Hepatitis B components

Abstract

A composition for treating chronic hepatitis B infection, comprising mRNA encoding a hepatitis B viral antigen, the mRNA being encapsulated in a lipid nanoparticle (LNP).

Description

[Technical Field]

[0001] The present invention relates to compositions for use in treating chronic hepatitis B, the compositions comprising mRNA encoding one or more hepatitis B antigens, and related embodiments. [Background technology]

[0002] Hepatitis B virus (HBV) infection is a major public health problem. The WHO estimated that 296 million people worldwide were living with chronic hepatitis B infection in 2019, with 1.5 million new cases occurring each year (WHO, 2021). The clinical course and outcome of HBV infection are largely determined by the age at which infection is acquired and the complex interplay between the virus and the individual's immune response. Therefore, exposure to HBV can result in acute hepatitis that resolves spontaneously, or it can progress to various forms of chronic infection, including inactive hepatitis B surface antigen (HBsAg) carrier state, chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). It is estimated that 15–40% of chronically infected individuals (defined as serum HBsAg levels detectable for ≥6 months) will develop liver sequelae, of which liver cirrhosis (LC), liver dysfunction, and hepatocellular carcinoma (HCC) are the main complications.

[0003] Although the implementation of universal hepatitis B vaccination for infants and young children has been highly effective in reducing the incidence and prevalence of hepatitis B in many endemic countries, it has not yet translated into strong reductions in the prevalence of chronic hepatitis B (CHB) in adolescents and adults, and is not expected to impact HBV-related deaths decades after its introduction. In 2019, the World Health Organization (WHO) estimated that 820,000 deaths were due to HBV-related causes, most of which were due to cirrhosis and hepatocellular carcinoma (primary liver cancer) (WHO, 2021).

[0004] Clinical management of chronic hepatitis B aims to improve survival and quality of life by preventing disease progression and, ultimately, the development of HCC. Current treatment strategies are primarily based on long-term suppression of HBV DNA replication to achieve stabilization of HBV-induced liver disease and prevent progression. Serum HBV DNA levels are the fundamental endpoint for all current treatment modalities. While achieving the disappearance of (detectable) hepatitis B e antigen (HBeAg) is an important biomarker, the disappearance of HBsAg, with or without anti-HBs seroconversion, is generally considered the optimal endpoint representing a "functional cure" because it indicates meaningful suppression of HBV replication and viral protein expression (Revill, 2019; Block, 2017; Cornberg, 2017). Currently, the two main treatment options for CHB patients are treatment with pegylated interferon α (PegIFNα) or nucleoside (nucleotide) analogs (NAs) (EASL, 2017). Although PegIFNα aims to induce long-term immune control with limited duration of treatment and can achieve sustained off-therapy control, sustained viral responses and hepatitis B surface antigen (HBsAg) clearance are achieved only in a small proportion of patients. Furthermore, poor tolerability and long-term safety concerns make this type of treatment ineligible for a significant number of patients.

[0005] NAs act by inhibiting HBV DNA replication through the inhibition of HBV polymerase reverse transcriptase activity. NAs approved for HBV treatment in Europe include those with a high barrier to HBV resistance, such as entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF), as well as those with a low barrier to HBV resistance, such as lamivudine (LAM), adefovir dipivoxil (ADV), and telbivudine (TBV). The main advantages of treatment with potent NAs with a high barrier to resistance are their predictable, long-term antiviral efficacy, leading to HBV DNA suppression in the majority of eligible patients, and their favorable safety profile. The drawback of NA treatment is the long-term treatment regimen, because NAs usually do not achieve HBV eradication, and HBV recurrence can occur if NAs are discontinued.

[0006] Due to the low rate of HBsAg seroclearance and the high risk of off-NA viral recurrence, most patients are maintained on long-term or indefinite NA treatment, which may lead to poor patient compliance, increased economic costs, and an increased risk of drug toxicity and drug resistance mutations due to long-term exposure. Therefore, new strategies to achieve a "functional cure" with a finite regimen are needed.

[0007] Messenger RNA (mRNA) is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by ribosomes during the process of producing proteins. mRNA-based vaccines offer an alternative vaccination strategy to traditional strategies, including attenuated / inactivated pathogens and subunit vaccines (Zhang, 2019). mRNA vaccines may utilize non-replicating mRNA (mRNA) or self-replicating RNA (also known as self-amplifying mRNA or SAM). Non-replicating mRNA-based vaccines typically encode the antigen of interest and contain 5' and 3' untranslated regions (UTRs), a 5' cap, and a poly(A) tail, while self-amplifying RNA also encodes the viral replication machinery, allowing for intracellular RNA amplification (Pardi, 2018).

[0008] The present invention aims to contribute to addressing the need for an HBV treatment that can clear HBsAg, allowing patients to safely discontinue NA treatment without viral or clinical relapse. Summary of the Invention

[0009] The present disclosure provides a composition for treating chronic hepatitis B infection, comprising mRNA encoding at least hepatitis B core antigen (HBc), wherein the mRNA is encapsulated in a lipid nanoparticle (LNP). In an embodiment, the HBc is fused to a human invariant chain (hIi).

[0010] In one aspect, the present disclosure provides a composition for treating chronic hepatitis B infection, comprising mRNA encoding at least one hepatitis B surface antigen (HBsAg), wherein the mRNA is encapsulated in a lipid nanoparticle (LNP). In an embodiment, the HBsAg is the hepatitis B small surface protein (HBs). In an embodiment, the HBsAg is fused with a human invariant chain (hIi).

[0011] In further embodiments, the mRNA is non-replicating. In other embodiments, the mRNA is a self-replicating mRNA (SAM).

[0012] In another aspect, the composition comprising mRNA is administered sequentially or simultaneously with one or more recombinant Hepatitis B polypeptides. In one embodiment, the recombinant Hepatitis B polypeptides include recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs). In a further embodiment, the one or more recombinant Hepatitis B polypeptides are administered with an adjuvant. The adjuvant can be AS01.

[0013] Also described herein is a method for treating chronic hepatitis B infection. The method may include a prime-boost regimen. An mRNA encoding a hepatitis B virus antigen may be administered as a priming dose, and one or more recombinant hepatitis B polypeptides may be administered as a booster dose. In one embodiment, one or more recombinant hepatitis B polypeptides may be administered as a booster dose with an adjuvant. The adjuvant may be AS01.

[0014] Also described herein is a method for treating chronic hepatitis B infection in a human, comprising the following steps: (a) administering to the human an adenoviral vector comprising a polynucleotide encoding a hepatitis B polypeptide; (b) administering to the human mRNA encoding a hepatitis B virus antigen; and (c) administering to the human at least one recombinant hepatitis B polypeptide. Such a method may be a heterologous prime-boost regimen comprising: (a) administering the adenoviral vector as a priming dose; (b) administering the mRNA as a booster dose; and (c) administering at least one recombinant hepatitis B polypeptide as one or more boost doses. In one embodiment, the adenoviral vector is a replication-deficient chimpanzee adenovirus (ChAd) vector.

[0015] The present invention also provides compositions comprising mRNA administered sequentially or simultaneously with one or more polypeptides. In a further embodiment, the one or more polypeptides are administered with an adjuvant. The adjuvant can be AS01.

[0016] In one aspect, there is a method comprising administering to a human an mRNA in combination with at least one polypeptide. The components (i.e., the mRNA and the polypeptide) may be administered sequentially in a heterologous prime-boost regimen. When using a heterologous prime-boost regimen, the mRNA is administered as a priming dose and the at least one polypeptide is administered as a booster dose. In another aspect, the at least one polypeptide is administered as a priming dose and the mRNA is administered as a booster dose. The at least one polypeptide may be administered with or without an adjuvant. In a particular embodiment, the polypeptide is administered with an adjuvant. In one embodiment, the mRNA is administered sequentially with the adjuvanted polypeptide. In another embodiment, the mRNA is administered simultaneously (e.g., simultaneously at different locations) with the adjuvanted polypeptide. The adjuvant is preferably AS01.

[0017] A brief description of arrays SEQ ID NO: 1: Amino acid sequence of HBs SEQ ID NO: 2: Amino acid sequence of truncated HBc SEQ ID NO: 3: Amino acid sequence of the spacer incorporating the 2A cleavage region of foot-and-mouth disease virus SEQ ID NO: 4: Nucleotide sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus SEQ ID NO: 5: Amino acid sequence of HBc-2A-HBs SEQ ID NO: 6: Nucleotide sequence encoding HBc-2A-HBs SEQ ID NO: 7: Amino acid sequence of hIi SEQ ID NO: 8: Nucleotide sequence encoding hIi SEQ ID NO: 9: Amino acid sequence of hIi-HBc-2A-HBs SEQ ID NO: 10: Nucleotide sequence encoding hIi-HBc-2A-HBs SEQ ID NO: 11: Amino acid sequence of HBc SEQ ID NO: 12: Amino acid sequence of hIi alternative variant SEQ ID NO: 13: Nucleotide sequence encoding hIi alternative variant SEQ ID NO: 14: Alternative nucleic acid sequence of hIi-HBc-2A-HBs SEQ ID NO: 15: Alternative amino acid sequence of hIi-HBc-2A-HBs SEQ ID NO: 16: Nucleic acid sequence of empty SAM vector SEQ ID NO: 17: Human codon-optimized (Genewiz) nucleic acid sequence encoding the hIi_HBc_2A_HBs SAM transgene SEQ ID NO: 18: hIi_HBc_2A_HBs SAM plasmid sequence SEQ ID NO: 19: Human codon-optimized (Genewiz) nucleic acid sequence encoding the HBc_2A_HBs SAM transgene SEQ ID NO: 20: HBc_2A_HBs in SAM plasmid sequence SEQ ID NO: 21: Amino acid sequence of hIi-HBc SEQ ID NO: 22: Nucleotide sequence encoding hIi-HBc SEQ ID NO: 23: hIi-HBc mRNA plasmid sequence (UTR4) SEQ ID NO: 24: Nucleotide sequence encoding HBs SEQ ID NO: 25: HBs mRNA plasmid sequence (UTR4) SEQ ID NO: 26: Amino acid sequence of hIi-HBs SEQ ID NO: 27: Nucleotide sequence encoding hIi-HBs SEQ ID NO: 28: hIi-HBs mRNA plasmid sequence (UTR4) SEQ ID NO: 29: IRES nucleotide sequence SEQ ID NO: 30: Human codon-optimized (CodeRNA2) nucleic acid sequence encoding the hIi_HBc mRNA transgene SEQ ID NO: 31: Human codon-optimized (CodeRNA2) nucleic acid sequence encoding the HBs mRNA transgene SEQ ID NO: 32: Human codon-optimized (CodeRNA2) nucleic acid sequence encoding the hIi_HBs mRNA transgene [Brief explanation of the drawings]

