragment OspA. OspA monoclonal antibody 1-15 was used for primary staining, with anti-human PE Conjugated Ab (JACKSON IMMUNORESEARCH LABS, 109-116-170) used for detection by Fortessa flow cytometer.

[0041] FIG. 7A-D depicts the expression of C-terminal fragment OspA on the surface of muscle skeletal cells. Representative confocal microscope images of C-terminal fragment OspA 5 ST1-2 expressed on the surface of muscle skeletal cells are shown. Cells were transfected with 120 ng C-terminal fragment OspA ST1-2 construct anchored by A / WSN / 33 or A / WSN / 33 plus stalk region domains of neuraminidase (NA). All images are at 40X magnification. C-terminal fragment OspA ST1-2 was stained with OspA specific monoclonal antibody 1-15, which recognizes epitope 239-GTKLEGSAVEI-249 of OspA ST1. Goat anti-Human Ig Alexa Fluor™ 488 secondary antibody was used for green fluorescence and nuclei were stained using 4’6-Diamidino-2- Phenylindole, Dilactate (DAPI). FIG. 7A: A / WSN / 33 total staining; FIG. 7B: A / WSN / 33 surface staining; FIG. 7C: A / WSN / 33 plus stalk total staining; FIG. 7D: A / WSN / 33 plus stalk surface staining.

[0042] FIG. 8 depicts the quantitation of OspA serotype-specific IgG response in immunized CD-1 mice. The figure displays OspA-specific IgG responses in serum from ST1-2 immunized CD-1 female mice vaccinated intramuscularly with modRNA LNPs at post-dose 2 (PD2) and post-dose 3 (PD3) timepoints. FIG. 8A: Week 6 (W6) anti-ST1 IgG; FIG. 8B: Week 10 (W10) anti-ST1 IgG; FIG. 8C: Week 6 (W6) anti-ST2 IgG; FIG. 8D: Week (10) anti-ST2 IgG. The saline (farthest left) data represents a negative control. The next six sets of data symbols represent modRNA LNPs with neuraminidase (NA) anchors, comprising a WSN33 NA anchor (0.1 pg or 1 pg) or WSN33 NA anchor + stalk (0.1 pg or 1 pg ), or secreted construct without an anchor (0.1 pg or 1 pg ), while filled symbols distinguish higher from lower doses. The lower horizontal dotted line represents lower limit of quantification (LLOQ) of the assay and the upper horizonal dotted line marks responder threshold from which responder rates were calculated and listed above each scatter plot alongside GMTs.

[0043] FIG. 9A-B depicts expression of secreted C-terminal fragment OspA ST9-10 from modRNA transcripts. FIG. 9A shows a diagram of the BMD576 modRNA transcript encoding secreted aglycosylated C-terminal fragment OspA ST9-10 used to transfect Expi293 cells. Sites above the diagram indicate asparagine residues that were mutated to prevent glycosylation in mammalian cells (N16D N27D N154S N196D). Sites below the diagram indicate residues that were mutated to cysteine to introduce the disulfide bonds necessary to maintain proper folding in C-terminal fragment OspA (E57C K143C E226C K314C). FIG. 9B shows the results of an anti- OspA Western blot with polyclonal Ab LYM1864 on transfected Expi293 cells and cell culture supernatant. “Cell” = cell pellet, “S / N” = supernatant, “R” = reduced with 50 mM DTT, “NR” = non- reduced. Expected molecular weight = 33.8 kDa. When preparing cell culture samples for Western Blot analysis, cell pellets were resuspended in an equivalent volume of spent media as the original sample, 50 pL. As a control, purified ST9-10 protein, “IST9-10”, was run in the last lane under reduced and non-reduced conditions.

[0044] FIG. 10A-B depicts percentage of Expi293 cells expressing C-terminal fragment OspA ST9-10 on the surface. C-terminal fragment OspA ST9-10 cell surface expression was detected by flow cytometry. FIG. 10A shows a diagram of the modRNA transcript encoding C-terminal fragment OspA ST9-10 with the neuraminidase N-terminal anchor used to transfect Expi293 suspension cells. Sites above the diagram indicate asparagine residues that were mutated to prevent glycosylation in mammalian cells (N16D N27D N154S N196D). Sites below the diagram indicate residues that were mutated to cysteine to introduce the disulfide bonds necessary for proper folding in C-terminal fragment OspA (E57C K143C E226C K314C). FIG. 10B shows expression of C-terminal fragment OspA ST9-10 (circles) and C-terminal fragment OspA ST1-2 (squares) on the surface of Expi293 suspension cells determined after overnight transfection with modRNA diluted five-fold over a range from 50.0 ng to 0.005 pg. Polyclonal Ab LYM1864 was used for primary staining, with anti-human PE Conjugated Ab (JACKSON IMMUNORESEARCH LABS, 109-116-170) used for detection by Fortessa flow cytometer. C-terminal fragment OspA ST1-2 modRNA was used as a control for comparison.

[0045] FIG. 11A-B depicts percentage of Expi293 cells expressing C-terminal fragment OspA ST1-2 on the surface using four different anchors, including haemagglutinin-neuraminidase of simian virus 5 I parainfluenza virus 5 (PIV5; circles), WSN33 NA(triangles), 66 N-terminal amino acids of human Respiratory Syncytial Virus Protein G (RSV-G(1-66); diamonds), and 73 N-terminal amino acids of RSV-G (RSV-G(1-73); squares). FIG. 11A shows percent ST1-2 positive cells for ST1 , mAb 1-15 (Expi293). FIG. 11B shows percent ST1-2 positive cells for ST2, mAb 2-30 (Expi293).

[0046] DETAILED DESCRIPTION

[0047] Lyme disease is the most common disease spread by ticks in the Northern Hemisphere and represents a tremendous public healthcare challenge. RNA-based vaccines have demonstrated success in combating global pandemics. Accordingly, described herein are mRNA-based Lyme disease vaccine compositions based on the OspA (outer surface protein A) lipoprotein.

[0048] Outer surface protein A (OspA) is a membrane-anchored lipoprotein expressed by Borrelia that is a critical factor required for stable persistence in the tick. It is robustly expressed by Borrelia during the tick cycle but turned off during transmission to the host. OspA is required for stable tick colonization via binding to TROSPA [Pal U. et al., TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi. Cell 2004; 119(4): 457-68], the receptor for OspA on the tick gut epithelium. OspA is attached to the spirochete outer membrane via an N-terminal lipid moiety, thus its C-terminus is accessible for anti-OspA antibodies. Consequently, antibodies that target the C-terminus are critical for protection [Golde et al., Reactivity with a specific epitope of outer surface protein A predicts protection from infection with the Lyme disease spirochete, Borrelia burgdorferi. Infect Immun 1997; 65(3): 882-9], OspA vaccines act through a novel mechanism involving neutralization of the Borrelia bacteria in the tick midgut, that prevents transmission to the animal host during tick feeding. OspA-based vaccines target spirochetes in the tick gut via neutralization by OspA-antibodies in a complement-independent manner [Dattwyler RJ, Gomes- Solecki M, The year that shaped the outcome of the OspA vaccine for human Lyme disease. NPJ Vaccines 2022; 7(1):10; Schwendinger MG, et al., Evaluation of OspA vaccination-induced serological correlates of protection against Lyme borreliosis in a mouse model. PLoS One 2013; 8(11): e79022]. Additionally, anti-OspA antibodies may also mediate complement-dependent bactericidal activity to kill Borrelia directly in the tick midgut. OspA variants are associated with individual genospecies: B. burgdorferi expresses OspA serotype (ST) 1 , B. afzelii expresses OspA ST2, European B. bavariensis isolates express OspA ST4, B. garinii are associated with OspA STs 3, 5, 6 and 7. VLA15, an investigational hexavalent OspA vaccine designed to prevent Lyme disease caused by Borrelia spp., is based on Outer Surface Protein A (OspA) STs 1-6 [Comstedt P, et al. Design and development of a novel vaccine for protection against Lyme borreliosis. PloS one 2014; 9(11): e113294],

[0049] A vaccine based on the C-terminal half of OspA conferred partial protection in a mouse model of Lyme infection, and introduction of mutations that stabilized the structure of the C-terminus increased both stability and protection to levels similar to that of the full length OspA protein [Koide S, Yang X, Huang X, Dunn J J, Luft BJ. Structure-based design of a second-generation Lyme disease vaccine based on a C-terminal fragment of Borrelia burgdorferi OspA. J Mol Biol 2005; 350(2): 290-9], The VLA15 vaccine candidate is composed of three fusion proteins, each comprised of two C-terminal fragments of OspA from different serotypes connected via a polypeptide linker (derived from OspA). Both of the C-terminal fragments of VLA15 contain a disulfide linkage that stabilizes the structure of each fragment; immunization with recombinant VLA15 protein was shown to be protective in mice [Comstedt P, et al. Design and development of a novel vaccine for protection against Lyme borreliosis. PloS one 2014; 9(11): e113294], The N-terminal subunit is lipidated, similar to full-length native OspA, and the lipidation serves as an adjuvant through TLR1 / 2 activation.

[0050] Accordingly, described herein are RNA molecules (e.g., RNA polynucleotides) comprising at least one open reading frame (ORF) encoding a Borrelia antigen. In some aspects, the Borrelia antigen is a Borrelia polypeptide. In some aspects, the Borrelia polypeptide is an outer surface protein A (OspA) polypeptide. In some aspects, disclosed herein are mRNA vaccine compositions for C-terminal fragment OspA and full length OspA. As described herein, full-length OspA and C-terminal fragment OspA proteins were expressed in mammalian cells as wild type versions as well as mutants wherein non-native glycosylation sites were removed by introduction of mutations to alternative amino acids. RNA molecules comprising modified nucleotides (modRNA) transcripts encoding secreted or membrane-tethered OspA and C-terminal fragment OspA were confirmed to drive the expression of proteins in the cell supernatant or the surface of Expi293 cells, respectively. Additionally, the design of mRNA vaccine constructs encoding secreted or membrane-anchored full length OspA and C-terminal fragment OspA are described. As described herein, OspA can be produced at high levels in in mammalian cells (as a full-length or C-terminal fragment presentation) and retains similar properties to protein produced in E. coli. In some aspects the RNA molecule comprises at least one of a 5' cap, 5' UTR, 3' UTR and poly- A tail. The present disclosure provides for an RNA molecule comprising modified nucleotides (e.g., modified RNA; modRNA).

[0051] The present disclosure provides for an immunogenic composition comprising any one of the RNA molecules encoding a OspA polypeptide described herein complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (RNA-LNPs). The present disclosure further provides for an immunogenic composition comprising any one of the RNA molecules comprising at least one RNA nucleic acid described herein complexed with, encapsulated in, and / or formulated with one or more lipids, and forming RNA-LNPs. The present disclosure further provides for a method of preventing, treating and / or ameliorating an infection, disease and / or condition {e.g., Lyme disease and Lyme borreliosis) in a subject via administering to a subject an effective amount of an RNA molecule, RNA-LNP and / or an immunogenic composition described herein. The present disclosure further provides for the use of the RNA molecule, RNA-LNP and / or an immunogenic compositions described herein as a vaccine.

[0052] CERTAIN DEFINITIONS

[0053] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some aspects, the term “about” may encompass a range of values that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less of the measurement or quantitation.

[0054] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0055] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0056] The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.

[0057] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. It is contemplated that aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.” Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of” (and any form of consisting of, such as “consist of’ and “consists of”) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0058] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” “a further embodiment,” “some embodiments”, “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” “a further aspect,” “some aspects” or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

[0059] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.

[0060] As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and / or determined substantially simultaneously and / or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and / or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.

[0061] The term “DNA,” as used herein, means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or thymine (T)). For example, DNA can contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. Without any limitation, DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.

[0062] The term “RNA,” as used herein, means a nucleic acid molecule that includes ribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or uracil (II) or N-1-methylpseudouridine). For example, RNA can contain all, or a majority of, ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. In one aspect, RNA can be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein. As known to those of skill in the art, mRNA generally contains a 5' untranslated region (5 -llTR), a polypeptide coding region, and a 3' untranslated region (3 -llTR). Without any limitation, RNA can encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, circular RNA, self-amplifying RNA (saRNA), guide RNA (gRNA), and modified RNA (modRNA).

[0063] The term “RNA drug substance,” as used herein, means a purified RNA that is solubilized in any form of aqueous solution appropriate to permit subsequent encapsulation of the RNA within encapsulating agents as described below.

[0064] The term “RNA drug product,” as used herein, means a purified RNA that has been encapsulated in any form of encapsulating agents (e.g., lipid nanoparticles) described herein to form a colloidal dispersion and where the resulting colloidal dispersion has been purified to stabilize the encapsulated RNA.

[0065] As contemplated herein, without any limitations, RNA can be used as a therapeutic modality to treat and / or prevent a number of conditions in mammals, including humans. Methods contemplated comprise administration of the RNA described herein to a mammal, such as a human. For example, in one aspect, such methods of use for RNA include an antigen-coding RNA vaccine to induce robust neutralizing antibodies and accompanying / concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses. The RNA administered is preferably in vitro transcribed RNA.

[0066] An “isolated RNA” is defined as an RNA molecule that can be recombinant or has been isolated from total genomic nucleic acid. A “modified RNA” or “modRNA” refers to an RNA molecule, e.g., an mRNA molecule, having at least one addition, deletion, substitution, and / or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations can refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5' and / or 3' end(s) of RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide. For example, a modified nucleotide can replace one or more uridine and / or cytidine nucleotides. For example, these replacements can occur for every instance of uridine and / or cytidine in the RNA sequence, or can occur for only select uridine and / or cytidine nucleotides. Such alterations to the standard nucleotides in RNA can include nonstandard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide can be replaced with 1 -methylpseudouridine in an RNA sequence. Other such altered nucleotides are known to those of skill in the art. Such altered RNAs are considered analogs of naturally-occurring RNA. In some aspects, the RNA is produced by continuous-flow in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, the RNA can be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA). In some aspects, the RNA can be guide RNA (gRNA) or circular RNA.

[0067] As used herein, a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues. As used herein, the term “wild-type” or “native” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0068] The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (e.g., when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (e.g., when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in the functional state and / or that is altered or removed from the natural state through human intervention. For example, a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered.

[0069] All patents, published patent applications, other publications, and databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0070] LYME DISEASE

[0071] The present disclosure provides for RNA molecules (e.g., RNA polynucleotides) comprising at least one open reading frame encoding a Borrelia antigen. In some aspects, the Borrelia antigen is a Borrelia polypeptide. In some aspects, the Borrelia polypeptide is an outer surface protein A (OspA) polypeptide. The present disclosure further provides for an immunogenic composition comprising at least one RNA molecule encoding a OspA polypeptide complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (LNPs).

[0072] Lyme disease, also known as Lyme borreliosis, is a vector-borne disease caused by Borrelia bacteria, which are spread by ticks in the genus Ixodes. Outer surface protein A (OspA) is an immunogenic lipoprotein of Borrelia. There are many existing serotypes of OspA. Serotype 1 is produced by B. burgdorferi, serotype 2 by B. afzelii, serotype 4 by B. bavariensis, and serotypes 3, 5-7 by B. garinii. As used herein, an OspA serotype can also be referred to as an OspA in silico type (1ST). OspA ISTs can be identified using a in silico sequence-based method for OspA typing using next-generation sequence data, such as a method using a compiled database of over 400 Borrelia genomes encompassing all major genospecies that defines boundaries for classification and assignment of OspA types based on sequence similarity [Lee et al., Development of a sequence-based in silico OspA typing method for Borrelia burgdorferi sensu lato Microb Genom 10(5):001252 (2024)].

[0073] Lyme disease occurs regularly in Northern hemisphere temperate regions. In Europe, Lyme disease is caused by infection with one or more pathogenic European genospecies of the spirochaete B. burgdorferi sensu lato, mainly transmitted by the tick Ixodes ricinus. The three B. burgdorferi s.l. species collectively responsible for most infections in Europe, B. afzelii, B. bavariensis, and B. garinii, are not found in the United States, where most infections are caused by B. burgdorferi sensu stricto. B. burgdorferi is also endemic however in Europe.