[0018] [Figure 1A]HBV core antigen (HBc)-specific CD4+ T cell responses in the spleen after priming with ChAd155-hIi-HBV and boosting with SAM-HBV (±hIi) are shown. Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) for assessment of HBc core (HBc)-specific CD4+ T cells by intracellular staining. Each point represents an individual animal, and the horizontal line represents the geometric mean (GM). [Figure 1B] HBV surface antigen (HBs)-specific CD4+ T cell responses in the spleen after priming with ChAd155-hIi-HBV and boosting with SAM-HBV (±hIi). Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) for assessment of HBs surface antigen (HBs)-specific CD4+ T cells by intracellular staining. Each point represents an individual animal, and the horizontal line represents the geometric mean (GM). [Figure 2A] HBV core antigen (HBc)-specific CD8+ T cell responses in the spleen after priming with ChAd155-hIi-HBV and boosting with SAM-HBV (±hIi) are shown. Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) for assessment of HBc core (HBc)-specific CD8+ T cells by intracellular staining. Each point represents an individual animal, and the horizontal line represents the geometric mean (GM). [Figure 2B] HBV surface antigen (HBs)-specific CD8+ T cell responses in spleens after priming with ChAd155-hIi-HBV and boosting with SAM-HBV (±hIi). Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) for assessment of HBs surface antigen (HBs)-specific CD8+ T cells by intracellular staining. Each point represents an individual animal, and the horizontal line represents the geometric mean (GM). [Figure 3A]HBV core antigen (HBc)-specific antibody responses after priming with ChAd155-hIi-HBV and boosting with SAM-HBV (±hIi) are shown. Serum samples were collected 12 / 13 days after the second immunization to assess anti-HBc IgG antibody titers by ELISA. For each group, each point represents the anti-HBc IgG antibody titer of an individual animal, and columns represent the geometric mean (GM) with 95% confidence intervals (CI). [Figure 3B] HBV surface antigen (HBs)-specific antibody responses after priming with ChAd155-hIi-HBV and boosting with SAM-HBV (±hIi) are shown. Serum samples were collected 12 / 13 days after the second immunization to assess anti-HBs IgG antibody titers by ELISA. For each group, each point represents the anti-HBs IgG antibody titer of an individual animal, and columns represent the geometric mean (GM) with 95% confidence intervals (CI). [Figure 4A] HBV core antigen (HBc)-specific CD8+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 14 days after the second immunization (14 dpII), spleens were harvested for assessment of HB core (HBc)-specific CD8+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 4B] HBV core antigen (HBc)-specific CD8+ T cell responses in the spleen at 22 dpIV (i.e., 22 days after the fourth dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Spleens were harvested 22 days after the fourth immunization (22 dpIV) for assessment of HB core (HBc)-specific CD8+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 5A]HBV surface antigen (HBs)-specific CD8+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 14 days after the second immunization (14 dpIV), spleens were harvested for assessment of HBs surface antigen (HBs)-specific CD8+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 5B] HBV surface antigen (HBs)-specific CD8+ T cell responses in the spleen at 22 dpIV (i.e., 22 days after the fourth dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Spleens were harvested 22 days after the fourth immunization (22 dpIV) for assessment of HBs surface antigen (HBs)-specific CD8+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 6A] HBV core antigen (HBc)-specific CD4+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 14 days after the second immunization (14 dpII), spleens were harvested for assessment of HB core (HBc)-specific CD4+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 6B]HBV core antigen (HBc)-specific CD4+ T cell responses in the spleen at 22 dpIV (i.e., 22 days after the fourth dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Spleens were harvested 22 days after the fourth immunization (22 dpIV) for assessment of HB core (HBc)-specific CD4+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 7A] HBV surface antigen (HBs)-specific CD4+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 14 days after the second immunization (14 dpII), spleens were harvested for assessment of HBs surface antigen (HBs)-specific CD4+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 7B] HBV surface antigen (HBs)-specific CD4+ T cell responses in the spleen at 22 dpIV (i.e., 22 days after the fourth dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Spleens were harvested 22 days after the fourth immunization (22 dpIV) to assess HBs surface antigen (HBs)-specific CD4+ T cells by intracellular staining. Each point represents an individual animal, and columns represent the geometric mean (GM). [Figure 8A]Anti-HBc binding antibody titers measured on 13 dp11 / 14 dp11 (i.e., 13 / 14 days after the second dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Serum samples were collected 13 / 14 days after the second immunization to assess anti-HBc IgG antibody titers by ELISA. For each group, each point represents the anti-HBc IgG antibody titer of an individual animal, and the columns represent the geometric mean (GM) with 95% confidence intervals (CI). [Figure 8B] Anti-HBc binding antibody titers measured 22 dpIV (i.e., 22 days after the fourth dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Serum samples were collected 22 days after the fourth immunization for assessment of anti-HBc IgG antibody titers by ELISA. For each group, each point represents the anti-HBc IgG antibody titer of an individual animal, and the columns represent the geometric mean (GM) with 95% confidence intervals (CI). [Figure 9A] Anti-HBs binding antibody titers measured on 13 / 14 dpII (i.e., 13 / 14 days after the second dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Serum samples were collected 13 / 14 days after the second immunization to assess anti-HBs IgG antibody titers by ELISA. For each group, each point represents the anti-HBs IgG antibody titer of an individual animal, and columns represent the geometric mean (GM) with 95% confidence intervals (CI). [Figure 9B]Anti-HBs binding antibody titers measured 22 dpIV (i.e., 22 days after the fourth dose) are shown for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). Serum samples were collected 22 days after the fourth immunization to assess anti-HBs IgG antibody titers by ELISA. For each group, each point represents the anti-HBs IgG antibody titer of an individual animal, and columns represent the geometric mean (GM) with 95% confidence intervals (CI). [Figure 10] The dynamics of circulating HBsAg titers detected in the different groups are shown. The geometric mean (GM) of circulating HBsAg titers is represented by a box with 95% confidence interval. [Figure 11A] Figure 1 shows circulating HBsAg titers after the second and fourth immunization compared to pre-immunization titers in different groups. Geometric mean ratios are represented by boxes with 90% confidence intervals. [Figure 11B] Continued from Figure 11A. [Figure 12] Figure 1 shows the dynamics of AST and ALT values ​​detected in the different groups. The geometric mean ratios (GMR) of AST and ALT titers compared to pre-immunization titers are represented by triangles with 95% confidence intervals. [Figure 13A] Cytokine co-expression profiles of HBc-specific CD8+ T cells are shown. The frequencies of HBc-specific CD8+ T cells expressing at least one, two, or three cytokines (IL-2, IFN-γ, and TNF-α) were assessed by intracellular staining 14 days after the second immunization. Median values ​​per group are plotted. [Figure 13B] Cytokine co-expression profiles of HBs-specific T cells are shown. The frequencies of HBs-specific CD8+ T cells expressing at least one, two, or three cytokines (IL-2, IFN-γ, and TNF-α) were assessed by intracellular staining 14 days after the second immunization. Median values ​​per group are plotted. [Figure 14]The SAM-HBV constructs used in the examples are shown. The SAM construct contains the genetic elements of VEEV TC-83 required for RNA amplification (nonstructural protein sequences, nsP1-4). The sequences encoding the structural proteins have been replaced with a transgene encoding an HBV polypeptide under the control of a subgenomic promoter. The empty SAM plasmid is shown in SEQ ID NO: 16. The insert begins after nucleotide 7561 of SEQ ID NO: 16. Two different HBV constructs are shown. In both constructs, the HBc and HBs proteins are separated by a 2A sequence. In one construct, the human invariant chain (hIi) is fused to HBc. [Figure 15] 1 shows the hIi_HBc_2A_HBs SAM plasmid map of the SAM plasmid sequence of SEQ ID NO: 18. [Figure 16] HBc_2A_HBs SAM plasmid map of the SAM plasmid sequence of SEQ ID NO: 20. [Figure 17A] HBV-specific CD8+ and CD4+ T cell responses after co-administration of three different mRNAs are shown. [Figure 17B] Continued from Figure 17A. [Figure 18A] HBc-specific and HBs-specific CD8+ T cell responses observed in Example 3 are shown. [Figure 18B] Continued from Figure 18A. [Figure 19A] FIG. 18 shows a comparison of the geometric mean ratio (GMR) of CD8+ T cell responses for the (hIi-HBc+hIi-HBs) mRNA construct combination versus hIi-HBc and hIi-HBs alone. [Figure 19B] Continued from Figure 19A. [Figure 20A] HBc-specific and HBs-specific CD4+ T cell responses observed in Example 3 are shown. [Figure 20B] Continued from Figure 20A. [Figure 21] HBc-specific IgG responses observed in Example 3 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0019] HBV antigen At least 10 HBV genotypes (A to J) have been identified (Liu, 2021). Within a particular HBV genotype, multiple subgenotypes have also been identified. For example, genotypes A, B, C, D, and F are further divided into subgenotypes. Antigens for use in the disclosed compositions and methods are selected to provide immunological coverage across all HBV genotypes. The HBV genome contains four overlapping open reading frames (ORFs) encoding (i) viral polymerase (Pol), (ii) viral surface proteins (L-HBs, M-HBs, and HBs), (iii) precore / core proteins (HBe and HBc), and (iv) the X protein (HBx).

[0020] Hepatitis B virus surface protein (HBsAg) consists of three related but distinct proteins: the large (L), middle (M), and small (S) surface proteins. HBV surface proteins (L, M, and S) are derived from alternative translations of the same ORF. The large surface protein consists of three domains: preS1 (108 / 118 / 119 amino acids depending on the genotype; the preS1 domain in genotype A is 119 amino acids), preS2 (55 amino acids), and the small surface protein (HBs, 226 amino acids). The middle surface protein consists of two domains: preS2 and the small surface protein (HBs). The small surface protein (HBs) does not contain preS1 or preS2 and is 226 amino acids long.

[0021] The hepatitis B core protein antigen (HBc) is highly conserved across genotypes and subtypes, and the sequence of the hepatitis B small surface protein antigen (HBs) is selected to contain important cross-genotypically conserved B-cell epitopes that enable the induction of a broad neutralizing response. Preferably, the HBc and HBs sequences for use in the disclosed methods and compositions are based on those of genotype / subtype A2.

[0022] Preferably, HBV surface protein antigens for use in the disclosed methods and compositions are derived from the small (S) surface antigen protein. In particular, HBV surface antigens for use herein may be derived from HBs.

[0023] In particular, a suitable HBV surface protein antigen comprises the small (S) protein (HBs) of genotype A HBV adw2 strain. For example, a suitable HBs antigen has at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, a suitable HBs antigen has 226 amino acids of the amino acid sequence of SEQ ID NO: 1. In one aspect, an HBs antigen can be fused to an hIi. In one embodiment, the hIi-HBs antigen has at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 26. In a preferred embodiment, the hIi-HBs antigen has the amino acid sequence of SEQ ID NO: 26.

[0024] Hepatitis B core protein (HBc) is the major component of the nucleocapsid shell that packages the viral genome. This protein (183-185 amino acids long) is expressed in the cytoplasm of infected cells. HBc contains a 149-residue assembly domain and a 34-36 residue RNA-binding domain at the C-terminus. HBc antigens used in the disclosed methods and compositions can be full-length or C-terminally truncated proteins (lacking the RNA-binding C-terminus), e.g., amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of wild-type hepatitis B core antigen protein. Truncated proteins retain the ability to assemble into nucleocapsid particles. An HBc antigen suitable for use in the disclosed methods and compositions has an amino acid sequence derived from the HBV adw2 strain of genotype A. When used as a recombinant polypeptide, the recombinant HBc protein is preferably truncated at the wild-type C-terminus. In particular, the recombinant HBc protein has at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:2. In a preferred embodiment, the recombinant HBc protein has the amino acid sequence of SEQ ID NO:2. When expressed from an mRNA or viral vector, the HBc antigen is preferably full-length HBc antigen. In particular, the HBc antigen has at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11. In one embodiment, the HBc antigen has the amino acid sequence of SEQ ID NO:11. In one aspect, the HBc antigen may be fused to an hIi. In one embodiment, the hIi-HBc has at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:21. In a preferred embodiment, the hIi-HBc has the amino acid sequence of SEQ ID NO:21.

[0025] immutable chain Antigens are substances that induce immune responses, particularly antibody production, in the body. Antigens can be foreign, i.e., pathogenic, or derived from the organism itself; these are called self-antigens or autoantigens. Antigens can be presented on the surface of antigen-presenting cells by MHC molecules. There are two classes of MHC molecules: MHC class I (MHC-I) and MHC class II (MHC-II). MHC-II molecules are membrane-bound receptors synthesized in the endoplasmic reticulum and exit the endoplasmic reticulum as MHC class II compartments. To prevent endogenous peptides, i.e., self-antigens, from binding to MHC-II molecules and being presented to trigger an immune response, nascent MHC-II molecules interact with another protein, the invariant chain, which blocks the peptide-binding cleft of the MHC-II molecule.

[0026] Human invariant chain (hIi, also known as CD74 when expressed on the plasma membrane) is an evolutionarily conserved type II membrane protein that plays several roles within cells and throughout the immune system (Borghese, 2011). When MHC class II compartments fuse with late endosomes containing phagocytosed and degraded foreign proteins, the invariant chain is cleaved, leaving only the CLIP domain bound to MHC-II molecules. In a second step, CLIP is removed by HLA-DM molecules, freeing MHC-II molecules to bind fragments of the foreign protein. These fragments are presented on the surface of antigen-presenting cells upon fusion of the MHC class II compartment with the plasma membrane, where they present foreign antigens to other cells, primarily T helper cells.

[0027] Adenoviral expression systems encoding invariant chain-antigen fusions are known to enhance immune responses to antigens when used for vaccination (see WO2007 / 062656, also published as US2011 / 0293704, incorporated by reference for the purposes of disclosing invariant chain sequences). That is, the invariant chain enhances the immunogenicity of the antigen. Furthermore, such adenoviral constructs have proven useful for priming immune responses in the context of prime-boost vaccination regimens (see WO2014 / 141176, also published as US2016 / 0000904; and WO2010 / 057501, also published as US2010 / 0278904, incorporated by reference for the purposes of disclosing invariant chain sequences and adenoviral vectors encoding invariant chain sequences).