[0074] In some aspects, the RNA molecules disclosed herein comprise an open reading frame encoding a Borrelia antigen. In some aspects, the Borrelia antigen is a Borrelia polypeptide. In some aspects, the Borrelia polypeptide is a Borrelia lipoprotein (e.g., outer surface protein A [OspA], outer surface protein B [OspB], outer surface protein C [OspC], outer surface protein D [OspD], outer surface protein E [OspE], outer surface protein F [OspF], Borrelial persistence in ticks A [BptA], BBA64 [also known as P35], decorin-binding protein A [DbpA], decorin-binding protein B [DbpB], BBK32, variable major protein like sequence [VIsE], and complement regulator-acquiring surface proteins [CRASPs, including OspE-related proteins, CspA, and CspZ]) or a fragment or a variant thereof. In some aspects, the RNA molecule encodes a Boreilia OspA polypeptide.

[0075] In some aspects, the Borrelia polypeptide is designed to avoid ER / golgi retention of polypeptides, leading to increased surface expression of the antigen. In some embodiments, the variant polypeptides are truncated to remove the ER retention portion (e.g., the anchor domain) and / or the cytoplasmic tail portion of the polypeptide. In some embodiments, the Borrelia polypeptides are mutated (e.g., mutations in one or more phosphorylated acidic motif(s)) to reduce Borrelia polypeptide localization to the ER / golgi / TGN. Such modifications inhibit ER trapping and, as such, expedite trafficking to the cell membrane. In some aspects, the Borrelia polypeptide comprises an additional sequence to aid secretion of the polypeptide.

[0076] In some aspects, the Borrelia polypeptide is a full-length Borrelia polypeptide. In some aspects, the Borrelia polypeptide is a truncated Borrelia polypeptide. In some aspects, the Borrelia polypeptide is a variant of an Borrelia polypeptide. In some aspects, the Borrelia polypeptide is a fragment of a Borrelia polypeptide.

[0077] In some aspects, the Borrelia polypeptide is a full-length OspA polypeptide. In some aspects, the Borrelia polypeptide is a truncated OspA polypeptide. In some aspects, the Borrelia polypeptide is a variant of a OspA polypeptide. In some aspects, the Borrelia polypeptide is a fragment of a OspA polypeptide.

[0078] In some aspects, the Borrelia polypeptide comprises at least one mutation. In some aspects, the Borrelia polypeptide is a Borrelia OspA polypeptide comprising at least one mutation.

[0079] In some aspects, the RNA molecule encodes a Borrelia OspA polypeptide comprising the amino acid sequence according to any one of GEN BANK® Accession Nos.: AAC66260.1 , WP_010890387.1 , ARS30922.1 , ARS32180.1 , and / or ARS32664.1 , or fragment or variant thereof, the respective sequences of which are herein incorporated by reference. In some aspects, the RNA molecule encodes a Borrelia OspA polypeptide comprising the amino acid sequence according to GEN BANK® Accession No. AAC66260.1 , or fragment or variant thereof, the sequence of which is herein incorporated by reference.

[0080] In some aspects, the RNA molecule encodes a Borrelia OspA polypeptide comprising an amino acid sequence of SEQ ID NO:1 , or fragment or variant thereof. In some aspects, the Borrelia OspA polypeptide may have at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1. In some aspects, the Borrelia OspA polypeptide consists of the amino acid sequence of SEQ I D NO: 1.

[0081] In some aspects, the RNA molecule sequence is transcribed from a DNA nucleic acid sequence (DNA polynucleotide). In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence of SEQ ID NO:4, or fragment or variant thereof. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence that may have at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic sequence of SEQ ID NO:4. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence that consists of the nucleic acid sequence of SEQ ID NO:4.

[0082] In some aspects, the RNA molecule comprises stabilized RNA. In some aspects, the RNA molecule comprises a nucleic acid sequence having at least one uridine replaced by N1- methylpseudouridine. In some aspects, the RNA molecule comprises a sequence having all uridines replaced by N1-methylpseudouridine (designated as ‘V). In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence of SEQ ID NO:4, wherein all uridines have been replaced by N1-methylpseudouridine (designated as ‘V).

[0083] RNA MOLECULE

[0084] In some aspects, the RNA molecule described herein is a coding RNA molecule. Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide. In some aspects, the coding RNA molecule includes at least one open reading frame (ORF) coding for at least one peptide or polypeptide. An open reading frame comprises a sequence of codons that is translatable into a peptide or protein. The coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) ORFs, which may be a sequence of codons that is translatable into a polypeptide or protein of interest.

[0085] The coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule, and / or self-amplifying RNA molecule (saRNA, also referred to as a replicon). In some aspects, the RNA molecule is an mRNA. Preferably, the RNA molecule of the present disclosure is an mRNA. In some aspects, the RNA molecule is a saRNA. In some aspects, the saRNA molecule may be a coding RNA molecule.

[0086] In some aspects, the RNA molecule described herein is a non-coding RNA molecule. A noncoding RNA (ncRNA) molecule includes a functional RNA molecule that is not translated into a peptide or polypeptide. Non-coding RNA molecules may include highly abundant and functionally important RNA molecules. In some aspects, the non-coding RNA is a functional mRNA molecule that is not translated into a peptide or polypeptide. The non-coding RNA may include modified nucleotides as described herein. Preferably, the RNA molecule is an mRNA.

[0087] The RNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten or more polypeptides. Alternatively, or in addition, one RNA molecule may also encode more than one polypeptide of interest, such as an antigen, e.g. , a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens. Bicistronic or multicistronic RNAs may include more than one polypeptide of interest with intervening sequences between the polypeptides of interest comprising an internal ribosome entry site (IRES) sequence(s) that allow for internal translation initiation between the polypeptides of interest, and / or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide. IRES sequences and 2A peptides may be used, in some aspects, to enhance expression of multiple proteins from the same vector. A variety of IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.

[0088] As used herein, the term “codon-optimized” refers to modification of codons in the coding region of a nucleic acid molecule to accommodate the codon bias a host organism without a corresponding modification to the amino acid sequence encoded by the nucleic acid molecule. Codon optimization, in some aspects, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability and / or reduce secondary structures; minimize tandem repeat codons and / or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert and / or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove and / or shuffle protein domains; insert and / or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; and / or reduce or eliminate problem secondary structures within the polynucleotide. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing uses of codon optimization can be excluded. Within the context of the present disclosure, in some aspects, coding regions are codon-optimized for optimal expression in a subject to be treated using an RNA polynucleotide described herein.

[0089] Codon-optimization is based on the finding that the translation efficiency may be determined by a different frequency in the occurrence of transfer RNAs (tRNAs) in cells. Thus, if so-called “rare codons” are present in the coding region of the inventive artificial nucleic acid molecule as defined herein, to an increased extent, the translation of the corresponding modified nucleic acid sequence is less efficient than in the case, where codons coding for relatively “frequent” tRNAs are present. Thus, the open reading frame of the RNA molecule is modified compared to the corresponding wild type coding region such that at least one codon of the wild type sequence, which is recognized by a tRNA, and which is relatively rare in the cell, is exchanged for a codon, which is recognized by a tRNA, and which is comparably frequent in the cell and carries the same amino acid as the relatively rare tRNA. By this modification, the open reading frame of the RNA molecule is modified such that codons for which frequently occurring tRNAs are available may replace codons that correspond to rare tRNAs. Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely, is known to a person skilled in the art (see, e.g., Akashi, Curr. Opin. Genet. Dev. 2001 , 1 1 (6): 660-666), and codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0090] The sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression and / or replication of the RNA, to provide additional stability and / or resistance to degradation, and / or to reduce immunogenicity, relative to an unmodified RNA molecule. For example, the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA. In some aspects, one or more of the foregoing reasons for modification of the RNA molecule can be excluded.

[0091] In some aspects, the RNA molecule of the present disclosure comprises an open reading frame having at least one codon modified sequence. A codon modified sequence relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type coding sequence. A codon modified sequence may show improved resistance to degradation, improved stability, and / or improved translatability.

[0092] In some aspects, G / C content of a coding region (e.g., of a gene of interest sequence; open reading frame (ORF)) of an RNA is increased compared to the G / C content of the corresponding coding sequence of a wild type RNA encoding the gene of interest, wherein in some aspects, the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine) / C (cytidine) content are more stable than sequences having an increased A (adenosine) / U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability may be determined (so-called alternative codon usage).

[0093] Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or II nucleosides may be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or II or contain a lower content of A and / or II nucleosides. Thus, in some aspects, G / C content of a coding region of an RNA described herein is increased by at least, at most, exactly, or between (inclusive or exclusive) any two of 10%, 20%, 30%, 40%, 50%, 55%, or even more compared to the G / C content of a coding region of a wild type RNA. In some aspects, the coding region of the VZV RNA described herein comprises a G / C content of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some aspects, the coding region of the VZV RNA described herein comprises a G / C content of or of about 50% to 75%, 55% to 70%, 50% to 60%, 60% to 70%, 70% to 80%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, or 75% to 80%. In some aspects, the coding region of the VZV RNA described herein comprises a G / C content of or of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%. In some aspects, the coding region of the VZV RNA described herein comprises a G / C content of or of about 58%, 66% or 62%.

[0094] In some aspects, the RNA molecule includes from or from about 20 to 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1 ,000, from 30 to 1 ,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1 ,000, from 100 to 1 ,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1 ,000, from 500 to 1 ,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1 ,000 to 1 ,500, from 1 ,000 to 2,000, from 1 ,000 to 3,000, from 1 ,000 to 5,000, from 1 ,000 to 7,000, from 1 ,000 to 10,000, from 1 ,000 to 25,000, from 1 ,000 to 50,000, from 1 ,000 to 70,000, from 1 ,000 to 100,000, from 1 ,500 to 3,000, from 1 ,500 to 5,000, from 1 ,500 to 7,000, from 1 ,500 to 10,000, from 1 ,500 to 25,000, from 1 ,500 to 50,000, from 1 ,500 to 70,000, from 1 ,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides).

[0095] In some aspects, the RNA molecule includes at least 100 nucleotides. For example, in some aspects, the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11 ,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; or between 7,000 and 25,000 nucleotides. In some aspects, the RNA molecule has at least, at most, exactly, between (inclusive or exclusive) any two of, or about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560,

[0096] 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940,

[0097] 960, 980, 1000, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350,

[0098] 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100,

[0099] 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850,

[0100] 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600,

[0101] 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350,

[0102] 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100,

[0103] 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850,

[0104] 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600,

[0105] 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350,

[0106] 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100,

[0107] 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850,

[0108] 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150,

[0109] 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800,

[0110] 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450,

[0111] 12500, 12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, 13000, 13050, 13100,

[0112] 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750,

[0113] 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400,

[0114] 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, 15000, 16000,

[0115] 18000, 20000, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000,

[0116] 44000, 46000, 48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000,

[0117] 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000,

[0118] 96000, 98000, or 100000 nucleotides.

[0119] The RNA molecules of the present disclosure may be prepared by any method know in the art, including chemical synthesis and in vitro methods, such as RNA in vitro transcription. In some of the aspects, the RNA of the present disclosure is prepared using in vitro transcription.

[0120] In some aspects, the RNA molecule of the present disclosure is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0121] In some aspects, the RNA molecule of the present disclosure is lyophilized to be temperature stable.

[0122] In some aspects of the present disclosure, an RNA is or comprises messenger RNA (mRNA) that relates to an RNA transcript that encodes a polypeptide. In some aspects, an RNA disclosed herein comprises: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5' UTR); a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3' UTR); and / or a polyadenylate (poly-A) sequence. In some aspects, an RNA disclosed herein comprises the following components in the 5' to 3' orientation: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5' UTR), a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3' UTR); and a poly-A sequence. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing dosing regimens can be excluded.

[0123] FUSION POLYPEPTIDES

[0124] In some aspects of the present disclosure, an RNA molecule is or comprises messenger RNA (mRNA) that relates to an RNA transcript that encodes a fusion polypeptide. In some aspects a fragment of an outer surface protein A (OspA) polypeptide is fused to a second fragment of an OspA polypeptide and is thus expressed as a fusion polypeptide of two OspA fragment polypeptides. In some aspects, the two OspA fragment polypeptides are different in silico types or different serotypes. In some aspects, the two OspA fragment polypeptides are selected from the group consisting of IST1-2, ST4-3, ST5-6, and IST8, 9, and 10.

[0125] In some aspects, a portion of the outer surface protein A polypeptide is expressed fusion to a membrane anchor and is thus expressed as a membrane-bound fusion polypeptide. In some aspects, the fusion polypeptide is operably linked to a transmembrane and / or a membrane anchor. In some aspects, the fusion polypeptide is operably linked to a signal peptide. Signal peptides are generally present at the N-terminus of membrane-presented or secreted polypeptides and initiate their passage into the endoplasmic reticulum. They generally comprise 15 or more essentially hydrophobic amino acids which are then removed by a specific ER-located endopeptidase to give the mature polypeptide. Transmembrane peptides are usually highly hydrophobic in nature and serve to anchor the polypeptides in the cell membrane. Transmembrane and signal peptides may be obtained from any membrane-anchored and / or secreted polypeptide (e.g. cellular or viral polypeptides) such as those of immunoglobulins, tissue plasminogen activator (tPA), insulin, rabies glycoprotein, the HIV virus envelope glycoprotein (Env), hepatitis C virus (HCV) envelope glycoproteins E1 and E2, herpes simplex virus (HSV) glycoprotein D (gD), influenza virus proteins (such as hemagglutinin or M2 protein), or the measles virus F protein or may be synthetic. In some aspects, the signal peptide is a mouse IgK secretion signal, or a native bacterial signal peptide, or a heterologous signal peptide from a mammalian cell (including interleukin-2, CD5, the immunoglobulin kappa light chain, trypsinogen, serum albumin, and prolactin). In some aspects, the transmembrane targeting sequence is from a type II signal anchored protein. In some aspects, the transmembrane targeting sequence is a type II signal anchored protein from a virus, including a type II signal anchored protein from influenza neuraminidase, simian virus 5 haemagglutinin-neuraminidase, the attachment protein G of human respiratory syncytial virus, or a type II transmembrane domain sequence.

[0126] MODIFIED NUCLEOBASES

[0127] In some aspects of the present disclosure, the RNA molecules are not chemically modified and comprise the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some aspects, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, and / or II). In some aspects, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, and / or dT).

[0128] In other aspects of the present disclosure, the RNA molecules may comprise modified nucleobases that may be incorporated into modified nucleosides and nucleotides. In some aspects, the RNA molecule may include one or more modified nucleotides. The terms "modification" and "modified", in regard to nucleic acids, refer to modification with respect to adenosine (A), guanosine (G), uridine (II), thymidine (T) and / or cytidine (C) ribonucleosides and / or deoxyribonucleosides in at least one of their position, pattern, percent and / or population. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides and / or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, and / or nucleobase portion of the nucleotide and / or nucleoside as are recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT / US2012 / 058519; PCT / US2013 / 075177; PCT / US2014 / 058897; PCT / U S2014 / 058891 ; PCT / US2014 / 070413; PCT / US2015 / 36773; PCT / US2015 / 36759; and PCT / U S2015 / 36771 ; or published international application No. PCT / IB2017 / 051367, all of which are incorporated by reference herein.

[0129] Hence, RNA molecules of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof. RNA molecules, in some aspects, comprise various (more than one) different types of standard and / or modified nucleotides and nucleosides. In some aspects, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0130] Modifications of RNA molecules include, without limitation, those described herein, and include, but are expressly not limited to, those modifications that comprise chemical modifications. RNA molecules may comprise modifications that are naturally-occurring or non-naturally-occurring, or the RNA molecule may comprise a combination of naturally-occurring and non-naturally- occurring modifications. RNA molecules may comprise non-natural modified nucleotides introduced during synthesis and / or post-synthesis of the RNA molecules to achieve desired functions and / or properties. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified. RNA molecules may include any useful modification, for example, of a sugar, a nucleobase, and / or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage and / or to the phosphodiester backbone).