[0028] In the present invention, the mRNA encoding the hepatitis B virus antigen comprises a nucleotide sequence encoding an invariant chain (Ii), preferably a human invariant chain (hIi). Two amino acid sequences of hIi are shown in SEQ ID NO: 7 and SEQ ID NO: 12. In a preferred embodiment, the invariant chain has SEQ ID NO: 12. Suitably, the nucleotide sequence encoding hIi is fused at the N-terminus to a nucleotide sequence encoding HBc antigen and / or HBs antigen.

[0029] The present invention provides a composition for treating chronic hepatitis B infection, which comprises at least mRNA encoding hepatitis B virus core antigen (HBc), the mRNA being encapsulated in a lipid nanoparticle (LNP), and the N-terminus of the nucleotide sequence encoding HBc being fused to a human invariant chain (hIi).

[0030] The present invention also provides a composition for treating chronic hepatitis B infection, comprising a first mRNA encoding hepatitis B virus core antigen (HBc) and a second mRNA encoding hepatitis B virus surface antigen (HBs), wherein the first and second mRNAs are encapsulated in lipid nanoparticles (LNPs), and the N-termini of the nucleotide sequences encoding HBc and HBs are fused to a human invariant chain (hIi).

[0031] In one embodiment, the mRNA encodes the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:15 (preferably SEQ ID NO:15), which is a fusion of hIi reported in SEQ ID NO:12, HBc reported in SEQ ID NO:11, 2A reported in SEQ ID NO:3, and HB reported in SEQ ID NO:1.

[0032] In certain embodiments, an adenoviral vector (Ad), e.g., a chimpanzee adenoviral vector (ChAd), for use in the methods and compositions disclosed herein, can contain a nucleotide sequence encoding an hIi. Two amino acid sequences of an hIi as contained in the disclosed adenoviral vectors are set forth in SEQ ID NO:7 and SEQ ID NO:12, and the nucleotide sequences encoding these amino acid sequences are set forth in SEQ ID NO:8 and SEQ ID NO:13, respectively. In a preferred embodiment, the invariant chain has SEQ ID NO:12. Suitably, the nucleotide sequence encoding the hIi is fused N-terminally to a nucleotide sequence encoding an HBc antigen.

[0033] messenger RNA (mRNA) non-replicating mRNA The present disclosure provides compositions comprising recombinant messenger RNA (mRNA) having an open reading frame encoding at least one hepatitis B virus antigen. As used herein, the term "recombinant messenger RNA (mRNA)" refers to any recombinantly produced polynucleotide that encodes at least one polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide of interest. mRNA typically includes a portion encoding the polypeptide of interest (i.e., a portion encoding a heterologous polypeptide such as a hepatitis B virus antigen), a 5' untranslated region (5' UTR), an optional 3' untranslated region (3' UTR), a 3' poly(adenosine monophosphate) (3' poly(A)) tail, and a 5' cap. The 5' UTR is upstream (i.e., 5') of the polypeptide of interest, while the 3' UTR is downstream (i.e., 3') of the polypeptide of interest. The 5' UTR begins at the transcription start site and ends one nucleotide before the translation initiation sequence (i.e., the sequence 5'-adenosine, uridine, guanosine-3' (5'-AUG-3')) of the coding region of the polypeptide of interest, and the 3' UTR follows the translation termination codon of the coding region of the polypeptide of interest.

[0034] In one embodiment, the mRNA of the present disclosure may be structurally or chemically modified. As used herein, a "structural" modification refers to a modification in which two or more linked nucleotides in a polynucleotide are inserted, deleted, duplicated, inverted, or randomized without significant chemical modification of the nucleotides themselves. Because structural modifications necessarily involve breaking and reforming chemical bonds, structural modifications are chemical in nature and are therefore chemical modifications. However, structural modifications result in changes to the nucleotide sequence. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G." The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG." In this case, the dinucleotide "CC" is inserted, resulting in a structural modification of the polynucleotide. In one embodiment, the mRNA of the present disclosure has all or any of the same nucleoside types uniformly chemically modified, or has all or any of the same nucleoside types chemically modified in measured proportions but randomly incorporated (e.g., all uridines replaced with uridine analogs, such as pseudouridines). In another embodiment, the mRNA can have two, three, or four of the same nucleotide types uniformly chemically modified throughout the polynucleoside (e.g., all uridines and all cysteines are similarly modified). When a polynucleotide of an mRNA of the present disclosure is chemically or structurally modified, the polynucleotide may be referred to as a "modified polynucleotide."

[0035] In one embodiment, the mRNA has the following configuration: 5' cap / 5' UTR / hIi / HBc / 3' UTR / polyA.

[0036] In one embodiment, the mRNA has the configuration 5' cap / 5' UTR / HBc / 3' UTR / polyA.

[0037] In one embodiment, the mRNA has the configuration 5'cap / 5'UTR / hIi / HBs / 3'UTR / polyA.

[0038] In one embodiment, the mRNA has the configuration 5' Cap / 5' UTR / HBs / 3' UTR / polyA.

[0039] In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the mRNA further comprises a 7-methylguanosine, a 5' first ribonucleoside, optionally a 5' second ribonucleoside, and optionally a triphosphate bridge. In some embodiments, the 7-methylguanosine is linked 5'-to-5' to the 5' first ribonucleoside directly or indirectly. In some embodiments, the 7-methylguanosine is linked 5'-to-5' to the 5' first ribonucleoside by a triphosphate bridge. In some embodiments, the 5' first ribonucleoside comprises a 2'-methylated ribose (2'-O-Me) (i.e., Cap-1 or Cap-2). In some embodiments, the 5' second ribonucleoside is attached to the 3' end of the 5' first ribonucleoside. In some embodiments, the 5' second ribonucleoside comprises a 2'-methylated ribose (2'-O-Me) (i.e., Cap-2). In some embodiments, the 5' first ribonucleoside comprises a 2'-methylated ribose (2'-O-Me) and the 5' second ribonucleoside comprises a 2'-methylated ribose (2'-O-Me) (i.e., Cap-2). The 5' cap comprises a guanosine linked to an RNA by an mRNA guanylyltransferase via a 5' to 5' triphosphate linkage, wherein the guanine of the guanosine is methylated at its 7-position. In this context and in some embodiments, a 5' to 5' triphosphate linkage occurs when the 5' end of the ribose of the guanosine is linked to the 5' end of the ribose of an mRNA by an mRNA guanylyltransferase via a triphosphate group. In some embodiments, the guanine of the guanosine is then methylated at position 7 by a (guanine-N7-) methyltransferase. In some embodiments, the addition of 7-methylguanosine 5'-to-5' to the 5' first ribonucleoside occurs in one step, without addition of the 7-guanosine and further methylation to obtain the 7-methylguanosine (i.e., CLEANCAP®).In some embodiments, the 5'-to-5' addition of a 7-methylguanosine to a 5' first ribonucleoside and the addition of a 5' first ribonucleoside containing a 2'-methylated ribose or a 5' second ribonucleoside containing a 2'-methylated ribose occur simultaneously (i.e., CLEANCAP®). In some embodiments, the cap structure is preformed (i.e., as cap-1, cap-2, or cap-0, with or without the addition of a 7-methyl group on the 5' guanosine / 7-methylguanosine) and added to the recombinant RNA molecule (i.e., by ligation). In some embodiments, the preformed cap structure is added with a 5'-AG-3' initiation sequence, as described in the CLEANCAP® AG product insert (Trilink catalog number N-7113), which is incorporated by reference.

[0040] A 7-methylguanosine linked 5'-to-5' to the first ribonucleoside at the 5' position without further methylation is known as Cap-0, and is expressed as 5'(m7Gp)(ppN)[pN]. N where "N" denotes the first (5') nucleobase of the mRNA, "pN" denotes an additional nucleotide in the RNA, and "[pN] N " in " [...] N The addition of " indicates the repeating polymeric structure of RNA, thereby collectively indicating each successive adjacent nucleotide in the RNA.

[0041] An additional oxygen-linked methylation by 2'-O-methyltransferase to the 2' carbon of the ribose of the mRNA nucleoside immediately adjacent to the 7-methylguanosine (i.e., the 5' first ribonucleoside) is 5'(m7Gp)(ppm2N)[pN] Nwhere the addition of "m2" indicates oxygen-linked methylation of the 2' carbon of the ribose of the nucleoside adjacent (via a triphosphate linkage) to the 7-methylguanosine. Further, additional methylation of the 5' second ribonucleoside (i.e., the next (3') nucleoside immediately adjacent to the 5' first nucleoside methylated in cap-1) results in a cap-2 structure represented as 5'(m7Gp)(ppm2N)(m2pN)[pN]n, where the addition of the latter "m2" indicates methylation of the nucleotide immediately adjacent to the nucleotide methylated in cap-1. This cap-2 methylation is also to the 2' carbon of the ribose of the immediately adjacent nucleotide (i.e., 2'-O-Me). In some embodiments, the 5' cap is cap-0, cap-1, or cap-2. In some embodiments, the 5' cap is cap-0. In some embodiments, the 5' cap is Cap-1. In some embodiments, the 5' cap is Cap-2.

[0042] In some embodiments, the 5' first ribonucleoside or the 5' second ribonucleoside is exogenously added to the mRNA. In some embodiments, the 5' first ribonucleoside or the 5' second ribonucleoside is endogenous to the mRNA (i.e., if the native sequence is 5'-UUAAT-3', the addition of m7Gp, if a triphosphate bridge is present, results in 5'-m7Gp(ppUUAAT-3'; if the native sequence is the same, a cap-1 structure results in 5-'(m7Gp)(ppm2U)UAAT-3', and a cap-2 structure results in 5-'(m7Gp)(ppm2U)(m2U)AAT-3').

[0043] Kits that provide all the materials for 5' capping, whether cap-1 or cap-2, and supplemental kits that add cap-1 and cap-2 volumes to the cap-0 kit may be used. The 5' capping method may be performed according to the manufacturer's instructions.

[0044] In some embodiments, the mRNA comprises a 3' poly(adenosine monophosphate) (poly(A)) tail. In some embodiments, the 3' poly(A) tail is 3' from the 3' UTR. In some embodiments, the 3' poly(A) tail is at the 3' end of the mRNA.

[0045] The mRNA disclosed herein may be modified. As used herein, the term "modified mRNA" or "RNA modification" may refer to chemical modifications, including backbone modifications, as well as sugar or base modifications. In this context, modified RNA molecules as defined herein may contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications. A backbone modification relevant to the present invention is a modification in which the phosphate of the backbone of a nucleotide contained in an RNA molecule as defined herein is chemically modified. A sugar modification relevant to the present invention is a chemical modification of the sugar of a nucleotide of an RNA molecule as defined herein. Furthermore, a base modification relevant to the present invention is a chemical modification of the base moiety of a nucleotide of an RNA molecule. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs applicable to transcription and / or translation.

[0046] In one aspect, modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can be modified at the sugar moiety. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CHCHO)CHCHOR; "locked" nucleic acids (LNAs) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, e.g., by a methylene bridge; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be attached to the sugar via a linker comprising one or more of the atoms C, N, and O. The sugar group can also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified RNA molecules can include nucleotides that contain, for example, arabinose as the sugar.

[0047] In another embodiment, the phosphate backbone can be further modified in modified nucleosides and nucleotides, which can be incorporated into modified RNA molecules as described herein. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can include complete replacement of the unmodified phosphate moiety with the modified phosphate described herein. Non-limiting examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In phosphorodithioates, both non-linked oxygens are replaced with sulfur. The phosphate linker can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), or carbon (bridged methylene phosphonates).

[0048] The modified nucleosides and nucleotides that can be used in the present invention can further be modified in the nucleobase portion.Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove surface.In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halo group.

[0049] Modified mRNAs may contain one or more modified nucleosides and nucleotides. Nucleosides and nucleotides, and the preparation of modified nucleotides and nucleosides, are well known in the art; see U.S. Patent Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642. Many modified nucleosides and nucleotides are commercially available.

[0050] Modified nucleobases that may be incorporated into modified nucleosides and nucleotides and present in mRNA molecules include pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6-glycinylcarbamoyl adenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyl adenosine, 1,2'-O-diaminobenz ... Methyladenosine, 1-methyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), 2-methyladenosine, 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine Adenosine;N6,N6-Dimethyladenosine;N6-Acetyladenosine;N6-Hydroxynorvalylcarbamoyladenosine;N6-Methyl-N6-threonylcarbamoyladenosine;2-Methyladenosine;2-Methylthio-N6-isopentenyladenosine;7-Deazaadenosine;N1-Methyladenosine;N6,N6(Dimethyl)adenine;N6-cis-Hydroxy-isopentenyladenosine;α-Thioadenosine;2-(Amino)adenine;2-(Aminopropyl)adenine;2-(Methylthio)N6(isopentenyl) Adenine; 2-(Alkyl)adenine; 2-(Aminoalkyl)adenine; 2-(Aminopropyl)adenine; 2-(Halo)adenine; 2-(Halo)adenine; 2-(Propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2'-Deoxy-2'-α-aminoadenosine TP; 2'-Deoxy-2'-α-azidoadenosine TP; 6-(Alkyl)adenine; 6-(Methyl)adenine; 6-(Alkyl)adenine; 6-(Methyl)adenine; 7-(Deaza)adenine; 8-(Alkenyl)adenine;8-(Alkynyl)adenine;8-(Amino)adenine;8-(Thioalkyl)adenine;8-(Alkenyl)adenine;8-(Alkyl)adenine;8-(Alkynyl)adenine;8-(Amino)adenine;8-(Halo)adenine;8-(Hydroxyl)adenine;8-(Thioalkyl)adenine;8-(Thioyl)adenine;8-Azido-adenosine;Azaadenine;Deazaadenine;N6-(Methyl)adenine;N6-(Isopentyl)adenine;7-Deaza-8-aza-adenosine;7-Methyladenine;1-Deazaadenosine TP;2'-Fu Fluoro-N6-Bz-deoxyadenosine TP;2'-OMe-2-amino-ATP;2'-O-methyl-N6-Bz-deoxyadenosine TP;2'-α-ethynyl adenosine TP;2-aminoadenine;2-aminoadenosine TP;2-amino-ATP;2'-α-trifluoromethyladenosine TP;2-azidoadenosine TP;2'-β-ethynyl adenosine TP;2-bromoadenosine TP;2'-β-trifluoromethyladenosine TP;2-chloroadenosine TP;2'-deoxy-2',2'-difluoroadenosine TP;2'-deoxy 2'-Deoxy-2'-a-mercaptoadenosine TP;2'-Deoxy-2'-a-thiomethoxyadenosine TP;2'-Deoxy-2'-b-aminoadenosine TP;2'-Deoxy-2'-b-azidoadenosine TP;2'-Deoxy-2'-b-bromoadenosine TP;2'-Deoxy-2'-b-chloroadenosine TP;2'-Deoxy-2'-b-fluoroadenosine TP;2'-Deoxy-2'-b-iodoadenosine TP;2'-Deoxy-2'-b-mercaptoadenosine TP;2'-Deoxy-2'-b-thiomethoxyadenosine TP;2- Fluoroadenosine TP; 2-iodoadenosine TP; 2-mercaptoadenosine TP; 2-methoxyadenine; 2-methylthioadenine; 2-trifluoromethyladenosine TP; 3-deaza-3-bromoadenosine TP; 3-deaza-3-chloroadenosine TP; 3-deaza-3-fluoroadenosine TP; 3-deaza-3-iodoadenosine TP; 3-deazaadenosine TP; 4'-azidoadenosine TP; 4'-carbocyclic adenosine TP; 4'-ethynyladenosine TP; 5'-homoadenosine TP; 8-azaATP; 8-bromoadenosine TP;8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-Aminopurine; 7-Deaza-2,6-diaminopurine; 7-Deaza-8-aza-2,6-diaminopurine; 7-Deaza-8-aza-2-aminopurine; 2,6-Diaminopurine; 7-Deaza-8-azaadenine, 7-Deaza-2-aminopurine; 2-Thiocytidine; 3-Methylcytidine; 5-Formylcytidine; 5-Hydroxymethylcytidine; 5-Methylcytidine; N4-Acetylcytidine; 2'-O-Methylcytidine; 2'-O-Methylcytidine; 5,2'-O-Dimethyl Cytidine; 5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-azacytidine; Pseudoisocytidine; Pyrrolocytidine; α-Thiocytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-α-aminocytidine TP; 2'-deoxy-2'-α-azidocytidine TP; 3(de Aza)5(aza)cytosine;3(methyl)cytosine;3-(alkyl)cytosine;3-(deaza)5(aza)cytosine;3-(methyl)cytidine;4,2'-O-dimethylcytidine;5(halo)cytosine;5(methyl)cytosine;5(propynyl)cytosine;5(trifluoromethyl)cytosine;5-(alkyl)cytosine;5-(alkynyl)cytosine;5-(halo)cytosine;5-(propynyl)cytosine;5-(trifluoromethyl)cytosine;5-bromocytidine;5-iodocytidine;5-propynylcytosine;6-(azo)cytosine;6-a Zecytidine; Azacytosine; Deazacytosine; N4(acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methylcytidine; 2-methoxy-cytidine; 2-thio-5-methylcytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine;Pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'-fluoro-N4-Bz-cytidine TP; 2'-fluoro-N4-acetyl-cytidine TP; 2'-O-methyl-N4-acetyl-cytidine TP; 2'-O-methyl-N4-Bz-cytidine TP; 2'-a-ethynylcytidine TP; 2'-a-trifluoromethylcytidine TP; 2'-b-ethynylcytidine TP; 2'-b-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP;2'-Deoxy-2'-α-mercaptocytidine TP;2'-Deoxy-2'-α-thiomethoxycytidine TP;2'-Deoxy-2'-β-aminocytidine TP;2'-Deoxy-2'-β-azidocytidine TP;2'-Deoxy-2'-β-bromocytidine TP;2'-Deoxy-2'-β-chlorocytidine TP;2'-Deoxy-2'-β-fluorocytidine TP;2'-Deoxy-2'-β-iodocytidine TP;2'-Deoxy-2'-β-mercaptocytidine TP;2'-Deoxy-2'-β-thiomethoxycytidine TP;2'-O-Me 5-(1-propynyl)cytidine TP; 3'-ethynylcytidine TP; 4'-azidocytidine TP; 4'-carbocyclic cytidine TP; 4'-ethynylcytidine TP; 5-(1-propynyl)aracytidine TP; 5-(2-chlorophenyl)-2-thiocytidine TP; 5-(4-aminophenyl)-2-thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylaracytidine TP; 5-ethynylcytidine TP; 5'-homocytidine TP; 5-methoxycytidine TP; 5-trifluoromethylcytidine TP; N4-aminocytidine N4-benzoylcytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archeosine; Methylwyosine (wyosine); N2,7-dimethylguanosine;N2,N2,2'-O-trimethylguanosine;N2,N2,7-trimethylguanosine;N2,N2-dimethylguanosine;N2,7,2'-O-trimethylguanosine;6-thioguanosine;7-deazaguanosine;8-oxoguanosine;N1-methylguanosine;α-thioguanosine;2-(propyl)guanine;2-(alkyl)guanine;2'-amino-2'-deoxy-GTP;2'-azido-2'-deoxy-GTP;2'-deoxy-2'-α-aminoguanosine TP;2'-deoxy-2'-α-azidoguanosine TP;6- (Methyl)guanine;6-(Alkyl)guanine;6-(Methyl)guanine;6-Methylguanosine;7(Alkyl)guanine;7(Deaza)guanine;7(Methyl)guanine;7-(Alkyl)guanine;7-(Deaza)guanine;7-(Methyl)guanine;8(Alkyl)guanine;8(Alkynyl)guanine;8(Halo)guanine;8(Thioalkyl)guanine;8-(Alkenyl)guanine;8-(Alkyl)guanine;8-(Alkynyl)guanine;8-(Amino)guanine;8-(Halo)guanine;8-(Hydroxyl)guanine;8 -(Thioalkyl)guanine; 8-(thiol)guanine; Azaguanine; Deazaguanine; N(methyl)guanine; N-(methyl)guanine, 1-methyl-6-thio-guanosine, 6-methoxy-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7-methyl-8-oxo-guanosine, N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'-fluoro-N2- Isobutyl-guanosine TP; 2'-O-methyl-N2-isobutyl-guanosine TP; 2'-a-ethynylguanosine TP; 2'-a-trifluoromethylguanosine TP; 2'-b-ethynylguanosine TP; 2'-b-trifluoromethylguanosine TP; 2'-deoxy-2',2'-difluoroguanosine TP; 2'-deoxy-2'-a-mercaptoguanosine TP; 2'-deoxy-2'-a-thiomethoxyguanosine TP; 2'-deoxy-2'-b-aminoguanosine TP; 2'-deoxy-2'-b-azidoguanosine TP;2'-Deoxy-2'-b-bromoguanosine TP; 2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5'-Homoguanosine TP; 8-Bromoguanosine TP; 9-Deazaguanosine TP; N2-Isobutylguanosine TP; 1-Methylinosine; Inosine; 1,2'-O-Dimethylinosine; 2'-O-Methylinosine; 7-Methylinosine; 2'-O-Methylinosine; Epoxyqueuosine; Galactosylqueuosine; Mannosylqueuosine; Queuosine; Allylaminothymidine; Azathymidine; Deazathymidine; Deoxythymidine; 2'-O-Methoxy 2-Thiouridine;3-Methyluridine;5-Carboxymethyluridine;5-Hydroxyuridine;5-Methyluridine;5-Taurinomethyl-2-thiouridine;5-Taurinomethyluridine;Dihydrouridine;(3-(3-amino-3-carboxypropyl)uridine;1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-Methylpseudouridine;1-Methylpseudouridine;2'-O-Methyluridine;2'-O-Methylpseudouridine;2'-O-Methyluridine;2-Thio-2'-O-methyl Thiuridine;3-(3-amino-3-carboxypropyl)uridine;3,2'-O-dimethyluridine;3-methylpseudo-uridine TP;4-thiouridine;5-(carboxyhydroxymethyl)uridine;5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine;5,6-dihydrouridine;5-aminomethyl-2-thiouridine;5-carbamoylmethyl-2'-O-methyluridine;5-carbamoylmethyluridine;5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyl Uridine methyl ester;5-carboxymethylaminomethyl-2'-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine;5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP;5-Methoxycarbonylmethyl-2'-O-methyluridine;5-Methoxycarbonylmethyl-2-thiouridine;5-Methoxycarbonylmethyluridine;5-Methoxyuridine;5-Methyl-2-thiouridine;5-Methylaminomethyl-2-selenouridine;5-Methylaminomethyl-2-thiouridine;5-Methylaminomethyluridine;5-Methyldihydrouridine;5-Hydroxyacetic acid-uridine TP;5-Hydroxyacetic acid-methyl ester-uridine TP;N1-Methyl-pseudouridine;N1-Ethyl-pseudouridine;Uridine 5-hydroxyacetic acid;Uridine 5-hydroxyacetic acid methyl ester;3-(3-amino-3-carboxypropyl)-uridine TP;5-(isopentenylaminomethyl)-2-thiouridine TP;5-(isopentenylaminomethyl) 1-(aminoalkylaminocarbonylethylenyl)-2'-O-methyluridine TP;5-(isopentenylaminomethyl)uridine TP;5-propynyluracil;α-thiouridine;1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouridine;1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouridine;1-(aminoalkylaminocarbonylethylenyl)-4(thio)pseudouridine;1-(aminoalkylaminocarbonylethylenyl)-pseudouridine;1-(aminocarbonylethylenyl)-2(thio)-pseudouridine;1-(aminoalkylaminocarbonylethylenyl)- 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouridine;1-(aminocarbonylethylenyl)-4(thio)pseudouridine;1-(aminocarbonylethylene)-pseudouridine;1-substituted 2(thio)-pseudouridine;1-substituted 2,4-(dithio)pseudouridine;1-substituted 4(thio)-pseudouridine;1-substituted pseudouridine;1-(aminoalkylaminocarbonylethylenyl)-2-(thio)-pseudouridine;1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP;1-methyl-3-(3-a (amino-3-carboxypropyl)pseudo-UTP; 1-methyl-pseudo-UTP; 2(thio)-pseudouridine; 2'-deoxyuridine; 2'-fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoroguanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azidodeoxyuridine TP; 2'-O-methylpseudouridine; 2'-deoxyuridine; 2'-fluorouridine; 2'-deoxy-2'-α-aminouridine TP;2'-Deoxy-2'-α-azidouridine TP;2-Methylpseudouridine;3(3-amino-3-carboxypropyl)uracil;4(Thio)pseudouridine;4-(Thio)pseudouridine;4-(Thio)uracil;4-Thiouracil;5(1,3-Diazol-1-alkyl)uracil;5(2-Aminopropyl)uracil;5(Aminoalkyl)uracil;5(Dimethylaminoalkyl)uracil;5(Guanidiniumalkyl)uracil;5(Methoxycarbonylmethyl)-2-(thio)uracil;5(Methoxycarbonylmethyl)uracil 5-(methyl)-2-(thio)uracil;5-(methyl)-2,4-(dithio)uracil;5-(methyl)-4-thiouracil;5-(methylaminomethyl)-2-(thio)uracil;5-(methylaminomethyl)-2,4-(dithio)uracil;5-(methylaminomethyl)-4-thiouracil;5-(propynyl)uracil;5-(trifluoromethyl)uracil;5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouridine;5-(alkyl)-2,4-(dithio)pseudouridine;5-(alkyl)-4-thio)pseudouridine;5- (Alkyl)pseudouridine;5-(Alkyl)uracil;5-(Alkynyl)uracil;5-(Allylamino)uracil;5-(Cyanoalkyl)uracil;5-(Dialkylaminoalkyl)uracil;5-(Dimethylaminoalkyl)uracil;5-(Guanidiniumalkyl)uracil;5-(Halo)uracil;5-(1,3-Diazol-1-alkyl)uracil;5-(Methoxy)uracil;5-(Methoxycarbonylmethyl)-2-(thio)uracil;5-(Methoxycarbonylmethyl)uracil;5-(Methyl)2(thio)uracil ;5-(Methyl)2,4(dithio)uracil;5-(Methyl)-4-(thio)uracil;5-(Methyl)-2-(thio)pseudouridine;5-(Methyl)-2,4-dithiopseudouridine;5-(Methyl)-4-(thio)pseudouridine;5-(Methyl)pseudouridine;5-(Methylaminomethyl)-2-(thio)uracil;5-(Methylaminomethyl)-2,4-(dithio)uracil;5-(Methylaminomethyl)-4-(thio)uracil;5-(Propynyl)uracil;5-(Trifluoromethyl)uracil;5-Aminoallyluridine;5-Bromouridine;5-Iodouridine;5-Uracil;6-(Azo)uracil;6-(Azo)uracil;6-Azauridine;Allylaminouracil;Azauracil;Deazauracil;N3(Methyl)uracil;Pseudo-UTP-1-2-ethanoic acid;Pseudouridine;4-Thiopseudo-UTP;1-Carboxymethylpseudouridine;1-Methyl-1-deazapseudouridine;1-Propynyluridine;1-Taurinomethyl-1-methyluridine;1-Taurinomethyl-4-thiouridine;1-Taurinomethylpseudouridine;2-Methoxy 4-Thiopseudouridine;2-Thio-1-methyl-1-deazapseudouridine;2-Thio-1-methylpseudouridine;2-Thio-5-azauridine;2-Thio-dihydropseudouridine;2-Thio-dihydrouridine;2-Thiopseudouridine;4-Methoxy-2-thiopseudouridine;4-Methoxy-pseudouridine;4-Thio-1-methyl-pseudouridine;4-Thio-pseudouridine;5-Aza-uridine;Dihydropseudouridine;(.±.)1-(2-hydroxypropyl)pseudouridine TP;(2R) -1-(2-hydroxypropyl)pseudouridine TP;(2S)-1-(2-hydroxypropyl)pseudouridine TP;(E)-5-(2-bromo-vinyl)ara-uridine TP;(E)-5-(2-bromo-vinyl)uridine TP;(Z)-5-(2-bromo-vinyl)ara-uridine TP;(Z)-5-(2-bromo-vinyl)uridine TP;1-(2,2,2-trifluoroethyl)-pseudouridine TP;1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP;1-(2,2-diethoxyethyl)pseudouridine Pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudo-UTP; 1-(2,4,6-trimethylphenyl)pseudo-UTP; 1-(2-amino-2-carboxyethyl)pseudo-UTP; 1-(2-aminoethyl)pseudo-UTP; 1-(2-hydroxyethyl)pseudouridine TP; 1-(2-methoxyethyl)pseudouridine TP; 1-(3,4-bistrifluoromethoxybenzyl)pseudouridine TP; 1-(3,4-dimethoxybenzyl)pseudouridine TP;1-(3-amino-3-carboxypropyl)pseudo-UTP;1-(3-aminopropyl)pseudo-UTP;1-(3-cyclopropyl-2-ynyl)pseudouridine TP;1-(4-amino-4-carboxybutyl)pseudo-UTP;1-(4-aminobenzyl)pseudo-UTP;1-(4-aminobutyl)pseudo-UTP;1-(4-aminophenyl)pseudo-UTP;1-(4-azidobenzyl)pseudouridine TP;1-(4-bromobenzyl)pseudouridine TP;1-(4-chlorobenzyl)pseudo Uridine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudo-UTP; 1-(4-Methoxyphenyl)pseudo-UTP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methylbenzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitrobenzyl)pseudo-UTP ;1-(4-Nitrophenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudo-uridine TP;1-(4-Trifluoromethoxybenzyl)pseudo-uridine TP;1-(4-Trifluoromethylbenzyl)pseudo-uridine TP;1-(5-Aminopentyl)pseudo-UTP;1-(6-Aminohexyl)pseudo-UTP;1,6-Dimethylpseudo-UTP;1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudo-uridine TP;1-{3-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudo-uridine TP (ethoxy)-ethoxy]-propionyl}pseudouridine TP; 1-acetylpseudouridine TP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-UTP; 1-alkyl-6-allyl-pseudo-UTP; 1-alkyl-6-ethynyl-pseudo-UTP; 1-alkyl-6-homoallyl-pseudo-UTP; 1-alkyl-6-vinyl-pseudo-UTP; 1-allylpseudouridine TP; 1-aminomethyl-pseudo-UTP; 1-benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinyl-pseudouridine TP; 1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP;1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP;1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP;1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP;1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP;1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP;1-Hexyl-pseudo-UTP;1-Homoallyl-pseudo-UTP Pseudouridine TP;1-Hydroxymethylpseudouridine TP;1-Isopropyl-pseudouridine TP;1-Me-2-thio-pseudo-UTP;1-Me-4-thio-pseudo-UTP;1-Me-α-thio-pseudo-UTP;1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-trifluoroethyl)-pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(substituted Phenyl)-pseudo-UTP;1-methyl-6-amino-pseudo-UTP;1-methyl-6-azido-pseudo-UTP;1-methyl-6-bromo-pseudo-UTP;1-methyl-6-butyl-pseudo-UTP;1-methyl-6-chloro-pseudo-UTP;1-methyl-6-cyano-pseudo-UTP;1-methyl-6-dimethylamino-pseudo-UTP;1-methyl-6-ethoxy-pseudo-UTP;1-methyl-6-ethylcarboxylate-pseudo-UTP;1-methyl-6-ethyl-pseudo-UTP;1-methyl-6- Fluoro-pseudo-UTP; 1-methyl-6-formyl-pseudo-UTP; 1-methyl-6-hydroxyamino-pseudo-UTP; 1-methyl-6-hydroxy-pseudo-UTP; 1-methyl-6-iodo-pseudo-UTP; 1-methyl-6-isopropyl-pseudo-UTP; 1-methyl-6-methoxy-pseudo-UTP; 1-methyl-6-methylamino-pseudo-UTP; 1-methyl-6-phenyl-pseudo-UTP; 1-methyl-6-propyl-pseudo-UTP; 1-methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP;1-Morpholinomethylpseudouridine TP;1-Pentyl-pseudo-UTP;1-Phenyl-pseudo-UTP;1-Pivaloylpseudouridine TP;1-Propyl-pseudo-UTP;1-Propynyl-pseudouridine;1-p-Tolyl-pseudo-UTP;1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP;1-Thiomorpho 2'-OMe-5-Me-UTP;2'-OMe-pseudo-UTP;2'-OMe-5-Me-UTP;2'-OMe-pseudo-UTP;2'-α-ethynyluridine TP;2'-α-trifluoromethyluridine TP;2'-β-ethynyluridine TP;2'-β-trifluoromethyluridine TP;2'-deoxyuridine TP;2'-deoxy-α-ethynyluridine TP;2'-β-trifluoromethyluridine TP;2'-deoxy-α-ethynyluridine TP;2'-β-trifluoromethyluridine TP;2'-deoxy-α-ethynyluridine TP 2'-Deoxy-2'-a-mercaptouridine TP;2'-Deoxy-2'-a-thiomethoxyuridine TP;2'-Deoxy-2'-b-aminouridine TP;2'-Deoxy-2'-b-azidouridine TP;2'-Deoxy-2'-b-bromouridine TP;2'-Deoxy-2'-b-chlorouridine TP;2'-Deoxy-2'-b-fluorouridine TP;2'-Deoxy-2'-b-iodouridine TP;2'-Deoxy-2'-b-mercaptouridine TP;2'-Deoxy-2'-b-aminouridine TP '-b-Thiomethoxyuridine TP; 2-Methoxy-4-thiouridine; 2-Methoxyuridine; 2'-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(1-Propynyl)aruridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homouridine TP; 5-Iodo-2'-fluorodeoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyluridine TP; 5-Vinylaruridine TP; 6-(2,2,2-Trifluoroethyl)-pseudoUTP; 6-(4-Morpholino)-pseudoUTP; 6-(4-Thiomorpholino)-pseudoUTP; 6-(Substituted Phenyl)-pseudoUTP; 6-Amino-pseudoUTP; 6-Azido-pseudoUTP; 6-Bromo-pseudoUTP; 6-Butyl-pseudoUTP; 6-Chloro-pseudoUTP; 6-Cyano-pseudoUTP; 6-Dimethylamino-pseudoUTP UTP;6-ethoxy-pseudo-UTP;6-ethylcarboxylate-pseudo-UTP;6-ethyl-pseudo-UTP;6-fluoro-pseudo-UTP;6-formyl-pseudo-UTP;6-hydroxyamino-pseudo-UTP;6-hydroxy-pseudo-UTP;6-iodo-pseudo-UTP;6-isopropyl-pseudo-UTP;6-methoxy-pseudo-UTP;6-methylamino-pseudo-UTP;6-methyl-pseudo-UTP;6-phenyl-pseudo-UTP;6-phenyl-pseudo-UTP;6-propyl-pseudo- UTP;6-tert-butyl-pseudo-UTP;6-trifluoromethoxy-pseudo-UTP;6-trifluoromethyl-pseudo-UTP;alpha-thio-pseudo-UTP;Pseudouridine 1-(4-methylbenzenesulfonic acid) TP;Pseudouridine 1-(4-methylbenzoic acid) TP;Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid;Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP 1-[3-{2-(2 -[2-{2-(2-ethoxy)-ethoxy)-ethoxy}-ethoxy]-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-methylphosphonic acid;Pseudouridine TP1-methylphosphonic acid diethyl ester;Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid;Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; wybutosin; hydroxywybutosin; isowyosin; peroxywybutosin; undermodified (modified) hydroxywybutosin; 4-demethylwyosin; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa-naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2'methyl, 2'amino, 2 'Azido, 2'fluoro-cytidine; 2'methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluorouridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloropurine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'-fluoro-2'-deoxyribose; 2'-fluoro-modified bases; 2'-O-methylribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo -Pyridopyrimidin-3-yl;2-Pyridinone;3-Nitropyrrole;3-(Methyl)-7-(propynyl)isocarbostyrilyl;3-(Methyl)isocarbostyrilyl;4-(Fluoro)-6-(methyl)benzimidazole;4-(Methyl)benzimidazole 4-(Methyl)indolyl;4,6-(Dimethyl)indolyl;5-Nitroindole;5-Substituted pyrimidines;5-(Methyl)isocarbostyrilyl;5-Nitroindole;6-(Aza)pyrimidine;6- (Azo)thymine;6-(methyl)-7-(aza)indolyl;6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aza)indolyl;7-(Guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(Guanidiniumalkylhydroxy)-1-(aza)-2 -(Thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Propynyl)isocarbostyrilyl;7-(Propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl;7-Deazyno Cinyl;7-Substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-Substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;9-(Methyl)-imidizopyridinyl;Aminoindolyl;Anthracenyl;Bis-ortho-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl;Bis-ortho-substituted-6-phenylpyrrolopyrimidin-2-one-3-yl;Difluorotolyl;Hypoxanthine;Imidizopyridinyl;Inosinyl;Isocarbostyrilyl;Isoguanisine;N2 -Substituted Purines;N6-Methyl-2-aminopurine;N6-Substituted Purines;N-Alkylated Derivatives;Naphthalenyl;Nitrobenzimidazolyl;Nitroimidazolyl;Nitroindazolyl;Nitropyrazolyl;Nubularine;O6-Substituted Purines;O-Alkylated Derivatives;ortho-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl;ortho-substituted-6-phenylpyrrolopyrimidin-2-one-3-yl;Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl;Para-substituted-6-phenylpyrrolopyrimidin-2-one-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; Propynyl-7-(aza)indolyl; Pyrenyl; Pyridopyrimidin-3-yl; Pyridopyrimidin-3-yl, 2-oxo-7-aminopyridopyrimidin-3-yl; Pyrrolopyrimidin-2-one-3-yl; Pyrrolopyrimidinyl; Pyrrolopyridinyl; Stilbenzyl; Substituted 1,2,4-triazoles; Tetracenyl; Tubercidin; Ki; Sanchin; Includes xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-amino-riboside-TP; formycin ATP; formycin BTP; pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; or N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0051] In some embodiments, the modified nucleotide capable of substituting adenosine is 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine(phosphate); 2-methyl Chiladenosine;2-Methylthio-N6-isopentenyladenosine;2-Methylthio-N6-hydroxynorvalylcarbamoyladenosine;2'-O-Methyladenosine;2'-O-Ribosyladenosine (phosphate);Isopentenyladenosine;N6-(cis-Hydroxyisopentenyl)adenosine;N6,2'-O-Dimethyladenosine;N6,2'-O-Dimethyladenosine;N6,N6,2'-O-Trimethyladenosine;N6,N6-Dimethyladenosine;N6-Acetyladenosine;N6-Hydroxynorvalylcarbamoyladenosine N6-Methyl-N6-threonylcarbamoyladenosine;2-Methyladenosine;2-Methylthio-N6-isopentenyladenosine;7-Deaza-adenosine;N1-Methyladenosine;N6,N6(dimethyl)adenine;N6-cis-Hydroxy-isopentenyl-adenosine;α-Thio-adenosine;2-(Amino)adenine;2(Aminopropyl)adenine;2(Methylthio)N6(isopentenyl)adenine;2-(Alkyl)adenines;2-(Aminoalkyl)adenines;2-(Aminopropyl)adenine;2-(Halo)adenine ;2-(Halo)adenine;2-(Propyl)adenine;2'-Amino-2'-deoxy-ATP;2'-Azido-2'-deoxy-ATP;2'-Deoxy-2'-α-aminoadenosine TP;2'-Deoxy-2'-α-azidoadenosine TP;6(Alkyl)adenine;6(Methyl)adenine;6-(Alkyl)adenine;6-(Methyl)adenine;7(Deaza)adenine;8(Alkenyl)adenine;8(Alkynyl)adenine;8(Amino)adenine;8(Thioalkyl)adenine;8-(Alkenyl)adenine;8-(Alkyl)adenine;8-(Alkynyl)adenine;8-(Amino)adenine;8-(Halo)adenine;8-(Hydroxyl)adenine;8-(Thioalkyl)adenine;8-(Thiolate)adenine;8-Azido-adenosine;Azaadenine;Deazaadenine;N6(Methyl)adenine;N6-(Isopentyl)adenine;7-Deaza-8-aza-adenosine;7-Methyladenine;1-Deazaadenosine TP;2'-Fluoro-N6-Bz-deoxyadenosine TP;2'-OMe-2-amino-ATP;2'-O-Methyl-N6-Bz-deoxyadenosine TP;2'-a -Ethynyl adenosine TP; 2-aminoadenine; 2-aminoadenosine TP; 2-amino-ATP; 2'-a-trifluoromethyl adenosine TP; 2-azidoadenosine TP; 2'-b-ethynyl adenosine TP; 2-bromoadenosine TP; 2'-b-trifluoromethyl adenosine TP; 2-chloroadenosine TP; 2'-deoxy-2',2'-difluoroadenosine TP; 2'-deoxy-2'-a-mercaptoadenosine TP; 2'-deoxy-2'-a-thiomethoxyadenosine TP; 2'-deoxy-2'-b-aminoadenosine TP 2'-Deoxy-2'-b-azidoadenosine TP;2'-Deoxy-2'-b-bromoadenosine TP;2'-Deoxy-2'-b-chloroadenosine TP;2'-Deoxy-2'-b-fluoroadenosine TP;2'-Deoxy-2'-b-iodoadenosine TP;2'-Deoxy-2'-b-mercaptoadenosine TP;2'-Deoxy-2'-b-thiomethoxyadenosine TP;2-Fluoroadenosine TP;2-Iodoadenosine TP;2-Mercaptaoadenosine TP;2-Methoxyadenine;2-Methylthioadenine;2-Trifluoromethan Chiladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'-Homoadenosine TP; 8-Aza-ATP; 8-Bromoadenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; Wybutosine; Hydroxywybutosine; Isowybutosine; Peroxywybutosine;Undermodified hydroxywybutosin; 4-demethylwybutosin; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2-(amino)purine; 2,4,5-(trimethyl)phenyl; 2'methyl, 2'amino, 2'azido, 2'fluorocytidine; 2'methyl, 2'amino, 2'-Azido, 2'-Fluoroadenine; 2'-Methyl, 2'-Amino, 2'-Azido, 2'-Fluorouridine; 2'-Amino-2'-deoxyribose; 2-Amino-6-chloropurine; 2-Aza-inosinyl; 2'-Azido-2'-deoxyribose; 2'-Fluoro-2'-deoxyribose; 2'-Fluoro-modified bases; 2'-O-Methyl-ribose; 2-Oxo-7-aminopyridopyrimidin-3-yl; 2-Oxo-pyridopyrimidin-3-yl; 2-Pyridinone; 3-Nitropyrrole; 3-(Methyl)-7-(propynyl)isocarbo Styrilyl;3-(Methyl)isocarbostyrilyl;4-(Fluoro)-6-(methyl)benzimidazole;4-(Methyl)benzimidazole;4-(Methyl)indolyl;4,6-(Dimethyl)indolyl;5-Nitroindole;5-Substituted pyrimidines;5-(Methyl)isocarbostyrilyl;5-Nitroindole;6-(Aza)pyrimidine;6-(Azo)thymine;6-(Methyl)-7-(aza)indolyl;6-Chloropurine;6-Phenyl-pyrrolo-pyrimidin-2-one-3-yl;7-(Aminoalkylhydroxy) -1-(Aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(Aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aza)indolyl;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl 7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Propynyl)isocarbostyril;7-(Propynyl)isocarbostyril, propynyl-7-(aza)indolyl;7-Deaza-inosinyl;7-Substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-Substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;9-(Methyl-imidizopyridinyl);Aminoindolyl ;Anthracenyl;Bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Difluorotolyl;Hypoxanthine;Imidizopyridinyl;Inosinyl;Isocarbostyrilyl;Isoguanisine;N2-Substituted purines;N6-Methyl-2-aminopurine;N6-Substituted purines;N-Alkylated derivatives;Naphthalenyl;Nitrobenzimidazolyl;Nitroimidazolyl;Nitroindazolyl;Nitropyrazolyl;Nublaryl O6-Substituted purines;O-Alkylated derivatives;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl;ortho-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl;Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl;para-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl;Pentacenyl;Phenanthracenyl;Phenyl;Propynyl-7-(aza)indolyl;Pyrenyl;Pyridopyrimidin-3-yl;Pyridopyrimidin-3-yl, 2-oxo-7-aminopyridopyrimidin-3-yl; Pyrrolopyrimidin-2-one-3-yl; Pyrrolopyrimidinyl; Pyrrolopyridinyl; Stilbenzyl; Substituted 1,2,4-triazoles; Tetracenyl; Tubercidin; Xanthine; Xanthosine-5'-TP; 2-Thio-Zebularine; 5-Aza-2-Thio-Zebularine; 7-Deaza-2-amino-Purine; Pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; or N6-(19-amino-pentaoxanonadecyl)adenosine TP;