[0131] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, and / or recombinantly, to include one or more modified and / or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

[0132] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and / or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard and / or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base and / or between two complementary non-standard base structures, such as, for example, in those polynucleotides having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar and / or linker may be incorporated into RNA molecules of the present disclosure.

[0133] In some aspects, the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, II, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some aspects, the RNA may include a 5' cap comprising a 7’-methylguanosine, and the first 1 , 2, or 3 5' ribonucleotides may be methylated at the 2’ position of the ribose.

[0134] 5’ CAP

[0135] In some aspects, the RNA molecule described herein includes a 5' cap which generally “caps” the 5' end of the RNA and stabilizes the RNA molecule.

[0136] In some aspects, the 5' cap moiety is a natural 5' cap. A “natural 5' cap” is defined as a cap that includes 7-methylguanosine connected to the 5' end of an mRNA molecule through a 5' to 5' triphosphate linkage. In some aspects, a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some aspects, a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'0MeG). In some aspects, a guanosine nucleoside included in a 5' cap comprises methylation at the 7- position of guanine (m7G). In some aspects, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3'0 methylation at a ribose (m7(3'OMeG)). The 5' cap may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some aspects, co-transcriptional capping with a cap disclosed herein improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference 5' cap. In some aspects, improving capping efficiency may increase the translation efficiency and / or translation rate of an RNA and / or increase expression of an encoded polypeptide. In some aspects, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.

[0137] In some aspects, an RNA described herein comprises a 5' cap or a 5' cap analog, e.g., a Cap 0, a Cap 1 or a Cap 2. In some aspects, a provided RNA does not have uncapped 5'-triphosphates. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide. In some aspects, the 5' cap moiety is a 5' cap analog. In some aspects, an RNA may be capped with a 5' cap analog. Cap structures include, but are not limited to, 7mG(5')ppp(5')N1pN2p (Cap 0), 7mG(5')ppp(5')N1 mpNp (Cap 1), and 7mG(5')ppp(5')N1 mpN2mp (Cap 2). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cap structures can be excluded from the RNA molecules disclosed herein.

[0138] In some aspects, an RNA described herein comprises a Cap 0. In some aspects, Cap 0 is a N7- methyl guanosine, and a Cap 0 structure comprises a guanosine nucleoside methylated at the 7- position of guanine (m7G). In some aspects, a Cap 0 structure is connected to an RNA via a 5' to 5'-triphosphate linkage and is also referred to herein as m7G, m7Gppp, and / or m7G(5')ppp(5'). A 5' cap may be methylated with the structure 7mG(5')ppp(5')N1pN2p (Cap 0) or a derivative thereof, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5'-end of an mRNA. An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyl- transferase and guanine-7-methytransferase, which catalyzes the construction of N7- monomethylated Cap 0 structures. Cap 0 structures play an important role in maintaining the stability and translational efficacy of the RNA molecule. In the cell, the Cap 0 structure is essential for efficient translation of the mRNA that carries the cap.

[0139] In some aspects, an RNA described herein comprises a Cap 1 , e.g., as described herein. The 5' cap of the RNA molecule may be further modified on the 2'0 position by a 2'-0-methyltransferase, which results in the generation of a Cap 1 structure (m7Gppp [m2'-0] N), which may further increase translation efficacy. In some aspects, a Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and a 2'0 methylated first nucleotide in an RNA (2'0MeN1). In some aspects, a Cap 1 structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as m7GpppNm, wherein Nm denotes any nucleotide with a 2'0 methylation, 7mG(5')ppp(5')N1mpNp, m7Gppp(2'OMeN1), and / or m7G(5')ppp(5')(2'OMeN1). In some aspects, N1 is chosen from A, C, G, or II. In some aspects, N1 is A. In some aspects, N1 is C. In some aspects, N1 is G. In some aspects, N1 is II. In some aspects, a m7G(5')ppp(5')(2'OMeN1) Cap 1 structure comprises a second nucleotide, N2, which is a cap proximal nucleotide at position 2 and is chosen from A, G, C, or II (m7G(5')ppp(5')(2'OMeN1)N2). In some aspects, N2 is A. In some aspects, N2 is C. In some aspects, N2 is G. In some aspects, N2 is II.

[0140] In some aspects, the RNA molecule of the present disclosure comprises at least one 5' cap structure. In some aspects, the RNA molecule of the present disclosure does not comprise a 5' cap structure.

[0141] Numerous synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, II., Jemielity, J., and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology 69 (Rabinovich, P.H. Ed), 2013). In one aspect, the 5' capping structure comprises a modified 5' Cap 1 structure (m7G+m3'-5'-ppp-5 - Am). In one aspect, the 5' capping structure comprises is (3'OMe)-m27,3'-OGppp(m12’-O)ApG (TriLink BioTechnologies). This molecule is identical to the natural RNA cap structure in that it starts with a guanosine methylated at N7, and is linked by a 5' to 5' triphosphate linkage to the first coded nucleotide of the transcribed RNA (in this case, an adenosine). This guanosine is also methylated at the 3' hydroxyl of the ribose to mitigate possible reverse incorporation of the cap molecule. The 2’ hydroxyl of the ribose on the adenosine is methylated, conferring a Cap 1 structure.

[0142] UNTRANSLATED REGIONS (UTRS)

[0143] The 5' UTR is a regulatory region situated at the 5' end of a protein open reading frame that is transcribed into mRNA but not translated into an amino acid sequence and / or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) may be present 5' (upstream) of an open reading frame (5' UTR) and / or 3' (downstream) of an open reading frame (3' UTR).

[0144] In some aspects, the UTR is derived from an mRNA that is naturally abundant in a specific tissue (e.g., lymphoid tissue), to which the mRNA expression is targeted. In some aspects, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR may increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and / or the rate at which ribosomes initiate translation on the message (message translation efficiency). Accordingly, the UTR sequence may prolong protein synthesis in a tissue-specific manner.

[0145] In some aspects, the regulatory features of a UTR can be incorporated into the RNAs of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5' UTR and the 3' UTR sequences are known and available in the art.

[0146] It should be understood that any UTR from any gene may be incorporated into the regions of the RNAs of the present disclosure. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation and / or location. Hence a 5' and / or 3' UTR may be inverted, shortened, lengthened, and / or made with one or more other 5' UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, 5' UTRs and / or 3' UTRs may be altered relative to a wild-type or native UTR by the change in orientation and / or location as taught above and / or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping, and / or transposition of nucleotides. Any of these changes produces an “altered” UTR (whether 5' and / or 3') including a variant UTR.

[0147] In some embodiments, a double, triple or quadruple UTR such as a 5' and / or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

[0148] RNAs may encode polypeptides of interest belonging to a family of proteins that are expressed in a particular cell, tissue and / or at some time during development. In some aspects, the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new RNA molecule. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, and / or expression pattern.

[0149] In some aspects, the 5' UTR and the 3' UTR sequences are computationally derived. In some aspects, the 5' UTR and the 3' UTRs are derived from a naturally abundant mRNA in a tissue. The tissue may be, for example, liver, a stem cell and / or lymphoid tissue. The lymphoid tissue may include, for example, any one of a lymphocyte (e.g., a B-lymphocyte, a helper T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, and / or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil and a reticulocyte. In some aspects, the 5' UTR and the 3' UTR are derived from an alphavirus. In some aspects, the 5' UTR and the 3' UTR are from a wild type alphavirus.

[0150] In some aspects, untranslated regions may also include translation enhancer elements (TEE). As a non- limiting example, the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.

[0151] 5’ UTRS

[0152] In some aspects, an RNA disclosed herein comprises a 5' UTR. A 5' UTR, if present, is located at the 5' end and starts with the transcriptional start site upstream of the start codon of a protein encoding region. A 5' UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. The 5' UTR may contain various regulatory elements, e.g., 5' cap structure, stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 5' UTR may harbor signatures like Kozak sequences, which are also involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG (SEQ ID NO: [[X]]), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5' UTRs may also form secondary structures involved in elongation factor binding.

[0153] In some aspects, a 5' UTR disclosed herein comprises a cap proximal sequence, e.g., as disclosed herein. In some aspects, a cap proximal sequence comprises a sequence adjacent to a 5' cap. In some aspects, a cap proximal sequence comprises nucleotides in positions +1 , +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0154] In some aspects, a Cap structure comprises one or more polynucleotides of a cap proximal sequence. In some aspects, a Cap structure comprises an m7 Guanosine cap and nucleotide +1 (N 1 ) of an RNA polynucleotide. In some aspects, a Cap structure comprises an m7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some aspects, a Cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (N1 and N2) of an RNA polynucleotide.

[0155] Those skilled in the art, reading the present disclosure, will appreciate that, in some aspects, one or more residues of a cap proximal sequence (e.g., one or more of residues +1 , +2, +3, +4, and / or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a Cap 1 structure, etc); alternatively, in some aspects, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified aspects where a (m27,3'-O)Gppp(m2’-O)ApG cap is utilized, +1 and +2 residues are the (m27,3'-O) A and G residues of the cap, and +3, +4, and +5 residues are added by polymerase (e.g., T7 polymerase).

[0156] Exemplary 5' UTRs include 5' UTRs derived from Xenopus or human alpha globin or beta globin, human cytochrome b-245 a, hydroxysteroid (17b) dehydrogenase, Tobacco etch virus, the CMV immediate-early 1 (I E 1 ) gene, TEV, HSP705', c-Jun, or a homolog, fragment, or variant of any of the foregoing. In some aspects, the 5' UTR is a fragment, homolog or variant of a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract), the 5' UTR derived from ribosomal protein Large 32 (L32) gene, the 5' UTR derived from the 5' UTR of an hydroxysteroid (17p) dehydrogenase 4 gene (HSD17B4), or the 5' UTR derived from the 5' UTR of ATP5A1. In some aspects, 5' UTRs are derived from UTR sequences disclosed in International Patent Application Publication No. WO2013 / 143700, the disclosure of which is incorporated herein by reference in its entirety, or a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing sequences. The sequence GGGAUCCUACC (SEQ ID NO:12) may also be used. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 5' UTR sequences may be excluded from the RNA molecules disclosed herein.

[0157] 3’ UTRS

[0158] In some aspects, an RNA disclosed herein comprises a 3' UTR. A 3' UTR, if present, is situated downstream of a protein coding sequence open reading frame, e.g., downstream of the termination codon of a protein-encoding region. A 3' UTR is typically the part of an mRNA which is located between the protein coding sequence and the poly-A tail of the mRNA. Thus, in some aspects, the 3' UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. The 3' UTR may be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.

[0159] In some aspects of the disclosure, a 3' UTR is a heterologous UTR, e.g., is a UTR found in nature associated with a different ORF. In another aspect, a 3' UTR is a synthetic UTR, e.g., does not occur in nature. In some aspects, the 3' UTR is functionally linked to the ORF, e.g., associated with the ORF such that it may exert a function, e.g., increasing, enhancing, stabilizing, and / or prolonging protein production from an RNA molecule and / or increasing protein expression and / or total protein production from an RNA molecule, compared to a reference RNA molecule comprising a reference 3' UTR or an RNA molecule lacking a 3' UTR. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR functions may be excluded.

[0160] Exemplary 3' UTRs include 3' UTRs derived from an albumin gene, an a-globin gene, a p-globin gene, a ribosomal protein gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (1) gene, or from a homolog, fragment, or variant of a 3' UTR of a gene comprising an albumin gene, an a-globin gene, a p- globin gene, a ribosomal protein gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and / or a collagen alpha gene, such as a collagen alpha 1 gene. In some aspects, the sequence UUUGAAUU (SEQ ID NO:12) is used. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR sequences may be excluded from the RNA molecules disclosed herein.

[0161] OPEN READING FRAME

[0162] The 5' and 3' UTRs may be operably linked to an open reading frame (ORF), which may be a sequence of codons that is capable of being translated into a polypeptide of interest. An open reading frame may be a sequence of several DNA or RNA nucleotide triplets, which may be translated into a peptide or protein. An ORF may begin with a start codon, e.g., a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG or AUG), at its 5' end and a subsequent region, which usually exhibits a length that is a multiple of 3 nucleotides. An open reading frame may terminate with at least one stop codon, including but not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof.

[0163] As stated herein, the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames.

[0164] In some aspects, the ORF encodes a structural viral gene. In some aspects, the ORF further includes one or more subgenomic promoters. In some aspects, the RNA molecule includes a subgenomic promoter operably linked to the ORF. In some aspects, a first RNA molecule does not include an ORF encoding any polypeptide of interest, whereas a second RNA molecule includes an ORF encoding a polypeptide of interest. In some aspects, the first RNA molecule does not include a subgenomic promoter.

[0165] The present disclosure provides for an RNA molecule comprising at least one open reading frame encoding a Borrelia polypeptide. In some aspects, an RNA molecule comprises at least one open reading frame encoding a Borrelia OspA polypeptide.

[0166] GENES OF INTEREST

[0167] The RNA molecules described herein may include a gene of interest. The gene of interest encodes a polypeptide of interest. Non-limiting examples of polypeptides of interest include, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties, those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery, or combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing polypeptides of interest may be excluded. The sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GENBANK®.

[0168] In some aspects, the RNA molecules include a coding region for a gene of interest. In some aspects, a gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some aspects, an antigenic polypeptide comprises one epitope from an antigen. In some aspects, an antigenic polypeptide comprises a plurality of distinct epitopes from an antigen. In some aspects, an antigenic polypeptide comprising a plurality of distinct epitopes from an antigen is polyepitopic. In some aspects, an antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing antigenic polypeptides may be excluded.

[0169] The term “antigen” may refer to a substance, which is capable of being recognized by the immune system, e.g., by the adaptive immune system, and which is capable of eliciting an antigen-specific immune response, e.g., by formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. An antigen may be or may comprise a peptide or protein, which may be presented by the MHC to T cells. An antigen may be the product of translation of a provided nucleic acid molecule, e.g., an RNA molecule comprising at least one coding sequence as described herein. In addition, fragments, variants and derivatives of an antigen, such as a peptide or a protein, comprising at least one epitope are understood as antigens.

[0170] In some aspects, an RNA encoding a gene of interest, e.g., an antigen, is expressed in cells of a subject treated to provide a gene of interest, e.g., an antigen. In some aspects, the RNA is transiently expressed in cells of the subject. In some aspects, expression of a gene of interest, e.g., an antigen, is at the cell surface. In some aspects, a gene of interest, e.g., an antigen, is expressed and presented in the context of MHC. In some aspects, expression of a gene of interest, e.g., an antigen, is into the extracellular space, e.g., the antigen is secreted.

[0171] In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen. In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen, that is derived from a pathogen associated with an infectious disease. In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen, that is derived from Borrelia.

[0172] In some aspects, the RNA molecule encodes a Borrelia OspA protein or a fragment or a variant thereof. In some aspects, the RNA molecule encodes a Borrelia protein comprising the amino acid sequence according to any one of GENBANK® Accession No.: AAC66260.1 , WP_010890387.1 , ARS30922.1 , ARS32180.1 , and / or ARS32664.1 , the respective sequences of which are herein incorporated by reference. In some aspects, the RNA molecule encodes a Borrelia OspA comprising the amino acid sequence according to GENBANK® Accession No. AAC66260.1 , the sequence of which is herein incorporated by reference.

[0173] In some aspects, an RNA polynucleotide described herein or a composition or medical preparation comprising the same comprises a nucleotide sequence disclosed herein. In some aspects, an RNA polynucleotide comprises a sequence having at least 80% identity to a nucleotide sequence disclosed herein. In some aspects, an RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some aspects, an RNA polynucleotide described herein or a composition or medical preparation comprising the same is transcribed by a DNA template. In some aspects, a DNA template used to transcribe an RNA polynucleotide described herein comprises a sequence complementary to an RNA polynucleotide. In some aspects, a gene of interest described herein is encoded by an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein. In some aspects, an RNA polynucleotide encodes a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some aspects, a polypeptide described herein is encoded by an RNA polynucleotide transcribed by a DNA template comprising a sequence complementary to an RNA polynucleotide.

[0174] In some aspects, the RNA molecule encodes a Boreilia OspA comprising the sequence of SEQ ID NO:1 , or a fragment or variant thereof.

[0175] In some aspects, the RNA molecule encodes a Boreilia OspA synthesized from the nucleic acid sequence of SEQ ID NO:4, or a fragment or variant thereof.

[0176] POLY-A TAIL

[0177] In some aspects, RNA molecules disclosed herein comprise a poly-adenylate (poly-A) sequence, e.g., as described herein. In some aspects, a poly-A sequence is situated downstream of a 3' UTR, e.g., adjacent to a 3' UTR. A “poly-A tail” or “poly-A sequence” refers to a stretch of consecutive adenine residues, e.g., of up to or up to about 400 adenosine nucleotides, e.g., from or from about 20 to about 400, preferably from or from about 50 to about 400, more preferably from or from about 50 to about 300, even more preferably from or from about 50 to about 250, most preferably from or from about 60 to about 250 adenosine nucleotides, which may be attached to the 3' end of the RNA molecule. In some embodiments, the poly-A tail comprises 80 adenosine nucleotides. Poly-A sequences are known to those of skill in the art and may follow the 3' UTR in the RNA molecules described herein. The poly-A tail may increase the stability, half-life, and / or translational efficiency of the RNA molecule.

[0178] Polyadenylation refers to the addition of a poly-A sequence to an RNA molecule, e.g., to a premature mRNA. Polyadenylation may be induced by a so-called polyadenylation signal. This signal may be located within a stretch of nucleotides close to or at the 3'-end of an RNA molecule to be polyadenylated. A polyadenylation signal may also be comprised by the 3' UTR of the artificial nucleic acid molecule. A polyadenylation signal typically comprises a hexamer consisting of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AALIAAA, though other sequences, preferably hexamer sequences, are also conceivable. Polyadenylation typically occurs during processing of a pre-mRNA (also called premature-mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) comprises the step of polyadenylation. Poly-A tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly-T tract into the DNA template or by post-transcriptional addition using poly-A polymerase. The term may relate to polyadenylation of RNA as a cellular process or to polyadenylation carried out by enzymatic reaction in vitro with a suitable enzyme, such as E. coli poly-A polymerase, or by chemical synthesis.

[0179] RNA molecules disclosed herein may have a poly-A sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. In some aspects, a poly-A sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.

[0180] The DNA sequence encoding a poly-A sequence (coding strand) is referred to as poly-A cassette. In some aspects, the poly-A cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such a random sequence may be at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. Such a cassette is disclosed in, e.g., WO 2016 / 005324 A1 , hereby incorporated by reference. Any poly-A cassette disclosed in WO 2016 / 005324 A1 may be used in the present disclosure. A poly-A cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides, shows, on a DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on an RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency. In some aspects, the poly-A sequence contained in an RNA polynucleotide described herein consists essentially of adenosine nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, II). Such a random sequence may be at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0181] The poly-A sequence may be located at any position within the 3' UTR. In some aspects, no nucleotides other than adenosine nucleotides flank a poly-A sequence at its 3'-end, e.g., the poly- A sequence is not masked or followed at its 3'-end by a nucleotide other than adenosine. In some aspects, the poly-A sequence may be located at the 3' terminus of the 3' UTR, e.g., the 3' UTR does not contain more than 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides located 3' of the poly- A sequence; more preferably the 3' UTR does not contain further elements located 3' to the poly- A sequence. In some aspects, poly-A sequence is located at the 3' terminus of the RNA molecule, e.g., the artificial nucleic acid molecule does not contain more than 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides located 3' of the poly-A sequence. Alternatively, the poly-A sequence may be located at the 5' terminus of the 3' UTR, e.g., immediately 3' of the ORF of the artificial nucleic acid molecule, or located within the 3' UTR, e.g., flanked on the 5' and on the 3' side by other 3' UTR elements. In some aspects, the poly-A sequence is flanked on the 3' side by a poly-C sequence and / or a histone stem-loop sequence. In addition or alternatively, the poly-A sequence can be flanked on the 5' side by a 3' UTR element derived from, e.g., a human albumin or globin gene.

[0182] In some aspects, the RNA molecule may further include an endonuclease recognition site sequence immediately downstream of the poly-A tail sequence. The RNA molecule may further include a poly-A polymerase recognition sequence (e.g., a polyadenylation signal) (e.g., AAUAAA) near its 3' end. In some aspects, the polyadenylation signal is located 3' of the poly-A sequence comprised in the 3' UTR. In some aspects, the poly-A sequence is separated from the polyadenylation signal by a nucleotide sequence comprising or consisting of at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides, wherein the nucleotide sequence does preferably not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides. In some aspects, the nucleotide sequence that separates the poly-A sequence and the polyadenylation signal comprises from or from about 1 to about 200 nucleotides, e.g., from 10 to 90, from 20 to 85, from 30 to 80, from 40 to 80, from 50 to 75 or from 55 to 85 nucleotides, more preferably from 55 to 80 nucleotides, and the nucleotide sequence does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides.

[0183] In some aspects, the polyadenylation signal comprises the consensus sequence NN(U / T)ANA, with N = A or U, preferably AA(U / T)AAA or A(U / T)(U / T)AAA. Such a consensus sequence may be recognized by most animal and bacterial cell-systems, for example, by the polyadenylationfactors, such as cleavage / polyadenylation specificity factor (CPSF) cooperating with CstF, PAP, PAB2, CFI and / or CFII. In some aspects, the polyadenylation signal (e.g., the consensus sequence NNLIANA) is located less than or less than about 50 nucleotides, e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, downstream of the 3'-end of the 3' UTR element as defined herein such that transcription of an RNA molecule will result in a premature-RNA containing the polyadenylation signal downstream of its 3' UTR and subsequent attachment of a poly-A sequence to the premature-RNA. Accordingly, a resulting RNA may comprise a 3' UTR, which comprises at least one poly-A sequence, and wherein the 3' UTR is followed by an additional poly-A sequence.

[0184] The poly-A sequence may be of any length. In some aspects, the poly-A tail may be 5 to 300 nucleotides in length. In some aspects, the RNA molecule includes a poly-A tail that comprises, consists essentially of, or consists of a sequence of or of about 25 to about 400 adenosine nucleotides, a sequence of or of about 50 to about 400 adenosine nucleotides, a sequence of or of about 50 to about 300 adenosine nucleotides, a sequence of or of about 50 to about 250 adenosine nucleotides, a sequence of or of about 60 to about 250 adenosine nucleotides, or a sequence of or of about 40 to about 100 adenosine nucleotides. In some aspects, the poly-A tail comprises, consists essentially of, or consists of at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,

[0185] 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,

[0186] 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445,

[0187] 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540,

[0188] 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635,

[0189] 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730,

[0190] 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825,

[0191] 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920,

[0192] 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 adenosine nucleotides. In this context, “consists essentially of” means that most nucleotides in the poly-A sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A sequence are adenosine nucleotides, but permits remaining nucleotides to be nucleotides other than adenosine nucleotides, such as uridine, guanosine, and / or cytosine. In this context, “consists of” means that all nucleotides in the poly-A sequence, i.e., 100% by number of nucleotides in the poly-A sequence, are adenosine nucleotides.

[0193] In some aspects, the RNA molecule includes a poly-A tail that includes a sequence of greater than 30 adenosine nucleotides. In some aspects, the RNA molecule includes a poly-A tail that includes or includes about 40 adenosine nucleotides. In some aspects, the RNA molecule includes a poly-A tail that includes about 80 adenosine nucleotides. In some aspects, the 3' poly- A tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some specific aspects, the RNA molecule includes or includes about 40 consecutive adenosine residues. In some aspects, the RNA molecule includes 80 consecutive adenosine residues. Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation.

[0194] In some aspects, a poly-A tail may be located within an RNA molecule or other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, e.g., an mRNA, e.g., by transcription of the vector. In some aspects, the RNA molecule may not include a poly-A tail.

[0195] In one aspect, a DNA encoding a poly-A tail disclosed herein comprises a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO:10. In one aspect, the DNA encoding the poly-A tail comprises a sequence of SEQ ID NO:10. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 10 + / - 2 adenosine (A) nucleotides. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 10 + / - 1 adenosine (A) nucleotides. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 10.

[0196] OTHER ELEMENTS

[0197] In some aspects of the present disclosure, the RNA molecules additionally include a chain terminating nucleoside. For example, a chain terminating nucleoside may include those nucleosides deoxygenated at the 2’ and / or 3' positions of their sugar group. Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxycytosine, 3' deoxyguanosine, 3' deoxythymine, and 2', 3' dideoxynucleosides, such as 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxycytosine, 2', 3' dideoxyguanosine, and 2', 3' dideoxythymine. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing chain terminating nucleosides may be excluded from the RNA molecules disclosed herein. In some aspects, incorporation of a chain terminating nucleotide into an mRNA, for example at the 3'-terminus, may result in stabilization of the mRNA, as described, for example, in International Patent Publication No. WO 2013 / 103659.

[0198] In some aspects of the present disclosure, the RNA molecules additionally include a stem loop, such as a histone stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in, before, or after an untranslated region (a 5' UTR or a 3' UTR), a coding region, or a poly-A sequence or tail. In some aspects, a stem loop may affect one or more function(s) of an mRNA, such as initiation of translation, translation efficiency, and / or transcriptional termination. Such histone stem-loop sequences may be histone stem-loop sequences disclosed in WO 2012 / 019780, the disclosure of which is incorporated herein by reference in its entirety. Other non-limiting examples of histone stem loop structures and nucleic acid sequences encoding such structures can be found in, e.g., WO 2016 / 091391 , the disclosure of which is incorporated by reference herein in its entirety.

[0199] In some aspects, the combination of a poly-A sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. In some aspects, the synergistic effect of the combination of poly-A and at least one histone stem-loop does not depend on the order of the elements and / or the length of the poly-A sequence.

[0200] In some aspects, the RNA does not comprise a histone downstream element (HDE). An HDE includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.

[0201] In some aspects, the histone stem-loop is generally derived from histone genes, and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and / or base composition of the paired region. In some aspects, wobble base pairing (non- Watson-Crick base pairing) may result. In some aspects, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0202] In some aspects, the RNA molecules include (e.g., within the 3' UTR) a poly(C) sequence. In some aspects, the poly-C sequences has at least, at most, exactly, or between (inclusive or exclusive) any two of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 cytidines. In some aspects, the poly-C sequences has or has about 30 cytidines.

[0203] In some aspects, the RNA molecules include an internal ribosome entry site (IRES) sequence or IRES-motif. In some aspects, an IRES sequence separates ORFs, e.g., if the RNA encodes two or more peptides or proteins. An IRES-sequence may therefore be useful if the RNA molecule is a bi- or multicistronic nucleic acid molecule.

[0204] In some aspects, the RNA does not comprise an intron. In some aspects, the RNA may instead or additionally include a microRNA binding site.

[0205] SELF-AMPLIFYING RNA (saRNA)

[0206] In some aspects, the RNA molecule may be an saRNA. “Self-amplifying RNA,” “saRNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A selfamplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, and / or may be transcribed to provide further transcripts with the same sense as the delivered RNA that are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNA molecules, and consequently, the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.

[0207] In some aspects, the self-amplifying RNA includes at least one or more genes including any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins, or combination thereof. In some aspects, 1 , 2, 3, or more of the foregoing genes may be excluded from the self-amplifying RNA molecules disclosed herein. In some aspects, the self-amplifying RNA may also include 5'- and 3'-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0208] In some aspects, a self-amplifying RNA molecule described herein encodes (i) an RNA- dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a Borrelia antigen. In some aspects, the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus proteins nsP1 , nsP2, nsP3, nsP4, or any combination thereof. In some aspects, 1 , 2, 3, or more of the foregoing alphavirus proteins may be excluded from the RNA molecules disclosed herein.

[0209] In some aspects, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame may encode a replicase; the second (3') open reading frame may encode a polypeptide comprising an antigen of interest. In some aspects the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides.

[0210] In some aspects, the saRNA molecule further includes (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some aspects, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

[0211] In some aspects, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.

[0212] In some aspects, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. In some aspects, the self-amplifying RNA described herein may encode epitopes capable of eliciting either a helper T cell response or a cytotoxic T cell response or both.

[0213] RNA TRANSCRIPTION

[0214] In some aspects, the RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.

[0215] According to the present disclosure, “transcription” comprises “in vitro transcription” or “IVT,” which refers to the process whereby transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for use in various applications, including, e.g., production of protein or polypeptides. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R. 1972. In vitro transcription, Ann Rev Biochem, 41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001. In vitro Transcription, Current Protocols in Cell Biology, 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B. 2010. Synthesis of RNA by In vitro Transcription in RNA, Methods in Molecular Biology, 703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology 530:101-114; all of which are incorporated herein by reference).

[0216] Cloning vectors may be applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term “vector.” According to specific aspects, the RNA used is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template, and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses, Methods 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012, each incorporated herein by reference). The promoter for controlling transcription may be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0217] Synthetic IVT RNA products may be translated in vitro or introduced directly into cells, where they may be translated. With respect to RNA, the term “expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein. Such synthetic RNA products include but are not limited to, e.g., mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, and the like. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing synthetic RNA products may be excluded. An IVT reaction typically utilizes a DNA template (e.g., a linear DNA template) as described and / or utilized herein, ribonucleotides (e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase.

[0218] In some aspects, an mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, an RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription may be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0219] In some aspects, starting material for IVT may include linearized DNA template, nucleotides, Rnase inhibitor, pyrophosphatase, and / or a polymerase (e.g., a T7 RNA polymerase). The nucleotides may be manufactured in house, may be obtained from a supplier, or may be synthesized. The nucleotides may be, but are not limited to, those described herein including natural and unnatural (modified) nucleotides. Any number of RNA polymerases or variants may be used, including, but not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing RNA polymerases may be excluded from. Some embodiments exclude the use of Dnase.

[0220] In some aspects, the IVT process is conducted in a bioreactor. The bioreactor may comprise a mixer. In some aspects, nucleotides may be added into the bioreactor throughout the IVT process.

[0221] In some aspects, one or more post-1 VT agents are added into the IVT mixture comprising RNA in the bioreactor after the IVT process. Exemplary post-IVT agents may include DNAse I configured to digest the linearized DNA template and / or proteinase K configured to digest DNAse I and T7 RNA polymerase. In some aspects, the post-IVT agents are incubated with the mixture in the bioreactor after IVT. In some aspects, the bioreactor may contain at least, at most, exactly, or between (inclusive or exclusive) any two of 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 ,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500 or more liters IVT mixture. The IVT mixture may have an RNA concentration that is or is not at least, at most, exactly, or between (inclusive or exclusive) any two of 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL or more RNA.

[0222] In some aspects, the IVT mixture may include residual spermidine, residual DNA, residual proteins, peptides, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+, Na+, Ca2+), RNA fragments, residual nucleotides, free phosphates, or any combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing can be excluded from the IVT mixture.

[0223] Isolation and / or purification of the nucleic acids described herein may include, but is not limited to, phenol / chloroform extraction and / or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid clean-up, quality assurance and quality control. Additional, non-limiting examples of purification procedures include AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo- T capture probes (EXIQON® Inc, Vedbaek, Denmark), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing purification may be excluded.

[0224] The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment and / or purification method.

[0225] In some aspects, at least a portion of the IVT mixture is filtered. The IVT mixture may be filtered via ultrafiltration and / or diafiltration to remove at least some impurities from the IVT mixture and / or to change buffer solution for the at least a portion of IVT mixture to produce a concentrated RNA solution as a retentate.