[0052] In some embodiments, the modified nucleotide that can substitute for uridine or thymidine is pseudouridine; N1-methylpseudouridine; N1-ethylpseudouridine; inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; epoxyqueuosine; galactosylqueuosine; mannosylqueuosine; queuosine; allylaminothymidine; azathymidine; deazathymidine; deoxythymidine; 2'-O-methyluridine; 2-thiouridine ;3-Methyluridine;5-Carboxymethyluridine;5-Hydroxyuridine;5-Methyluridine;5-Taurinomethyl-2-thiouridine;5-Taurinomethyluridine;Dihydrouridine;(3-(3-amino-3-carboxypropyl)uridine;1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-Methylpseudouridine;1-Methylpseudouridine;2'-O-Methyluridine;2'-O-Methylpseudouridine;2'-O-Methyluridine;2-Thio-2'-O-methyluridine;3-( 3-Amino-3-carboxypropyl)uridine;3,2'-O-dimethyluridine;3-Methylpseudo-uridine TP;4-Thiouridine;5-(Carboxyhydroxymethyl)uridine;5-(Carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine;5,6-Dihydro-uridine;5-Aminomethyl-2-thiouridine;5-Carbamoylmethyl-2'-O-methyluridine;5-Carbamoylmethyluridine;5-Carboxyhydroxymethyluridine;5-Carboxyhydroxymethyluridine methyl ethyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine;5-carboxymethylaminomethyluridine;5-carbamoylmethyluridine TP;5-methoxycarbonylmethyl-2'-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine;5-methoxycarbonylmethyluridine;5-methoxyuridine;5-methyl-2-thiouridine;5-Methylaminomethyl-2-selenouridine;5-Methylaminomethyl-2-thiouridine;5-Methylaminomethyluridine;5-Methyldihydrouridine;5-Hydroxyacetic acid-uridine TP;5-Hydroxyacetic acid-methyl ester-uridine TP;N1-Methyl-pseudouridine;N1-Ethyl-pseudouridine;Uridine 5-hydroxyacetic acid;Uridine 5-hydroxyacetic acid methyl ester;3-(3-amino-3-carboxypropyl)-uridine TP;5-(isopentenylaminomethyl)-2-thiouridine TP;5-(isopentenylaminomethyl) )-2'-O-Methyluridine TP;5-(Isopentenylaminomethyl)uridine TP;5-Propynyluracil;α-Thiouridine;1(Aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouridine;1(Aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouridine;1(Aminoalkylaminocarbonylethylenyl)-4(thio)pseudouridine;1(Aminoalkylaminocarbonylethylenyl)-pseudouridine;1(Aminocarbonylethylenyl)-2(thio)-pseudouridine;1(Amino 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouridine;1-(aminocarbonylethylenyl)-4(thio)pseudouridine;1-(aminocarbonylethylenyl)-pseudouridine;1-substituted 2(thio)-pseudouridine;1-substituted 2,4-(dithio)-pseudouridine;1-substituted 4(thio)pseudouridine;1-substituted pseudouridine;1-(aminoalkylaminocarbonylethylenyl)-2-(thio)-pseudouridine;1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP;1-methyl-3-(3-amino (3-carboxypropyl)pseudo-UTP; 1-methyl-pseudo-UTP; 2(thio)pseudouridine; 2'deoxyuridine; 2'fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'methyl, 2'amino, 2'azido, 2'fluoroguanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2'deoxyuridine; 2'fluorouridine; 2'-deoxy-2'-α-aminouridine TP;2'-Deoxy-2'-α-azidouridine TP;2-Methylpseudouridine;3(3-amino-3-carboxypropyl)uracil;4(thio)pseudouridine;4-(thio)pseudouridine;4-(thio)uracil;4-Thiouracil;5(1,3-Diazol-1-alkyl)uracil;5(2-aminopropyl)uracil;5(Aminoalkyl)uracil;5(Dimethylaminoalkyl)uracil;5(Guanidiniumalkyl)uracil;5(Methoxycarbonylmethyl)-2-(thio)uracil;5(Methoxycarbonylmethyl)uracil 5-(methyl)-2-(thio)uracil;5-(methyl)-2,4-(dithio)uracil;5-(methyl)-4-thiouracil;5-(methylaminomethyl)-2(thio)uracil;5-(methylaminomethyl)-2,4-(dithio)uracil;5-(methylaminomethyl)-4-thiouracil;5-(propynyl)uracil;5-(trifluoromethyl)uracil;5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouridine;5-(alkyl)-2,4-(dithio)pseudouridine;5-(alkyl)-4-thiopseudouridine;5-(aminopropyl)uracil 5-(Alkyl)pseudouridine;5-(Alkyl)uracil;5-(Alkynyl)uracil;5-(Allylamino)uracil;5-(Cyanoalkyl)uracil;5-(Dialkylaminoalkyl)uracil;5-(Dimethylaminoalkyl)uracil;5-(Guanidiniumalkyl)uracil;5-(Halo)uracil;5-(1,3-Diazol-1-alkyl)uracil;5-(Methoxy)uracil;5-(Methoxycarbonylmethyl)-2-(thio)uracil;5-(Methoxycarbonylmethyl)uracil;5-(Methyl)2(thio)uracil;5-( 5-(Methyl)-2,4(dithio)uracil;5-(Methyl)-4(thio)uracil;5-(Methyl)-2-(thio)pseudouridine;5-(Methyl)-2,4(dithio)pseudouridine;5-(Methyl)-4(thio)pseudouridine;5-(Methyl)pseudouridine;5-(Methylaminomethyl)-2(thio)uracil;5-(Methylaminomethyl)-2,4(dithio)uracil;5-(Methylaminomethyl)-4-(thio)uracil;5-(Propynyl)uracil;5-(Trifluoromethyl)uracil;5-Aminoallyluridine;5-Bromouridine5-Iodouridine;5-Uracil;6-(Azo)uracil;6-(Azo)uracil;6-Azauridine;Allylaminouracil;Azauracil;Deazauracil;N3(Methyl)uracil;Pseudo-UTP-1-2-ethanoic acid;Pseudouridine;4-Thio-pseudo-UTP;1-Carboxymethyl-pseudouridine;1-Methyl-1-deaza-pseudouridine;1-Propynyl-uridine;1-Taurinomethyl-1-methyl-uridine;1-Taurinomethyl-4-thio-uridine;1-Taurinomethyl-pseudouridine;2-Methoxy-4-thio O-pseudouridine;2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine;2-thio-5-aza-uridine;2-thio-dihydropseudouridine;2-thio-dihydrouridine;2-thio-pseudouridine;4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine;4-thio-1-methyl-pseudouridine;4-thio-pseudouridine;5-aza-uridine;Dihydropseudouridine;(.±.)1-(2-hydroxypropyl)pseudouridine TP;(2R)-1 -(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP;(E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP;(Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudouridine TP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP ;1-(2,4,6-trimethylbenzyl)pseudouridine TP;1-(2,4,6-trimethylbenzyl)pseudo-UTP;1-(2,4,6-trimethylphenyl)pseudo-UTP;1-(2-amino-2-carboxyethyl)pseudo-UTP;1-(2-aminoethyl)pseudo-UTP;1-(2-hydroxyethyl)pseudouridine TP;1-(2-methoxyethyl)pseudouridine TP;1-(3,4-bistrifluoromethoxybenzyl)pseudouridine TP;1-(3,4-dimethoxybenzyl)pseudouridine TP;1-(3-amino-3-carboxypropyl)pseudo-UTP;1-(3-aminopropyl)pseudo-UTP;1-(3-cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-amino-4-carboxybutyl)pseudo-UTP;1-(4-aminobenzyl)pseudo-UTP;1-(4-aminobutyl)pseudo-UTP;1-(4-aminophenyl)pseudo-UTP;1-(4-azidobenzyl)pseudouridine TP;1-(4-bromobenzyl)pseudouridine TP;1-(4-chlorobenzyl)pseudouridine Lysine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudo-UTP; 1-(4-Methoxyphenyl)pseudo-UTP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methylbenzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitrobenzyl)pseudo-UTP; 1 -(4-Nitrophenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Aminopentyl)pseudo-UTP;1-(6-Aminohexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP;1-{3-[2-(2-amino (noethoxy)-ethoxy]-propionyl}pseudouridine TP; 1-acetylpseudouridine TP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-UTP; 1-alkyl-6-allyl-pseudo-UTP; 1-alkyl-6-ethynyl-pseudo-UTP; 1-alkyl-6-homoallyl-pseudo-UTP; 1-alkyl-6-vinyl-pseudo-UTP; 1-allylpseudouridine TP; 1-aminomethyl-pseudo-UTP; 1-benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinyl-pseudouridine TP; 1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP; 1-Cycloheptylmethyl-pseudo-UTP;1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP;1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP;1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP;1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP;1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP;1-Hexyl-pseudo-UTP;1-Homoallyl-pseudo-UTP Pseudouridine TP;1-Hydroxymethylpseudouridine TP;1-Isopropyl-pseudouridine UTP;1-Me-2-thio-pseudo-UTP;1-Me-4-thio-pseudo-UTP;1-Me-α-thio-pseudo-UTP;1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-trifluoroethyl)pseudouridine TP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP;1-Methyl-6-(substituted phenyl)pseudo-UTP TP;1-methyl-6-amino-pseudo-UTP;1-methyl-6-azido-pseudo-UTP;1-methyl-6-bromo-pseudo-UTP;1-methyl-6-butyl-pseudo-UTP;1-methyl-6-chloro-pseudo-UTP;1-methyl-6-cyano-pseudo-UTP;1-methyl-6-dimethylamino-pseudo-UTP;1-methyl-6-ethoxy-pseudo-UTP;1-methyl-6-ethylcarboxylate-pseudo-UTP;1-methyl-6-ethyl-pseudo-UTP;1-methyl-6-fluoro-pseudo-UTP;1-methyl-6-pho 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP;1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-isopropyl-pseudo-UTP;1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP;1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP;1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudouridine TP;1-Morpholinomethyl-pseudouridine TP;1-Pentyl-pseudouridine TP;1-Phenyl-pseudouridine TP;1-Pivaloyl-pseudouridine TP;1-Propyl-pseudouridine TP;1-Propynyl-pseudouridine;1-p-Tolyl-pseudouridine TP;1-tert-Butyl-pseudouridine TP;1-Thiomethoxymethyl-pseudouridine TP;1-Thiomorpholinomethyl-pseudouridine TP;1-Trifluoroacetyl-pseudouridine T P;1-Trifluoromethyl-pseudouridine TP;1-Vinylpseudouridine TP;2,2'-Anhydrouridine TP;2'-Bromodeoxyuridine TP;2'-F-5-Methyl-2'-deoxy-UTP;2'-OMe-5-Me-UTP;2'-OMe-pseudo-UTP;2'-α-Ethynyluridine TP;2'-α-Trifluoromethyluridine TP;2'-b-Ethynyluridine TP;2'-b-Trifluoromethyluridine TP;2'-Deoxy-2',2'-difluorouridine TP;2'-Deoxy-2'-α-mercaptouridine T P;2'-Deoxy-2'-a-thiomethoxyuridine TP;2'-Deoxy-2'-b-aminouridine TP;2'-Deoxy-2'-b-azidouridine TP;2'-Deoxy-2'-b-bromouridine TP;2'-Deoxy-2'-b-chlorouridine TP;2'-Deoxy-2'-b-fluorouridine TP;2'-Deoxy-2'-b-iodouridine TP;2'-Deoxy-2'-b-mercaptouridine TP;2'-Deoxy-2'-b-thiomethoxyuridine TP;2-Methoxy-4-thiouridine;2-Methoxyuridine;2' -O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP; 5-Iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-uridine TP;5-Vinylaruridine TP;6-(2,2,2-trifluoroethyl)-pseudo-UTP;6-(4-morpholino)-pseudo-UTP;6-(4-thiomorpholino)-pseudo-UTP;6-(substituted phenyl)-pseudo-UTP;6-amino-pseudo-UTP;6-azido-pseudo-UTP;6-bromo-pseudo-UTP;6-butyl-pseudo-UTP;6-chloro-pseudo-UTP;6-cyano-pseudo-UTP;6-dimethylamino-pseudo-UTP;6-ethoxy-pseudo-UTP;6-ethylcarboxylate-pseudo- UTP;6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP;6-Formyl-pseudo-UTP;6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP;6-Iodo-pseudo-UTP;6-Iso-propyl-pseudo-UTP;6-Methoxy-pseudo-UTP;6-Methylamino-pseudo-UTP;6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP;6-Phenyl-pseudo-UTP;6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP;6-Trifluoro Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine 1-[3-(2-ethoxy)]propionic acid; Pseudouridine 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine 1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy)]-ethoxy}-ethoxy]-ethoxy Pseudouridine TP1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy)-ethoxy}-ethoxy]-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-methylphosphonic acidPseudouridine TP 1-methylphosphonic acid diethyl ester; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butanoic acid; pseudo-UTP-N1-5-pentanoic acid; pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; wybutosin; hydroxywybutosin; isowybutosin; peroxywybutosin; insufficiently modified hydroxywybutosin; 4-demethylwybutosin; 2,6-(Diamino)purine;1-(Aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene;2(amino)purine;2,4,5-(trimethyl)phenyl;2'Methyl,2'amino,2'azido,2'fluoro-cytidine;2'Methyl,2'amino,2'azido,2'fluoro-adenine;2'Methyl,2'amino,2' Azido, 2'-fluoro-uridine;2'-amino-2'-deoxyribose;2-amino-6-chloropurine;2-Aza-inosinyl;2'-azido-2'-deoxyribose;2'-fluoro-2'-deoxyribose;2'-fluoro-modified bases;2'-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl;2-oxo-pyridopyrimidin-3-yl;2-pyridinone;3-nitropyrrole;3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl;4-(fluoro) -6-(Methyl)benzimidazole;4-(Methyl)benzimidazole;4-(Methyl)indolyl;4,6-(Dimethyl)indolyl;5-Nitroindole;5-Substituted pyrimidines;5-(Methyl)isocarbostyrilyl;5-Nitroindole;6-(Aza)pyrimidine;6-(Azo)thymine;6-(Methyl)-7-(aza)indolyl;6-Chloro-purine;6-Phenyl-pyrrolo-pyrimidin-2-one-3-yl;7-(Aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(Aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Aza)indolyl;7-(Guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-fu 7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl -(Diaza)-2-(oxo)-phenoxazin-1-yl;7-(Propynyl)isocarbostyril;7-(Propynyl)isocarbostyril, propynyl-7-(aza)indolyl;7-Deaza-inosinyl;7-Substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-Substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;9-(Methyl)-imidizopyridinyl;Aminoindolyl;Anthracenyl;Bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one- 3-yl;Bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Difluorotolyl;Hypoxanthine;Imidizopyridinyl;Inosinyl;Isocarbostyrilyl;N2-Substituted Purines;N6-Methyl-2-aminopurine;N6-Substituted Purines;N-Alkylated Derivatives;Naphthalenyl;Nitrobenzimidazolyl;Nitroimidazolyl;Nitroindazolyl;Nitropyrazolyl;Nubularine;O6-Substituted Purines;O-Alkylated Derivatives;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl;Ortho-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl; para-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; Propynyl-7-(aza)indolyl; Pyrenyl; Pyridopyrimidin-3-yl; Including pyridopyrimidin-3-yl, 2-oxo-7-aminopyridopyrimidin-3-yl; pyrrolopyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyresinyl; stilbenzyl; substituted 1,2,4-triazoles; tetracenyl; tubercidin; xanthine; xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-amino-riboside-TP; formycin A TP; formycin B TP; pyrrolosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TPTP; or 5-(2-carbomethoxyvinyl)uridine TP.