[0226] In some aspects, both “ultrafiltration” and “diafiltration” refer to a membrane filtration process. Ultrafiltration typically uses membranes having pore sizes of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 pm. In some aspects, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO may be at least, at most, exactly, or between (inclusive or exclusive) any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa,

[0227] 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa,

[0228] 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa,

[0229] 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa,

[0230] 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, and 10000 kDa. A skilled artisan will understand that filtration membranes may comprise different suitable materials, including, e.g., polymers, cellulose, ceramic, etc., depending upon the application. In some aspects, membrane filtration may be more desirable for large volume purification process.

[0231] In some aspects, ultrafiltration and diafiltration of the IVT mixture for purifying RNA may include (1) Direct Flow Filtration (DFF), also known as “dead-end” filtration, that applies a feed stream perpendicular to the membrane face and attempts to pass 100% of the fluid through the membrane, and / or (2) Tangential Flow Filtration (TFF), also known as crossflow filtration, where a feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is retained and / or recirculated back to the feed tank.

[0232] In some aspects, the filtering of the IVT mixture is conducted via TFF comprising an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some aspects, the first diafiltration step is conducted in the presence of ammonium sulfate. The first diafiltration step may be configured to remove a majority of impurities from the IVT mixture. In some aspects, the second diafiltration step is conducted without ammonium sulfate. The second diafiltration step may be configured to transfer the RNA into a DS buffer formulation.

[0233] A filtration membrane with an appropriate MWCO may be selected for ultrafiltration in the TFF process. The MWCO of a TFF membrane determines which solutes may pass through the membrane into the filtrate and which are retained in the retentate. The MWCO of a TFF membrane may be selected such that substantially all of the solutes of interest (e.g., desired synthesized RNA species) remain in the retentate, whereas undesired components (e.g., excess ribonucleotides, small nucleic acid fragments such as digested or hydrolyzed DNA template, peptide fragments such as digested proteins and / or other impurities) pass into the filtrate. In some aspects, the retentate comprising desired synthesized RNA species may be re-circulated to a feed reservoir to be re-filtered in additional cycles. In some aspects, a TFF membrane may have a MWCO of at least, at most, exactly, or between (inclusive or exclusive) any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or more. In some aspects, a TFF membrane may have a MWCO of at least, at most, exactly, or between (inclusive or exclusive) any two of 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or more. In some aspects, a TFF membrane may have a MWCO of or of about 250-350 kDa. In some aspects, a TFF membrane (e.g., a cellulose-based membrane) may have a MWCO of or of about 30-300 kDa; 50-300 kDa, 100-300 kDa, or 200-300 kDa.

[0234] Diafiltration may be performed either discontinuously, or alternatively, continuously. For example, in continuous diafiltration, a diafiltration solution may be added to a sample feed reservoir at the same rate as filtrate is generated. In this way, the volume in the sample reservoir remains constant but small molecules (e.g., salts, solvents, etc.) that may freely permeate through a membrane are removed. Using solvent removal as an example, each additional diafiltration volume (DV) reduces the solvent concentration further. In discontinuous diafiltration, a solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the desired concentration of small molecules (e.g., salts, solvents, etc.) remaining in the reservoir is reached. Each additional diafiltration volume (DV) reduces the small molecule (e.g., solvent) concentration further. Continuous diafiltration typically requires a minimum volume for a given reduction of molecules to be filtered. Discontinuous diafiltration, on the other hand, permits fast changes of the retentate condition, such as pH, salt content, and the like. In some aspects, the first diafiltration step is conducted with at least, at most, exactly, or between (inclusive or exclusive) any two of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more diavolumes. In some aspects, the second diafiltration step is conducted with at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more diavolumes. In some aspects, the first diafiltration step is conducted with 5 diavolumes, and second diafiltration step is conducted with 10 diavolumes.

[0235] In some aspects, for ultrafiltration and / or diafiltration, the I T mixture is filtered at a rate of at least, at most, exactly, or between (inclusive or exclusive) any two of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L / m2 of filter area per hour, or more. The concentrated RNA solution may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 2.0, 2.1 , 2.2, 2.3, 2.4, or 2.5 mg / mL single stranded RNA.

[0236] The bioburden of the concentrated RNA solution via filtration to obtain an RNA product solution may also be reduced, in some aspects. The filtration for reducing bioburden may be conducted using one or more filters. The one or more filters may include a filter with a pore size that is or is not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.2 pm, 0.45 pm, 0.65 pm, 0.8 pm, or any other pore size configured to remove bioburdens.

[0237] As one example, reducing the bioburden may include draining a retentate tank containing retentate obtained from the ultrafiltration and / or diafiltration to obtain the retentate. Reducing the bioburden may include flushing a filtration system for ultrafiltration and / or diafiltration using a wash buffer solution to obtain a wash pool solution comprising residue RNA remaining in the filtration system. The retentate may be filtered to obtain a filtered retentate. The wash pool solution may be filtered using a first 0.2 pm filter to obtain a filtered wash pool solution. The retentate may be filtered using the first 0.2 pm filter or another 0.2 pm filter.

[0238] The filtered wash pool solution and the filtered retentate may be combined to form a combined pool solution. The combined pool solution may be filtered using a second 0.2 pm filter to obtain a filtered combined pool solution, which is further filtered using a third 0.2 pm filter to produce an RNA product solution.

[0239] A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, and / or analytical HPLC.

[0240] In some aspects, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[0241] In some aspects, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size and / or to assess degradation. Degradation of the nucleic acid may be assessed by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing assessment methods may be excluded.

[0242] CHARACTERIZATION AND ANALYSIS OF THE RNA MOLECULE

[0243] The RNA molecule produced by the methods described herein may be analyzed and characterized using various methods. Analysis may be performed before or after capping. Alternatively, analysis may be performed before or after purification via poly(A) capture-based affinity, TFF, SPTFF, or other chromatographic and filtration purification methods. In another aspect, analysis may be performed before or after additional purification steps, e.g., anion exchange chromatography and the like. RNA transcript integrity may be determined using electrophoresis (e.g., using the fragment analyzer capillary or Bioanalyzer chip systems) or through a reverse phase HPLC method. In other aspects, RNA purity is analyzed using analytical reverse phase HPLC. Capping efficiency may be analyzed using, e.g., total nuclease digestion followed by LC-LIV or LC-MS quantitation of the dinucleotide cap species vs. uncapped GTP species. The level of residual DNA template (resDNA) can be measured using quantitative polymerase chain reaction (qPCR). The concentration of residual NTPs and / or 5’ cap analogs can be measured using anion-exchange chromatography on an HPLC-UV system. Next- Generation Sequencing (also referred to as massively parallel sequencing) which refers to nonSanger sequencing technologies enables the determination of nucleic acid order which can be used to confirm the sequence identity of the mRNA transcripts as well as determine the location and frequency of sequence variants within said transcripts. In vitro efficacy may be analyzed by, e.g., transfecting RNA molecules into a human cell line. Protein expression of the polypeptide of interest may be quantified using methods such as ELISA or flow cytometry. Immunogenicity may be analyzed by, e.g., transfecting RNA molecules into cell lines that indicate innate immune stimulation, e.g., PBMCs. Cytokine induction may be analyzed using, e.g., methods such as ELISA to quantify a cytokine, e.g., Interferon-a.

[0244] The methods of continuous production of an RNA molecule described herein may produce an RNA molecule that is at least 30% full-length transcript, or at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% full-length transcript, or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measured by, e.g., peak area of full-length RNA molecule relative to total peak area.

[0245] ENTRAPMENT OF RNA IN NANOPARTICLES AND PROCESSING TO DRUG PRODUCT

[0246] In some aspects, the in vitro transcribed RNA molecules within the RNA drug substance of the present disclosure may be encapsulated to form colloidal dispersions (e.g. RNA-loaded LNP dispersion) comprising at least one encapsulating agent. In one aspect, the encapsulating agent comprises one or more lipids, a lipid nanoparticle (LNP), lipoplexes, one or more polymers, polymeric particles, polyplexes, monolithic delivery systems, or a combination thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing elements may be excluded as an encapsulating agent.

[0247] In one aspect, the encapsulating agent is a lipid, and produced is an RNA-loaded LNP dispersion. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and / or the cationic polymer combine together with the nucleic acid to form colloidally stable dispersions.

[0248] A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. As encapsulating agent, a lipid is a substance that is insoluble or partially insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids and are encompassed by the compositions and methods of the present disclosure. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently.

[0249] In some aspects, LNPs may be designed to protect RNA molecules with unmodified and / or modified nitrogenous bases and various sizes (e.g., mRNA, modified mRNA [modRNA], saRNA, gRNA and / or circRNA) from extracellular RNases and / or may be engineered for systemic delivery of the RNA to target cells. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intravenously administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intramuscularly administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intradermally administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intranasally administered to a subject in need thereof.

[0250] In one aspect, the RNA in the RNA drug substance is at a concentration of < 1 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.05 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 1 mg / mL. In another aspect, the RNA concentration is from or from about 0.05 mg / mL to about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least 10 mg / mL. In another aspect, the RNA is at a concentration of at least 50 mg / mL. In some aspects, the RNA is or is not at a concentration of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or more.

[0251] The present disclosure provides for an RNA drug substance and a lipid preparation mixture or compositions thereof comprising at least one RNA encoding, e.g., an antigen complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes and / or nanoliposomes. In some aspects, the composition comprises a lipid nanoparticle.

[0252] A lipid nanoparticle or LNP refers to particles of any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of RNA. In some aspects, lipid nanoparticles are included in a formulation that may be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some aspects, the lipid nanoparticles of the present disclosure comprise a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids, or combinations thereof. In some aspects, the LNPs comprise at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and / or at least one polymer conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, 1 , 2, 3, or more of the foregoing excipients may be excluded from the LNPs.

[0253] In some aspects, the LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipid(s). For example, the LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30- 40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise or do not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 45 to 55 mole percent (mol%) cationic (e.g., ionizable) lipid(s). For example, LNPs may comprise or not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipid(s).

[0254] In some aspects, the LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipid(s). For example, the LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 5 to 15 mol% neutral (e.g., non-cationic) lipid(s). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 mol% neutral (e.g., non-cationic) lipid(s).

[0255] In some aspects, the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid). For example, the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, SO- 55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 35 to 40 mol% structural lipid(s) (e.g., a steroid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, or 40 mol% structural lipid(s) (e.g., a steroid).

[0256] In some aspects, the LNPs comprise 0.5-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). For example, the lipid nanoparticles (LNPs) may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% polymer conjugated lipid(s) (e.g., a PEG- conjugated lipid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). In some aspects, the LNPs comprise 1 to 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)- conjugated lipid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 1.5, or 2 mol% polymer conjugated lipid(s) (e.g., a PEG- conjugated lipid).

[0257] In some aspects, the LNPs comprise 20-75 mol% cationic (e.g., ionizable) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%), 0.5-25 mol% neutral (e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11%, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25%, and 25%), 5-55 mol% structural lipid(s) (e.g., a sterol) e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5-20 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11%, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some aspects, 1 , 2, 3, or more of the lipids may be excluded from the LNPs.

[0258] In some non-limiting aspects, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 60 / 7.5 / 31 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.5 / 7.5 / 31.5 / 3.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 50 / 10 / 35 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer conjugated lipid).

[0259] In some aspects, the active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle and / or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. The nucleic acid (e.g., mRNA) or a portion thereof may also be associated and complexed with the lipid nanoparticle. A lipid nanoparticle may comprise any lipid capable of forming a particle to which the nucleic acids are attached, and / or in which the one or more nucleic acids are encapsulated.

[0260] In some aspects, provided RNA molecules (e.g., mRNA, modRNA, saRNA, gRNA and / or circRNA) may be formulated with LNPs. In some aspects, the lipid nanoparticles may or may not have a mean diameter of or of about 1 to 500 nm (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some aspects, the lipid nanoparticles have a mean diameter of or of from about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, exactly, or between (inclusive or exclusive) of 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. The term “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, “mean diameter,” “diameter,” or “size” for particles is used synonymously with the value of the Z-average.

[0261] LNPs described herein may exhibit a polydispersity index less than or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, exactly, or between (inclusive or exclusive) of 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. The polydispersity index is, in some aspects, calculated based on dynamic light scattering measurements by the so-called cumulant analysis referred to in the definition of “average diameter.” Under certain prerequisites, it may be taken as a measure of the size distribution of an ensemble of nanoparticles.

[0262] In some aspects, an LNP of the disclosure comprises or does not comprise a molar ratio of positively chargeable nitrogen of tertiary amine in the cationic lipid to negatively charged phosphates of mRNA backbone (known as N:P ratio) of or of from about 2:1 to about 30:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 6:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 3:1.

[0263] In some aspects, an LNP of the disclosure comprises or does not comprise a wt / wt ratio of the cationic lipid component to the RNA of or of from about 5:1 to about 100:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1,

[0264] 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1,

[0265] 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,

[0266] 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1,

[0267] 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, or 100:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 20:1. In some aspects, an LNP of the disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of or of about 10:1.

[0268] In certain aspects, nucleic acids (e.g., RNA molecules), when present in provided LNPs, are resistant in aqueous solution to degradation with a nuclease. In some aspects, LNPs are livertargeting lipid nanoparticles. In some aspects, LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some aspects, cationic LNPs may comprise at least one cationic lipid, at least one polymer conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

[0269] In certain aspects, the mixture of RNA and lipid preparations or compositions thereof may have at least, at most, exactly, between (inclusive or exclusive) of, or about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

[0270] 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

[0271] 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,

[0272] 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,

[0273] 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a particular lipid, lipid type, or non-lipid component such as lipid-like materials and / or cationic polymers and / or an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or as would be known to one of skill in the art.

[0274] LNPs described herein can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551 ; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety. For example, methods of preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term “colloid” as used herein relates to a type of mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal dispersion. Sometimes the term “colloid” refers only to the particles in the mixture and not the entire dispersion.

[0275] While methods for preparing a colloid containing an organic solvent are described herein, other methods having organic solvent-free characteristics may also be used according to the present disclosure.

[0276] In some aspects, an RNA-loaded LNP dispersion may be produced by inline mixing of an RNA solution or adjusted RNA solution described herein (e.g., an RNA drug substance) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some aspects, suitable buffering agents comprise tris, histidine, citrate, acetate, phosphate, and / or succinate. In some aspects, 1 , 2, 3, or more of the foregoing buffering agents are excluded. The pH of a liquid formulation relates to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which, for example, in some aspects, may result in greater encapsulation efficiency than is achieved in the absence of interactions between nucleic acids and at least one of the lipid components. In certain aspects, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

[0277] Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0278] Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species, such as RNA species. In an LNP formulation, it is possible that each nucleic acid species is separately formulated as an individual LNP formulation. In that case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

[0279] The resulting dispersion following the formation of LNPs can be filtered as liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system and recovering the retentate (containing the LNPs) and permeate from the system in separate containers without recirculation through the SPTFF system, thereby filtering the liquid feed.

[0280] In some aspects, the SPTFF system comprises a plurality of filtration modules that are fluidly connected. Each of the filtration modules comprises a manifold segment that includes a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module. The filtration modules are fluidly connected through the manifold segments to provide a serial flow path between filtration modules, by coupling of the first manifold in a manifold segment to the second manifold of a manifold segment in an adjacent module, such that the retentate of one module serves as the feed for the next module. The manifold segment in each module is also fluidly connected to a plurality of TFF cassettes that are stacked on one or both faces of the manifold segment. In addition, the SPTFF system comprises a feed inlet on the first module in the system and a retentate outlet on the last module in the system.

[0281] In another aspect, the SPTFF system consists of one filtration module. The filtration module comprises a feed inlet, a retentate outlet, a manifold segment that comprises a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module, wherein the flow path through the manifold segment is serial, and a plurality of TFF cassettes that are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes.