[0053] In some embodiments, the modified nucleotide that can substitute for cytosine is 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; lysidine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine; 4-methylcytidine ;5-Aza-cytidine;Pseudoisocytidine;Pyrrolo-cytidine;α-Thio-cytidine;2-(Thio)cytosine;2'-Amino-2'-deoxy-CTP;2'-Azido-2'-deoxy-CTP;2'-Deoxy-2'-α-aminocytidine TP;2'-Deoxy-2'-α-azidocytidine TP;3(Deaza)5(Aza)cytosine;3(Methyl)cytosine;3-(Alkyl)cytosine;3-(Deaza)5(Aza)cytosine;3-(Methyl)cytidine;4,2'-O-Dimethylcytidine;5(Halo)cytosine;5(Methyl)cytosine;5(Propyne) (I)cytosine;5(trifluoromethyl)cytosine;5-(alkyl)cytosine;5-(alkynyl)cytosine;5-(halo)cytosine;5-(propynyl)cytosine;5-(trifluoromethyl)cytosine;5-bromocytidine;5-iodocytidine;5-propynylcytosine;6-(azo)cytosine;6-azacytidine;azacytosine;deazacytosine;N4(acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine;1-methyl-pseudoisocytidine;2-methoxy-5-methylcytidine;2-methoxy-cytidine;2-thio- 5-Methylcytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'-fluoro-N4-Bz-cytidine TP; 2'-fluoro-N4-acetyl-cytidine TP;2'-O-methyl-N4-acetyl-cytidine TP; 2'-O-methyl-N4-Bz-cytidine TP; 2'-a-ethynylcytidine TP; 2'-a-trifluoromethylcytidine TP; 2'-b-ethynylcytidine TP; 2'-b-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP; 2'-deoxy-2'-a-mercaptocytidine TP; 2'-deoxy 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine 2'-Deoxy-2'-b-thiomethoxycytidine TP; 2'-O-methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(1-Propynyl)aracytidine TP; 5-(2-Chlorophenyl)-2-thiocytidine TP; 5-(4-Aminophenyl)-2 -thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylaracytidine TP; 5-ethynylcytidine TP; 5'-homocytidine TP; 5-methoxycytidine TP; 5-trifluoromethylcytidine TP; N4-aminocytidine TP; N4-benzoylcytidine TP; pseudoisocytidine; 2'-fluorocytidine; or 2'-OH-aracytidine TP;