[0282] In another embodiment, described herein are methods of filtering a liquid feed, comprising passing a liquid feed through a tangential flow filtration (TFF) system, recovering permeate and a portion of the retentate from the system in separate containers without recirculation through the TFF system, and recirculating the remainder of the retentate through the TFF system at least once, thereby filtering the liquid feed.

[0283] In another aspect, the TFF system comprises a plurality of filtration modules that are fluidly connected. Each of the filtration modules comprises a manifold segment that includes a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module. The filtration modules are fluidly connected through the manifold segments to provide a serial flow path between filtration modules, by coupling of the first manifold in a manifold segment to the second manifold of a manifold segment in an adjacent module, such that the retentate of one module serves as the feed for the next module. The manifold segment in each module is also fluidly connected to a plurality of TFF cassettes that are stacked on one or both faces of the manifold segment. In addition, the TFF system comprises a feed inlet on the first module in the system, a retentate outlet on the last module in the system, a recirculation loop (e.g., a pump) for recirculating retentate through all or part of the system, and at least one conduit for recirculating retentate.

[0284] In another aspect, the TFF system consists of one filtration module. The filtration module comprises a feed inlet, a retentate outlet, a recirculation loop (e.g., a pump) for recirculating retentate through all or part of the system, at least one conduit for recirculating retentate, a manifold segment that comprises a first manifold for receiving and carrying the feed into the filtration module, a second manifold for receiving and carrying retentate out of the filtration module, and a third manifold for receiving and carrying permeate through the filtration module, wherein the flow path through the manifold segment is serial, and a plurality of TFF cassettes that are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes.

[0285] The processes described herein can be performed using SPTFF and TFF systems that lack diverter plates, thereby reducing the length of the flow path required to achieve effective serial processing and providing other advantages, such as improved flow distribution and multiplication of system size with a compact design and minimal external piping.

[0286] The method of filtration of a liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system is disclosed in, e.g., U.S. Patent Publication No. 2023 / 11 , 679, 349B2 and PCT Pub. No. WO 2016 / 033546A1 , the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0287] In some aspects, the described SPTFF systems can be used to purify and adjust the concentration of the RNA-loaded LNP dispersion. The resulting composition of the post-SPTFF dispersion can be continuously adjusted through inline bifurcating mixers, followed by passing through filters for bioburden reduction and sterilization to obtain RNA drug product. In some aspects, the RNA drug product can be passed to a surge tank for transferring of the RNA drug product into containers / closure systems.

[0288] In some aspects, a composition such as a pharmaceutical composition comprises more than one individual LNP formulation. Respective pharmaceutical compositions are referred to as mixed LNP formulations. Mixed LNP formulations according to the invention are obtainable by forming, separately, individual LNP formulations, as described above, followed by a step of mixing of the individual LNP formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing LNPs is obtainable. Individual LNP populations may be together in one container, comprising a mixed population of individual LNP formulations.

[0289] Alternatively, it is possible that different nucleic acid species are formulated together as a combined LNP formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. As opposed to a mixed LNP formulation, a combined LNP formulation will typically comprise LNPs that comprise more than one RNA species. In a combined LNP composition, different RNA species are typically present together in a single particle.

[0290] CATIONIC POLYMERIC MATERIALS

[0291] Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L- lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some aspects herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. In some aspects, such synthetic materials may be suitable for use as cationic materials herein.

[0292] A “polymeric material,” as used herein, is given its ordinary meaning, e.g., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some aspects, such repeat units may all be identical; alternatively, in some cases, there may be more than one type of repeat unit present within the polymeric material. In some cases, a polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties may also be present in the polymeric material, for example targeting moieties such as those described herein.

[0293] Those skilled in the art are aware that, when more than one type of repeat unit is present within a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is said to be a “copolymer.” In some aspects, a polymer (or polymeric moiety) utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer may be arranged in any fashion. For example, in some aspects, repeat units may be arranged in a random order; alternatively or additionally, in some aspects, repeat units may be arranged in an alternating order, or as a “block” copolymer, e.g., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0294] In certain aspects, a polymeric material for use in accordance with the present disclosure is biocompatible. Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations. In certain aspects, a biocompatible material is biodegradable, e.g., is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body. In certain aspects, a polymeric material may be or comprise protamine or polyalkylene imine, in particular protamine.

[0295] As those skilled in the art are aware, the term “protamine” is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (e.g., fish). In particular, the term “protamine” is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0296] In some aspects, the term “protamine” as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and / or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides that are artificial and designed for specific purposes and cannot be isolated from native or biological sources.

[0297] In some aspects, a polyalkylene imine comprises polyethylenimine and / or polypropylenimine. In some aspects, the polyalkylene imine is polyethyleneimine (PEI). In some aspects, the polyalkylene imine is a linear polyalkylene imine, e.g., linear polyethyleneimine (PEI).

[0298] Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some aspects, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid may be associated, e.g., by forming complexes with the nucleic acid and / or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0299] In some aspects, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and / or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.

[0300] LIPIDS & LIPID-LIKE MATERIALS

[0301] The terms “lipid” and “lipid-like material” are used herein to refer to molecules that comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0302] The term “lipid” refers to a group of organic compounds that are characterized by being insoluble or partially soluble in water but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids as well as sterol-containing metabolites such as cholesterol, and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramides phosphosphingolipids (e.g., sphingomyelins, phosphocholine), and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives. In some aspects, 1 , 2, 3, 4, 5, or more of the lipids may be excluded from the LNPs of the present disclosure.

[0303] The term “lipid-like material,” “lipid-like compound,” or “lipid-like molecule” relates to substances that structurally and / or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, and includes surfactants or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules that comprise hydrophilic and hydrophobic moieties with different structural organization that may or may not be similar to that of lipids.

[0304] In some aspects, the mixture of RNA and lipid preparations or compositions thereof may comprise cationic lipids, neutral lipids, cholesterol, and / or polymer (e.g., polyethylene glycol)-conjugated lipids which form lipid nanoparticles that encompass the RNA molecules. Therefore, in some aspects, the LNP may comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer conjugated lipids (e.g., PEG-lipid), or combinations thereof. In some aspects, 1 , 2, 3, or more of the foregoing excipients may be excluded from the LNPs of the present disclosure. In some aspects, the lipids are present in a composition in an amount that is effective to form a lipid nanoparticle and deliver a therapeutic agent, e.g., an RNA molecule, for treating a particular disease or condition of interest. In some aspects, the LNPs encompass, or encapsulate, the nucleic acid molecules.

[0305] CATIONIC LIPIDS

[0306] Cationic or cationically ionizable lipids or lipid-like materials refer to a lipid or lipid-like material capable of being positively charged and able to electrostatically bind nucleic acid. As used herein, a “cationic lipid” or “cationic lipid-like material” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl, or more acyl chains, and the head group of the lipid typically carries the positive charge. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Cationic lipids may encapsulate negatively charged RNA.

[0307] In some aspects, cationic lipids are ionizable such that they may exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. Without wishing to be bound by theory, this ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH. For purposes of the present disclosure, such “cationically ionizable” lipids or lipid-like materials are comprised by the term “cationic lipid” or “cationic lipid-like material” unless contradicted by the circumstances.

[0308] In some aspects, a cationic lipid may comprise from or from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle. In some aspects, a cationic lipid may or may not be at least, at most, exactly, or between (inclusive or exclusive) of 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, or 100 mol %, or any range or value derivable therein, of the total lipid present in the particle.

[0309] Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl)azanediyl)bis(hexane-

[0310] 6.1-diyl)bis(2-hexyldecanoate), 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), N,N- dimethyl-2,3-dioleyloxypropylamine (DODMA), 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-( N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP),

[0311] 1.2-diacyloxy-3-dimethylammonium propanes, 1 ,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE),

[0312] 1.2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DM EPC), 1 ,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl- 1-propanamium trifluoroacetate (DOSPA), 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l- (cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy- N,N-dimethylpropylamine (DLinDAP), 1 ,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1 ,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]- dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin- KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3- DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1- propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-

[0313] 2.3-bis(tetradecyloxy)-1-propanaminium bromide (bAE-DMRIE), N-(4-carboxybenzyl)-N,N- dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1 -amine (Octyl- CLinDMA), 1 ,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1 ,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1 ,2-dioleoyl-sn- glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1 -amine (DM DMA), Di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2- dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2- [bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid 02-200); C 12-200; or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cationic lipids may be excluded from the LNPs of the present disclosure.

[0314] In some aspects, an ionizable cationic lipid of the disclosure comprises a compound of Formula (I): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0315] Ri is a C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, or -R”M’R’;

[0316] R2 and R3 are independently a H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, or-R*OR”, and / or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0317] R4 is a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted Ci-e alkyl, where Q is a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, - CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, - N(R)C(S)N(R)2, -N(R)R8I-O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2I-C(=NR9)R, - C(O)N(R)OR, or -C(R)N(R)2C(O)OR, and / or each n is independently a 1 , 2, 3, 4, or 5; each R5 is independently a C1.3 alkyl, C2-3 alkenyl, or H; each Re is independently a C1.3 alkyl, C2-3 alkenyl, or H;

[0318] M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group;

[0319] R? is a C1.3 alkyl, C2-3 alkenyl, or H;

[0320] Rs is a C3-6 carbocycle or heterocycle;

[0321] Rg is a H, CN, NO2, Ci-e alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle, or heterocycle; each R is a C1.3 alkyl, C2-3 alkenyl, or H; each R' is a C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, or H; each R” is a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1-12 alkyl or C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently a F, Cl, Br, or I; and m is a 5, 6, 7, 8, 9, 10, 11 , 12, or 13. In some aspects, a subset of compounds of Formula (I) includes those in which when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when n is 1 , 2, 3, 4, or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

[0322] In some aspects, another subset of compounds of Formula (I) includes those in which R1 is a C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, or -R”M’R’;

[0323] R2 and R3 are independently an H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0324] R4 is a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted Ci-e alkyl, where Q is a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms comprising N, O, or S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, - CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - CRN(R)2C(O)OR, -N(R)R8I-O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, - OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2I-C(=NR9)R, - C(O)N(R)OR, or a 5- to 14-membered heterocycloalkyl having one or more heteroatoms comprising N, O, and S which is substituted with one or more substituents comprising oxo (=0), OH, amino, mono- or di- alkylamino, or C1.3 alkyl, and / or each n is independently 1 , 2, 3, 4, or 5; each R5 is independently a C1.3 alkyl, C2-3 alkenyl, or H; each Re is independently a C1.3 alkyl, C2-3 alkenyl, or H;

[0325] M and M' are independently a -C(0)0-, -0C(0)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(0)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(0)2-, -S-S-, an aryl group, or a heteroaryl group;

[0326] R? is a C1.3 alkyl, C2-3 alkenyl, or H;

[0327] Rs is a C3-6 carbocycle or heterocycle;

[0328] Rg is a H, CN, NO2, Ci-e alkyl, -OR, -S(0)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle or heterocycle; each R is independently a C1.3 alkyl, C2-3 alkenyl, or H; each R’ is independently a C1.18 alkyl, C2-18 alkenyl, -R* YR”, -YR”, or H; each R” is independently a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1-12 alkyl or C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11 , 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof.

[0329] In some aspects, another subset of compounds of Formula (I) includes those in which:

[0330] R1 is a C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, or -R”M’R’; R2 and R3 are independently an H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0331] R4 is a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted Ci-e alkyl, where Q is a C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms comprising N, O, or S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, - CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - CRN(R)2C(O)OR, -N(R)R8I-O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, - OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, or - C(=NR9)N(R)2, and / or each n is independently 1 , 2, 3, 4, or 5; and / or when Q is a 5- to 14- membered heterocycle and (i) R4 is -(CH2)nQ in which n is 1 or 2, or (ii) R4 is -(CH2)nCHQR in which n is 1 , or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R5 is independently a C1.3 alkyl, C2-3 alkenyl, or H; each Re is independently a C1.3 alkyl, C2-3 alkenyl, or H;

[0332] M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group;

[0333] R? is a C1.3 alkyl, C2-3 alkenyl, or H;

[0334] Rs is C3-6 carbocycle or heterocycle;

[0335] Rg is H, CN, NO2, C1.6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle, or heterocycle; each R is independently a C1.3 alkyl, C2-3 alkenyl, or H; each R’ is independently a C1.18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1-12 alkyl or C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11 , 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof.

[0336] In some aspects, another subset of compounds of Formula (I) includes those in which:

[0337] R1 is a C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, or -R”M’R’;

[0338] R2 and R3 are independently an H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0339] R4 is a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, or unsubstituted Ci-e alkyl, where Q is a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms comprising N, O, or S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, - CXH2, -CN, -C(O)N(R)2I-N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - CRN(R)2C(O)OR, -N(R)R8I-O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, - OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, or - C(=NR9)N(R)2, and / or each n is independently 1 , 2, 3, 4, or 5; each Rs is independently a C1.3 alkyl, C2.3 alkenyl, or H; each Re is independently a C1.3 alkyl, C2.3 alkenyl, or H;

[0340] M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group;

[0341] R? is a C1.3 alkyl, C2.3 alkenyl, or H;

[0342] Rs is a C3-6 carbocycle or heterocycle;

[0343] R9is an H, CN, NO2, Ci-e alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2.6 alkenyl, C3-6 carbocycle, or heterocycle; each R is independently a C1.3 alkyl, C2.3 alkenyl, or H; each R' is independently a C1.18 alkyl, C2-is alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1.12 alkyl or C2.12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11 , 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof.

[0344] In some aspects, another subset of compounds of Formula (I) includes those in which:

[0345] R1 is a C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, or -R”M’R’;

[0346] R2and R3 are independently an H, C2.14 alkyl, C2.14 alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0347] R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2, and / or n is 3, 4, or 5; each R5 is independently a C1.3 alkyl, C2.3 alkenyl, or H; each Re is independently a C1.3 alkyl, C2.3 alkenyl, or H;

[0348] M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group;

[0349] R? is a C1.3 alkyl, C2.3 alkenyl, or H; each R is independently a C1.3 alkyl, C2.3 alkenyl, or H; each R’ is independently a C1.18 alkyl, C2-ie alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1.12 alkyl or C1.12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11 , 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof.

[0350] In some aspects, another subset of compounds of Formula (I) includes those in which:

[0351] Ri is a C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, or -R”M’R’;

[0352] R2 and R3 are independently a C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, or -R*OR”, and / or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0353] R4 is a -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, where Q is -N(R)2, and / or n is 1 , 2, 3, 4, or 5; each R5 is independently a C1.3 alkyl, C2-3 alkenyl, or H; each Re is independently a C1.3 alkyl, C2-3 alkenyl, or H;

[0354] M and M' are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R’)C(O)-, -C(O)-, -C(S)- , -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, or a heteroaryl group;

[0355] R? is a C1.3 alkyl, C2-3 alkenyl, or H; each R is independently a C1.3 alkyl, C2-3 alkenyl, or H; each R' is independently a C1.18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, or H; each R” is independently a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1-12 alkyl or C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently a F, Cl, Br, or I; and m is 5, 6, 7, 8, 9, 10, 11 , 12, or 13, and / or pharmaceutically acceptable salts, tautomers, prodrugs, or stereoisomers thereof.

[0356] In some aspects, a subset of compounds of Formula (I) includes those of Formula (IA): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein I is 1 , 2, 3, 4, or 5; m is 5, 6, 7, 8, or 9; Mi is a bond or M’; R4 is unsubstituted C1.3 alkyl, or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, - N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, - S-S-, an aryl group, or a heteroaryl group; and R2 and R3 are independently a H, C1-14 alkyl, or C2-14 alkenyl. In some aspects, a subset of compounds of Formula (I) includes those of Formula (II): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein I is 1 , 2, 3, 4, or 5; Mi is a bond or M’; R4 is unsubstituted C1.3 alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently a -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR’)O-, - S-S-, an aryl group, or a heteroaryl group; and R2and R3 are independently a H, C1-14 alkyl, or C2.14 alkenyl. In some aspects, a subset of compounds of Formula (I) includes those of Formula (Ila), (lib), (lie), or (lie): or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein R4 is as described herein. In some aspects, a subset of compounds of Formula (I) includes those of Formula (lid):

[0357] (lid): (TId), or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2 through Re are as described herein. For example, each of R2 and R3 may be independently a C5-14 alkyl or C5-14 alkenyl.

[0358] In some aspects, an ionizable cationic lipid of the disclosure comprises a compound having structure: (Compound 1).

[0359] In some aspects, an ionizable cationic lipid of the disclosure comprises a compound having structure: (Compound 11).

[0360] In some aspects, an ionizable cationic lipid of the disclosure comprises a compound having structure: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, - NRaC(=O)-, -C(=O) =NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, - C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- oradirect bond; G1and G2are each independently unsubstituted C1-C12 alkylene or Ci- C12 alkenylene; G is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0361] Rais H or C1-C12 alkyl;

[0362] R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0363] R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;

[0364] R4is C1-C12 alkyl;

[0365] R5is H or Ci-Ce alkyl; and x is 0, 1 , or 2.

[0366] In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0367] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

[0368] R6is, at each occurrence, independently H, OH or C1-C24 alkyl; and n is an integer ranging from 1 to 15.

[0369] In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein y and z are each independently integers ranging from 1 to 12.

[0370] In any of the foregoing aspects, one of L1or L2is -OCCO)-. For example, in some aspects, each of L1and L2are -O(C=O)-. In some aspects of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some aspects, each of L1and L2is - (C=O)O-.

[0371] In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0372] In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures:

[0373] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0374] In some of the foregoing aspects, n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some aspects, n is 3, 4, 5, or 6. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6.

[0375] In some of the foregoing aspects, y and z are each independently an integer ranging from 2 to 10. For example, in some aspects, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0376] In some of the foregoing aspects, R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other aspects, R6is OH. In some embodiments, G is unsubstituted. In other aspects, G3is substituted. In various different aspects, G3is linear C1-C24 alkylene or linear Ci- 024 alkenylene.

[0377] In some other foregoing embodiments, R1or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: wherein:

[0378] R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7b, and a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0379] In some of the foregoing aspects, at least one occurrence of R7ais H. For example, in some aspects, R7ais H at each occurrence. In other different aspects of the foregoing, at least one occurrence of R7bis Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0380] In different aspects, R1or R2, or both, has one of the following:

[0381] In some of the foregoing aspects, R is OH, CN, -C(=O)OR4-OC(=O)R4or -NHC(=O)R4. In some aspects, R4is methyl or ethyl.

[0382] It is understood that any aspect of the compounds set forth above, and any specific substituent and / or variable in the compounds set forth above, may be independently combined with other aspects and / or substituents and / or variables of compounds to form aspects of the inventions not specifically set forth above. In addition, in the event that a list of substituents and / or variables is listed for any particular substituent and / or variable in a particular embodiment and / or claim, it is understood that each individual substituent and / or variable may be deleted from the particular aspect and / or claim and that the remaining list of substituents and / or variables will be considered to be within the scope of the disclosure. It is understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in stable compounds.

[0383] In some embodiments, the cationic lipid is

[0384] In some embodiments, the cationic lipid is

[0385] In some aspects, the lipid nanoparticles comprise one or more cationic lipids. In one aspect, the lipid nanoparticles comprise (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315), having the formula: Exemplary cationic lipids are disclosed in, e.g., U.S. 10,166,298, the full disclosure of which is herein incorporated by reference in its entirety for all purposes. Representative cationic lipids include:

[0386]

[0387] In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cationic lipids may be excluded from the LNPs of the present disclosure.

[0388] In some aspects, the RNA-loaded LNPs comprise a cationic lipid, an RNA molecule as described herein, and one or more of neutral lipids, steroids, pegylated lipids, or combinations thereof. In one aspect, the cationic lipid is or is not present in the LNP in an amount such as at least, at most, exactly, between (inclusive or exclusive) of, or about 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent (mol %). In some aspects, two or more cationic lipids are incorporated within the LNP. If more than one cationic lipid is incorporated within the LNP, the foregoing percentages apply to the combined cationic lipids. In some aspects, an ionizable cationic lipid of the disclosure comprises a compound of Formula

[0389] (I)

[0390] Formula (I) or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof, wherein m, n, o, and p are each independently 1 - 3;

[0391] R2and R3are each independently H, Ci-ealkyl, Cs-scycloalkyl, or aryl; or R2and R3together with the nitrogen atom to which they are attached form a heterocyclic ring;

[0392] R4is H, Ci-ealkyl or Cs-scycloalkyl;

[0393] R5is Ci-ealkyl or Cs-scycloalkyl;

[0394] R6is H, CN, NO2, Ci-ealkyl, OR5, S(O)2R5, S(O)2N(R2)R3; q is 2 - 6; r is 1 - 6;

[0395] X is N or CH;

[0396] G2and G3are each independently Ci.^alkylene or C2-i2alkenylene;

[0397] L1and L2are each independently -C(=O)OR7, -OC(=O)R7, -OC(=O)(CH2)rC(=O)OR7, - OC(=O)(CH2)rOC(=O)R7, -OC(=O)N(R4)R7, -N(R4)C(=O)OR7, -N(R4)C(=O)N(R4)R7, -

[0398] OC(=O)OR7, or -S-SR7; and

[0399] R7is Ce-24alkyl, Ce-24alkenyl, or Ce-24alkynyl where each is optionally substituted by F, C1- ealkoxy, Cs-scycloalkyl, or Cs-scycloalkenyl.

[0400] In some aspects, the compound of Formula (I) has the structure of Formula (la)

[0401] Formula (la) or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein m, n, o, and p are each independently 1 or 2.

[0402] In some aspects, the compound of Formula (I) has the structure of Formula (lb)

[0403] Formula (lb) or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein m, n, o, and p are each independently 1 or 2.

[0404] In some aspects, the compound of Formula (I) has the structure of Formula (Ic)

[0405] Formula (Ic) or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein m and n are each independently 1 or 2; and o and p are each 1.

[0406] In some aspects, the compound of Formula (I) has a structure wherein

[0407] R2and R3are each independently H, Ci-ealkyl, or Cs-scycloalkyl; or R2and R3together with the nitrogen atom to which they are attached form a heterocyclic ring;

[0408] R4is H, Ci-ealkyl or Cs-scycloalkyl;

[0409] G2and G3are each independently Ci. ^alkylene;

[0410] L1and L2are each -OC(=O)R7; and

[0411] R7is Ce-24alkyl, C^alkenyl, or C^alkynyl wherein each is optionally substituted by F, Ci-ealkoxy, Cs-scycloalkyl, or Cs-scycloalkenyl.

[0412] In some aspects, the compound of Formula (I) has a structure wherein R7has the following structure:

[0413]

[0414] In some aspects, the compound of Formula (I) has a structure wherein R1is OH. In some aspects, the compound of Formula (I) has a structure wherein R1is

[0415] In some aspects, the compound of Formula (I) has a structure wherein G1is C2-C5 alkylene. In some aspects, the compound of Formula (I) has a structure wherein G1is C3-C5 alkylene. In some aspects, the compound of Formula (I) is selected from the group consisting of:

[0416] (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene) bis(2- heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl bis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1 ,3-diyl bis(2- heptylnonanoate); and 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1 ,3-diyl bis(2- heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0417] In some aspects, an ionizable cationic lipid of the disclosure comprises a compound of Formula (II) Formula (II) or a pharmaceutically acceptable salt, tautomer, N-oxide, or stereoisomer thereof, wherein

[0418] G1, G2and G3are independently C1-12 alkylene or C2-12 alkenylene;

[0419] L1and L2are independently -C(=O)O- or -OC(=O)-;

[0420] R1is Ce-24 alkyl, Ce-24 alkenyl, or Ce-24 alkynyl where each is substituted by W-Y;

[0421] R2is Ce-24 alkyl, Ce-24 alkenyl, or Ce-24 alkynyl where each is optionally substituted by W-Y;

[0422] W is independently Ci-e alkylene or a bond;

[0423] Y is independently C3-i2cycloalkyl, Cs-^cycloalkenyl, or a 4-6 membered heterocycloalkyl containing one oxygen atom, with the proviso that when R2 is Ce-24 alkyl, R1 is Ce-24 alkyl substituted by W-Y, and W is a bond, then Y is not cyclopropyl;

[0424] R4and R5are each independently H, Ci-e alkyl, C3-8 cycloalkyl, or aryl; or R4and R5together with the nitrogen atom to which they are attached form a heterocyclic ring;

[0425] R6is H, C1.6 alkyl or C3-8 cycloalkyl;

[0426] R7is C1.6 alkyl or C3-8 cycloalkyl; q is 2 - 6; r is 1 - 6.

[0427] In some aspects, the compound of Formula (II) has the structure of Formula (Ila)

[0428] Formula (Ila) or a pharmaceutically acceptable salt, tautomer, N-oxide, or stereoisomer thereof, wherein R8is independently C4-16 alkyl.

[0429] In some aspects, the compound of Formula II has a structure wherein

[0430] G1, G2, and G3are independently Ci- 12 alkylene;

[0431] L1is independently -C(=O)O- or -OC(=O)-; L2is independently -C(=0)0- or -OC(=O)-;

[0432] R1is Ce-24 alkyl substituted by W-Y;

[0433] R2is Ce-24 alkyl optionally substituted by W-Y;

[0434] W is independently -CH2- or -CH2CH2-;

[0435] Y is independently C3-i2cycloalkyl, Cs-^cycloalkenyl, or a 4-6 membered heterocycloalkyl containing one oxygen atom;

[0436] R4and R5are each independently H, Ci-e alkyl, C3-8 cycloalkyl, or aryl; or R4and R5together with the nitrogen atom to which they are attached form a heterocyclic ring;

[0437] R6is H, C1-6 alkyl or C3-8 cycloalkyl.

[0438] In some aspects, the compound of Formula (II) has a structure wherein Y has the following structure:

[0439] In some aspects, the compound of Formula (II) has a structure wherein R3is -OH. In some aspects, the compound of Formula (II) has a structure wherein R3is

[0440] In some aspects, the compound of Formula (II) has a structure wherein G3is C2-5 alkylene.

[0441] In some aspects, the compound of Formula (II) has a structure wherein R1and R2have the following structure:

[0442] In some aspects, the compound of Formula (II) is ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1- diyl) bis(2-(cyclobutylmethyl)decanoate), or a pharmaceutically acceptable salt thereof.

[0443] In some aspects of the disclosure, the LNP comprises a combination or mixture of any the lipids described above.

[0444] POLYMER CONJUGATED LIPIDS

[0445] In some aspects, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid (e.g., polyethylene glycol-lipid, PEG-lipid). In certain aspects, the LNP comprises an additional, stabilizing lipid that is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.

[0446] Pegylated lipids are known in the art and include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and mixtures thereof. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, PEG-DSG, PEG-DPG, and PEG-s-DMG (1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol). In one aspect, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one aspect, the polyethylene glycol-lipid is PEG-2000-DMG. In one aspect, the polyethylene glycol-lipid is PEG- c-DOMG. In other aspects, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((O-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate. PEG-lipids are disclosed in, e.g., U.S. 9,737,619, the full disclosures of which is herein incorporated by reference in its entirety for all purposes. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing pegylated lipids may be excluded from the LNPs of the present disclosure.

[0447] In some aspects, the composition comprises a pegylated lipid having the following structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0448] R8and R9are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some aspects, R8and R9are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some aspects, w has a mean value ranging from 43 to 53. In other aspects, the average w is or is about 45. In other different embodiments, the average w is or is about 49.

[0449] In some aspects, the lipid nanoparticles comprise a polymer conjugated lipid. In one aspect, the lipid nanoparticle comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159), having the formula:

[0450] In some aspects, the lipid nanoparticles comprise a polymer conjugated lipid compound of Formula (III): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0451] R1and R2are each independently hydrogen or methyl;

[0452] R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor;

[0453] R5is hydrogen or halogen; m is an integer from 1 to 6;

[0454] Y is a polymer having Formula (IV): n is an integer from 1 to 1000; x is an integer from 2 to 6; and w is an integer from 2 to 4.

[0455] In a preferred embodiment, m is 3, x is 2 and w is 2.

[0456] In another embodiment, R5is -Br.

[0457] In some aspects, the polymer conjugated lipid compound of Formula (III) is Formula (Illa): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0458] R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor; R5is hydrogen or halogen; and n is an integer from 1 to 1000. In some aspects, the polymer conjugated lipid compound of Formula (III) is Formula (111 b): or a pharmaceutically acceptable salt or stereoisomer thereof.

[0459] In some aspects, the polymer conjugated lipid compound of Formula (III) comprises a structure wherein (i) n ranges from 15 to 19, (ii) n ranges from 20 to 24, (iii) n ranges from 25 to 29, (iv) n ranges from 30 to 33, or (v) n ranges from 34 to 38. In some aspects, the polymer conjugated lipid compound of Formula (III) comprises a structure wherein the polymer conjugated lipid compound has a polydispersity index of about 1.0 to about 2.0. In some aspects, the polymer conjugated lipid compound of Formula (III) comprises a structure wherein the polymer conjugated lipid compound has a polydispersity index of between 1 .0 to 1.4. In a preferred embodiment, the polydispersity index is 1.2. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between 100 Daltons and 100,000 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between 4,500 Daltons and 12,000 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between about 4,800 Daltons and about 5,300 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between about 8,300 Daltons and about 8,900 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) has a molecular weight of between about 11 ,200 Daltons and about 11 ,800 Daltons. In some aspects, the polymer conjugated lipid compound of Formula (III) is selected from the group consisting of:

[0460] (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]; and (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11k], or a pharmaceutically acceptable salt or stereoisomer thereof.

[0461] In various aspects, the molar ratio of the cationic lipid to the pegylated lipid ranges from or from about 100:1 to about 20:1 , e.g., 20:1 , 25:1 , 30:1 , 35:1 , 40:1 , 45:1 , 50:1 , 55:1 , 60:1 , 65:1 , 70:1 , 75: 1 , 80: 1 , 85: 1 , 90: 1 , 95: 1 , or 100: 1 , or any range or value derivable therein.

[0462] In certain aspects, the PEG-lipid is or is not present in the LNP in an amount from or from about 1 to about 10 mole percent (mol %) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %), relative to the total lipid content of the nanoparticle.

[0463] In some aspects, the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulations.

[0464] ADDITIONAL LIPIDS

[0465] In certain aspects, the LNP comprises one or more additional lipids or lipid-like materials that stabilize particles during their formation. Suitable stabilizing or structural lipids include noncationic lipids, e.g., neutral lipids and anionic lipids. Without being bound by any theory, optimizing the formulation of LNPs by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable / cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery.

[0466] As used herein, an “anionic lipid” refers to any lipid that is negatively charged at a selected pH. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. In some aspects, additional lipids comprise one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

[0467] Representative neutral lipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, ceramides, sphingomyelins, dihydro-sphingomyelins, cephalins, and cerebrosides. Exemplary phospholipids include, for example, phosphatidylcholines, e.g., diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DM PC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC); and phosphatidylethanolamines, e.g., diacylphosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), 1 ,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroyl- phosphatidylethanolamine (DLPE), distearoyl-phosphatidylethanolamine (DSPE), 1-phytanoyl- phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearoyl-2-oleoylphosphatidyethanolamine (SOPE), 1 ,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (transDOPE), 1 ,2- dilinolenoyl-sn-glycero-3-phosphocholine,1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2- didocosahexaenoyl-sn-glycero-3- phosphocholine, 1 ,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure.

[0468] In one aspect, the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), having the formula:

[0469] In some aspects, the LNPs comprise a neutral lipid, and the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and / or SM. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure.