[0054] In some embodiments, the modified nucleotides are 7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; wyosine; 1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine; 2'-O-ribosylguanosine(phosphate); 2'-O-methylguanosine; 2'-O-ribosylguanosine(phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; archaeosine; methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethyl N2,N2,7-Trimethylguanosine;N2,N2-Dimethylguanosine;N2,7,2'-O-Trimethylguanosine;6-Thioguanosine;7-Deazaguanosine;8-Oxoguanosine;N1-Methylguanosine;α-Thioguanosine;2-(Propyl)guanine;2-(Alkyl)guanines;2'-Amino-2'-deoxy-GTP;2'-Azido-2'-deoxy-GTP;2'-Deoxy-2'-α-aminoguanosine TP;2'-Deoxy-2'-α-azidoguanosine TP;6-(Methyl)guanine;6-(Alkyl)guanine )guanine;6-(Methyl)guanine;6-Methylguanosine;7-(Alkyl)guanine;7-(Deaza)guanine;7-(Methyl)guanine;7-(Alkyl)guanine;7-(Deaza)guanine;7-(Methyl)guanine;8-(Alkyl)guanine;8-(Alkynyl)guanine;8-(Halo)guanine;8-(Thioalkyl)guanine;8-(Alkenyl)guanine;8-(Alkyl)guanine;8-(Alkynyl)guanine;8-(Amino)guanine;8-(Halo)guanine;8-(Hydroxyl)guanine;8-(Thioalkyl)guanine N-methyl-6-thioguanosine; 8-(thiol)guanine; Azaguanine; Deazaguanine; N-(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thioguanosine; 6-methoxyguanosine; 6-thio-7-deaza-8-azaguanosine; 6-thio-7-deazaguanosine; 6-thio-7-methylguanosine; 7-deaza-8-azaguanosine; 7-methyl-8-oxoguanosine; N2,N2-dimethyl-6-thioguanosine; N2-methyl-6-thioguanosine; 1-Me-GTP; 2'-fluoro-N2-isobutyl-guanosine TP;2'-O-methyl-N2-isobutyl-guanosine TP;2'-a-ethynylguanosine TP;2'-a-trifluoromethylguanosine TP;2'-b-ethynylguanosine TP;2'-b-trifluoromethylguanosine TP;2'-deoxy-2',2'-difluoroguanosine TP;2'-deoxy-2'-a-mercaptoguanosine TP;2'-deoxy-2'-a-thiomethoxyguanosine TP;2'-deoxy-2'-b-aminoguanosine TP;2'-deoxy-2'-b-azidoguanosine TP;2'-deoxy-2'- b-Bromoguanosine TP; 2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP; 5'-Homoguanosine TP; 8-Bromoguanosine TP; 9-Deazaguanosine TP; N2-Isobutylguanosine TP; 1-Methylisothiazolinone Nosine;Inosine;1,2'-O-Dimethylinosine;2'-O-Methylinosine;7-Methylinosine;2'-O-Methylinosine;Epoxyqueusine;Galactosylqueusine;Mannosylqueusine;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(Guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3 -(Aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl;7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl;7-Deazainosinyl;7-Substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-Substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;9-(Methyl)-imidizopyridinyl;Aminoindolyl;Anthracenyl;Bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl;Difluorotolyl ;Hypoxanthine;Imidizopyridinyl;Inosinyl;Isocarbostyrilyl;Isoguanisine;N2-Substituted Purines;N6-Methyl-2-aminopurine;N6-Substituted Purines;N-Alkylated Derivatives;Naphthalenyl;Nitrobenzimidazolyl;Nitroimidazolyl;Nitroindazolyl;Nitropyrazolyl;Nubularine;O6-Substituted Purines;O-Alkylated Derivatives;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidine-2- On-3-yl; ortho-substituted-6-phenyl-pyrrolopyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolopyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; Propynyl-7-(aza)indolyl; Pyrenyl; Pyridopyrimidin-3-yl; Pyridopyrimidin-3-yl, 2 -oxo-7-aminopyridopyrimidin-3-yl; pyrrolopyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyridinyl; stilbenzyl; substituted 1,2,4-triazole; tetracenyl; tubercidin; xanthine; xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-amino-riboside-TP; formycin A TP; formycin B TP; pyrrolosine TP; or 2'-OH-araguanosine TP. Many of these modified nucleobases and the corresponding ribonucleosides are commercially available.