[0470] In various aspects, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0471] In certain aspects, the steroid or steroid analogue is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing steroid or steroid analogues may be excluded from the LNPs of the present disclosure. In certain aspects, the steroid or steroid analogue is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cholesterol derivaties may be excluded from the LNPs of the present disclosure. In one aspect, the cholesterol has the formula:

[0472] Without being bound by any theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some aspects, the molar ratio of the cationic lipid to the neutral lipid ranges from or from about 2: 1 to about 8:1 , or from or from about 10:0 to about 1 :9, about 4: 1 to about 1 :2, or about 3: 1 to about 1 :1.

[0473] In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and / or cholesterol), may comprise from or from about 0 mol % to about 90 mol %, from or from about 0 mol % to about 80 mol %, from or from about 0 mol % to about 70 mol %, from or from about 0 mol % to about 60 mol %, or from or from about 0 mol % to about 50 mol %, of the total lipid present in the particle. In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and / or cholesterol), may or may not be at least, at most, exactly, or between (inclusive or exclusive) of 0 mol %, 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, or 90 mol % of the total lipid present in the particle.

[0474] The following sequences are useful in the compositions and methods disclosed herein (FL = full- length):

[0475]

[0476]

[0477]

[0478] Embodiments of the disclosure are further described in the following numbered embodiments:

[0479] E1) A ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction:

[0480] (a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide and / or a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and

[0481] (b) an open reading frame encoding an outer surface protein A (OspA) polypeptide.

[0482] E2) The RNA molecule of embodiment 1 , further comprising a linker sequence located between the open reading frame encoding an influenza NA structural domain polypeptide, a PIV5 structural domain polypeptide and / or a RSV-G structural domain polypeptide and the open reading frame encoding an OspA polypeptide.

[0483] E3) The RNA molecule of embodiment 1 , wherein the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types.

[0484] E4) The RNA molecule of embodiment 1 , wherein the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25.

[0485] E5) The RNA molecule of embodiment 1 , wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3.

[0486] E6) The RNA molecule of embodiment 1 , wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49 or 63-69.

[0487] E7) The RNA molecule of embodiment 1 , wherein the open reading frame encoding an OspA polypeptide comprises one or more linkers.

[0488] E8) The RNA molecule of embodiment 1 , wherein the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides.

[0489] E9) The RNA molecule of embodiment 8, wherein the at least two OspA fragment polypeptides are different in silico types.

[0490] E10) The RNA molecule of embodiment 8, wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51 , or 54.

[0491] E11) The RNA molecule of embodiment 1 , wherein the NA structural domain polypeptide comprises a cytoplasmic domain, a transmembrane domain and an extracellular stalk.

[0492] E12) The RNA molecule of embodiment 1 , wherein the NA structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:14-19 or 62, the NA structural domain polypeptide and linker have at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:70-75, the PIV5 structural domain polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:59 and / or the RSV-G structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ I D NQs:60- 61.

[0493] E13) The RNA molecule of embodiment 1 , wherein the RNA molecule encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52.

[0494] E14) The RNA molecule of embodiment 1 , wherein the RNA molecule has at least 90% identity to any one of SEQ ID NQs:20-27, 53, 55, or 76-78.

[0495] E15) The RNA molecule of embodiment 1 , further comprising a 5' untranslated region (5' UTR).

[0496] E16) The RNA molecule of embodiment 15, wherein the 5' UTR comprises SEQ ID NO:28.

[0497] E17) The RNA molecule of embodiment 1 , further comprising a 3' untranslated region (3' UTR).

[0498] E18) The RNA molecule of embodiment 17, wherein the 3' UTR comprises SEQ ID NO:29.

[0499] E19) The RNA molecule of embodiment 2, wherein the linker sequence comprises SEQ ID NQ:30.

[0500] E20) The RNA molecule of embodiment 1 , wherein the RNA molecule further comprises a 3' poly-A tail.

[0501] E21) The RNA molecule of embodiment 20, wherein the poly-A tail comprises SEQ ID NO:31.

[0502] E22) The RNA molecule of embodiment 1 , wherein the RNA molecule further comprises a 5’ cap moiety.

[0503] E23) The RNA molecule of embodiment 1 , wherein the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%.

[0504] E24) The RNA molecule of embodiment 1 , wherein the RNA comprises at least one modified nucleotide.

[0505] E25) The RNA molecule of embodiment 1 , wherein at least one uridine is replaced by N1- methylpseudouridine (^P).

[0506] E26) The RNA molecule of embodiment 1 , wherein each uridine is replaced by N1- methylpseudouridine (^P).

[0507] E27) A composition comprising the RNA molecule of embodiment 1 , wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0508] E28) The composition of embodiment 27, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog.

[0509] E29) The composition of embodiment 28, wherein the cationic lipid is (4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0510] E30) The composition of embodiment 28, wherein the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycollipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3'- di(tetradecanoyloxy)propyl-1-O-((o- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>- methoxy(polyethoxy)ethyl)carbamate.

[0511] E31) The composition of embodiment 28, wherein the neutral lipid is 1 ,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE).

[0512] E32) The composition of embodiment 28, wherein the steroid or steroid analog is cholesterol.

[0513] E33) A method of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of the RNA molecule of embodiment 1.

[0514] E34) A method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of the RNA molecule of embodiment 1.

[0515] E35) The method of embodiment 34, wherein the infection, disease, or condition is Lyme disease.

[0516] E36) The method of embodiment 33 or 34, wherein the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older.

[0517] E37) The method of embodiment 33 or 34, wherein the RNA molecule is administered as a vaccine.

[0518] E38) The method of embodiment 33 or 34, wherein the subject is administered a single dose, two doses, three doses, four doses or more, and optionally, a booster dose of the RNA molecule.

[0519] E39) A method of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of the composition of embodiment 27.

[0520] E40) A method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of the composition of embodiment 27.

[0521] E41) A ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction:

[0522] (a) a 5' untranslated region (5’ UTR);

[0523] (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide;

[0524] (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide;

[0525] (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide, wherein the extracellular stalk is truncated compared to wild-type;

[0526] (e) a linker sequence;

[0527] (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide;

[0528] (g) a 3’ untranslated region (3’ UTR); and

[0529] (h) a 3’ poly-A tail.

[0530] E42) The RNA molecule of embodiment 41 , wherein the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types.

[0531] E43) The RNA molecule of embodiment 41 , wherein the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25.

[0532] E44) The RNA molecule of embodiment 41, wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3.

[0533] E45) The RNA molecule of embodiment 41 , wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49 or 63-69.

[0534] E46) The RNA molecule of embodiment 41 , wherein the open reading frame encoding an OspA polypeptide comprises one or more linkers.

[0535] E47) The RNA molecule of embodiment 41 , wherein the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides.

[0536] E48) The RNA molecule of embodiment 47, wherein the at least two OspA fragment polypeptides are different in silico types.

[0537] E49) The RNA molecule of embodiment 46, wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51, or 54.

[0538] E50) The RNA molecule of embodiment 41 , wherein the NA structural domain polypeptide has at least 90% identity to any one of SEQ ID NOs:14-19 or 62.

[0539] E51) The RNA molecule of embodiment 41 , wherein the RNA molecule encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52 and / or the RNA molecule has at least 90% identity to any one of SEQ ID NQs:20-27, 53, or 55.

[0540] E52) The RNA molecule of embodiment 41, wherein the 5' UTR comprises SEQ ID NO:28.

[0541] E53) The RNA molecule of embodiment 41, wherein the 3' UTR comprises SEQ ID NO:29.

[0542] E54) The RNA molecule of embodiment 41, wherein the linker sequence comprises SEQ ID NQ:30.

[0543] E55) The RNA molecule of embodiment 41, wherein the poly-A tail comprises SEQ ID NO:31.

[0544] E56) The RNA molecule of embodiment 41, wherein the RNA molecule further comprises a 5’ cap moiety.

[0545] E57) The RNA molecule of embodiment 41 , wherein the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%.

[0546] E58) The RNA molecule of embodiment 41, wherein the RNA comprises at least one modified nucleotide.

[0547] E59) The RNA molecule of embodiment 41, wherein at least one uridine is replaced by N1- methylpseudouridine (^P).

[0548] E60) The RNA molecule of embodiment 41 , wherein each uridine is replaced by N1- methylpseudouridine (^P).

[0549] E61) A composition comprising the RNA molecule of embodiment 41 , wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0550] E62) The composition of embodiment 61 , wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog.

[0551] E63) The composition of embodiment 62, wherein the cationic lipid is (4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0552] E64) The composition of embodiment 62, wherein the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycollipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3'- di(tetradecanoyloxy)propyl-1-O-((o- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>- methoxy(polyethoxy)ethyl)carbamate.

[0553] E65) The composition of embodiment 62, wherein the neutral lipid is 1 ,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero- 3-phosphoethanolamine (transDOPE).

[0554] E66) The composition of embodiment 62, wherein the steroid or steroid analog is cholesterol.

[0555] E67) A method of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of the RNA molecule of embodiment 41.

[0556] E68) A method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of the RNA molecule of embodiment 41.

[0557] E69) The method of embodiment 68, wherein the infection, disease, or condition is Lyme disease.

[0558] E70) The method of embodiment 67 or 68, wherein the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older.

[0559] E71) The method of embodiment 67 or 68, wherein the RNA molecule is administered as a vaccine.

[0560] E72) The method of embodiment 67 or 68, wherein the subject is administered a single dose, two doses, three doses, four doses or more, and optionally, a booster dose of the RNA molecule.

[0561] E73) A method of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of the composition of embodiment 61.

[0562] E74) A method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of the composition of embodiment 61.

[0563] E75) A ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction:

[0564] (a) a 5' untranslated region (5’ UTR);

[0565] (b) an open reading frame encoding an influenza neuraminidase (NA) cytoplasmic domain polypeptide;

[0566] (c) an open reading frame encoding an influenza neuraminidase (NA) transmembrane domain polypeptide;

[0567] (d) an open reading frame encoding an influenza neuraminidase (NA) extracellular stalk polypeptide;

[0568] (e) a linker sequence;

[0569] (f) an open reading frame encoding an outer surface protein A (OspA) polypeptide;

[0570] (g) a 3’ untranslated region (3’ UTR); and

[0571] (h) a 3’ poly-A tail.

[0572] E76) The RNA molecule of embodiment 75, wherein the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more in silico types.

[0573] E77) The RNA molecule of embodiment 75, wherein the OspA polypeptide is selected from the group consisting of in silico type (1ST) 1-25.

[0574] E78) The RNA molecule of embodiment 75, wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:1-3.

[0575] E79) The RNA molecule of embodiment 75, wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:33-49 or 63-69.

[0576] E80) The RNA molecule of embodiment 75, wherein the open reading frame encoding an OspA polypeptide comprises one or more linkers.

[0577] E81) The RNA molecule of embodiment 75, wherein the open reading frame encoding an OspA polypeptide comprises at least two OspA fragment polypeptides.

[0578] E82) The RNA molecule of embodiment 81 , wherein the at least two OspA fragment polypeptides are different in silico types.

[0579] E83) The RNA molecule of embodiment 81 , wherein the OspA polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:4-7, 50-51, or 54.

[0580] E84) The RNA molecule of embodiment 75, wherein the NA structural domain polypeptide has at least 90% identity to any one of SEQ ID NOs:14-19 or 62.

[0581] E85) The RNA molecule of embodiment 75, wherein the RNA molecule encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 8-13 or 52 and / or the RNA molecule has at least 90% identity to any one of SEQ ID NQs:20-27, 53, or 55.

[0582] E86) The RNA molecule of embodiment 75, wherein the 5' UTR comprises SEQ ID NO:28.

[0583] E87) The RNA molecule of embodiment 75, wherein the 3' UTR comprises SEQ ID NO:29.

[0584] E88) The RNA molecule of embodiment 75, wherein the linker sequence comprises SEQ ID NQ:30.

[0585] E89) The RNA molecule of embodiment 75, wherein the poly-A tail comprises SEQ ID NO:31.

[0586] E90) The RNA molecule of embodiment 75, wherein the RNA molecule further comprises a 5’ cap moiety.

[0587] E91) The RNA molecule of embodiment 75, wherein the open reading frame encoding an OspA polypeptide comprises a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or of about 50% to 75% or 55% to 70%.

[0588] E92) The RNA molecule of embodiment 75, wherein the RNA comprises at least one modified nucleotide.

[0589] E93) The RNA molecule of embodiment 75, wherein at least one uridine is replaced by N1- methylpseudouridine (^P).

[0590] E94) The RNA molecule of embodiment 75, wherein each uridine is replaced by N1- methylpseudouridine (^P).

[0591] E95) A composition comprising the RNA molecule of embodiment 75, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

[0592] E96) The composition of embodiment 95, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog.

[0593] E97) The composition of embodiment 96, wherein the cationic lipid is (4- hydroxyb...

Claims

CLAIMS1. A ribonucleic acid (RNA) molecule comprising in the 5’ to 3’ direction:(a) an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, a paramyxovirus simian virus 5 haemagglutinin-neuraminidase (PIV5) structural domain polypeptide and / or a respiratory syncytial virus (RSV) protein G (RSV-G) structural domain polypeptide; and(b) an open reading frame encoding an outer surface protein A (OspA) polypeptide.

2. The RNA molecule of claim 1, wherein the OspA polypeptide is selected from the group consisting of full-length polypeptides, fragment polypeptides, full-length fusion polypeptides, and fragment fusion polypeptides from one or more of in silico types 1-25.

3. The RNA molecule of claim 1, wherein the OspA polypeptide comprises an OspA C- terminal fragment from one or more of in silico types 1-25.

4. The RNA molecule of claim 1, wherein the OspA polypeptide comprises an aglycosylated OspA C-terminal fragment from one or more of in silico types 1-25.

5. The RNA molecule of claim 1, wherein the OspA polypeptide has at least 90% identity to an OspA C-terminal fragment of any one of the amino acid sequences of SEQ ID NOs:33-49 or 79-86.

6. The RNA molecule of claim 1, wherein the open reading frame encoding an OspA polypeptide comprises at least two OspA C-terminal fragment polypeptides.

7. The RNA molecule of claim 1, wherein the open reading frame encoding an OspA polypeptide comprises at least two OspA C-terminal fragment polypeptides of different in silico types.

8. The RNA molecule of claim 1, wherein the OspA polypeptide has at least 90% identity to the OspA polypeptide portion of any one of the amino acid sequences of SEQ ID NOs:1- 13, 50-52, 54, or 63-69.

9. The RNA molecule of claim 1, wherein the influenza NA structural domain polypeptide has at least 90% identity to any one of SEQ ID NOs:14-19 or 62, the influenza NA structural domain polypeptide and a linker have at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:70-75, the PIV5 structural domain polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:59 and / or the RSV- G structural domain polypeptide has at least 90% identity to any one of the amino acid sequences of SEQ ID NQs:60-61.

10. The RNA molecule of claim 1, wherein the RNA molecule encodes a polypeptide having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs:8-13 or 52.

11. The RNA molecule of claim 1 , wherein the RNA molecule has at least 90% identity to any one of SEQ ID NQs:20-27, 53, 55 or 76-78.

12. The RNA molecule of claim 1, wherein the RNA molecule further comprises a linker between the influenza NA, PIV5 and / or RSV-G structural domain polypeptide and the OspA polypeptide.

13. The RNA molecule of claim 1, wherein the RNA molecule further comprises a 5' cap moiety, a 5’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR) and 3’ poly- A tail.

14. The RNA molecule of claim 1, wherein the RNA comprises at least one modified nucleotide.

15. The RNA molecule of claim 1, wherein at least one uridine is replaced by N1- methylpseudouridine (^P).

16. A composition comprising the RNA molecule of claim 1 , wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

17. The composition of claim 16, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, a steroid, and a steroid analog.

18. A method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with Borrelia in a subject, comprising administering to a subject an effective amount of the RNA molecule of claim 1.

19. The method of claim 18, wherein the infection, disease, or condition is Lyme disease.

20. The method of claim 18, wherein the RNA molecule is administered as a vaccine.

21. A method of inducing an immune response against Borrelia in a subject, comprising administering to the subject an effective amount of the composition of claim 16.

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