[0055] Self-amplifying mRNA (SAM) The mRNA disclosed herein can be replicative, i.e., self-amplifying.Self-amplifying mRNA molecules can be mRNA replicons derived from alphaviruses.MRNA amplification can also be achieved by providing a non-replicative mRNA encoding an antigen in combination with another mRNA encoding a replication mechanism.

[0056] Self-replicating RNA molecules are well known in the art and can be produced, for example, by using replication elements from alphaviruses to replace viral structural proteins with nucleotide sequences encoding proteins of interest. Self-replicating RNA molecules are typically positive-stranded molecules that can be directly translated after delivery to cells, providing an RNA-dependent RNA polymerase to produce both antisense and sense transcripts from the delivered RNA. In this way, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, like collinear subgenomic transcripts, can either be translated to result in the in situ expression of the encoded antigen, or they can be transcribed into additional transcripts with the same sense as the delivered RNA, which can then be translated to result in the in situ expression of the antigen. As a result of this round of transcription, the number of introduced replicon RNAs is greatly increased, and the encoded antigen becomes the major polypeptide product of the cell.

[0057] Suitable alphavirus replicons may use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type viral sequences may be used; for example, the attenuated TC83 mutant of VEEV has been used in the replicon. See WO2005 / 113782.

[0058] In certain embodiments, the self-replicating RNA molecules described herein encode (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an antigen. The polymerase may be, for example, an alphavirus replicase comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4 (where nsP represents a nonstructural protein).

[0059] While naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, the self-replicating RNA molecules do not encode alphavirus structural proteins. Thus, while the self-replicating RNA can produce its own genomic RNA copies within cells, it cannot produce RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecules cannot persist in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are absent from the self-replicating RNA of the present disclosure, and instead contain genes encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the alphavirus virion structural proteins.

[0060] A self-replicating RNA molecule useful in the present invention may have two open reading frames: a first (5') open reading frame encoding a replicase and a second (3') open reading frame encoding one or more HBV antigens.

[0061] In certain embodiments, the self-replicating RNA molecules disclosed herein have a 5' cap (e.g., 7-methylguanosine). This cap can facilitate in vivo translation of the RNA. In some embodiments, the 5' sequence of the self-replicating RNA molecule must be selected to ensure compatibility with the encoded replicase.

[0062] The self-replicating RNA molecule may have a 3' polyA tail and may contain a polyA polymerase recognition sequence (e.g., AAUAAA) near the 3' end.

[0063] Self-replicating RNA molecules can vary in length, but are typically 5,000 to 25,000 nucleotides in length. Self-replicating RNA molecules are typically single-stranded. Single-stranded RNA generally binds to TLR7, TLR8, RNA helicase, and / or PKR to exert an adjuvant effect. RNA delivered as double-stranded RNA (dsRNA) can bind to TLR3, and this receptor can also be activated by dsRNA formed during single-stranded RNA replication or within the secondary structure of single-stranded RNA.

[0064] In another embodiment, the self-replicating RNA can comprise two separate RNA molecules, each comprising a nucleotide sequence derived from an alphavirus. One RNA molecule comprises an RNA construct for expressing an alphavirus replicase, and the other RNA molecule comprises an RNA replicon that can be replicated in trans by the replicase. The RNA construct for expressing the alphavirus replicase comprises a 5' cap. See WO2017 / 162265.

[0065] Self-replicating RNA can be conveniently prepared by in vitro transcription (IVT). IVT can use templates (cDNA) produced and propagated in bacteria as plasmids or synthetically produced (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering). For example, DNA-dependent RNA polymerases (e.g., bacteriophage T7, T3, or SP6 RNA polymerases) can be used to transcribe self-replicating RNA from DNA templates. Appropriate capping and poly(A) addition reactions can be used, if necessary. (However, the poly(A) of the replicon is usually encoded within the DNA template.) These RNA polymerases can have strict requirements for the 5' nucleotide transcribed, and in some embodiments, these requirements must match those of the encoded replicase. This is to ensure that IVT-transcribed RNA functions efficiently as a substrate for the self-encoded replicase.

[0066] The self-replicating RNA may contain one or more nucleotides with modified nucleobases (in addition to any 5' cap structure). The RNA used in the present invention ideally contains only phosphodiester linkages between nucleosides, but in some embodiments may contain phosphoramidate and / or methylphosphonate linkages.

[0067] The self-replicating RNA molecule can encode a single heterologous polypeptide antigen (i.e., antigen), or two or more heterologous polypeptide antigens linked (e.g., in tandem) such that each sequence retains its identity when expressed as an amino acid sequence. Heterologous polypeptides generated from self-replicating RNA can be produced as fusion polypeptides or can be engineered to yield distinct polypeptide or peptide sequences.

[0068] The self-replicating RNA molecules described herein can be engineered to express multiple nucleotide sequences, thereby allowing for the co-expression of proteins, such as one, two, or more HBV antigens (e.g., surface and core antigens). The RNA molecules can express these proteins together with cytokines or other immunoregulatory factors to enhance the generation of an immune response.

[0069] If desired, self-replicating RNA molecules can be screened or analyzed to confirm their therapeutic and prophylactic properties using various in vitro or in vivo testing methods known to those skilled in the art. For example, vaccines containing self-replicating RNA molecules can be tested for their effect on the proliferation or effector function of specific lymphocyte types of interest, such as B cells, T cells, T cell lines, and T cell clones. For example, spleen cells from immunized mice can be isolated and tested for the ability of cytotoxic T lymphocytes to lyse autologous target cells containing self-replicating RNA molecules encoding antigens. Furthermore, T helper cell differentiation can be analyzed by measuring the proliferation or production of TH1 (IL-2 and IFN-γ) and / or TH2 (IL-4 and IL-5) cytokines using ELISA or directly in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry.

[0070] Self-replicating RNA molecules encoding antigens can also be tested for their ability to induce a humoral immune response, as evidenced, for example, by the induction of B cell production of antibodies specific to the antigen of interest. These assays can be performed, for example, using peripheral B lymphocytes from immunized individuals. Such assay methods are known to those skilled in the art. Other assays that can be used to characterize self-replicating RNA molecules include detecting the expression of the encoded antigen by target cells. For example, FACS can be used to detect antigen expression on the cell surface or intracellularly. Another advantage of FACS selection is the ability to select based on different expression levels. In some cases, lower expression may be desirable. Other suitable methods for identifying cells expressing a specific antigen include panning on a plate using a monoclonal antibody or capturing using monoclonal antibody-coated magnetic beads.

[0071] In one embodiment, the self-replicating RNA of the present disclosure may include a sequence encoding a self-cleaving peptide. The self-cleaving peptide may be, but is not limited to, the 2A cleavage region (referred to herein as "2A") of foot-and-mouth disease virus (FMDV). The 2A peptide has the amino acid sequence of SEQ ID NO: 3. In one aspect, the 2A peptide cleaves between the last glycine and the last proline. The 2A peptide causes the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A peptide, resulting in separation ("cleavage") between the end of the 2A sequence and the next downstream peptide. In one embodiment, the 2A peptide may be used to separate the coding regions of two or more polypeptides of interest. As a non-limiting example, the nucleotide sequence encoding the 2A peptide may be between a first coding region A (e.g., encoding hIi-HBc) and a second coding region B (e.g., encoding HBs).

[0072] In another embodiment, the self-replicating RNA of the present disclosure may contain a sequence encoding an internal ribosome entry site (IRES). The IRES element functions like an additional ribosome recruitment site, allowing translation at an internal region of the mRNA, so that the downstream ORF is translated separately from the upstream ORF. In one embodiment, an IRES may be used to separate the coding regions of two or more polypeptides of interest. As a non-limiting example, a nucleotide sequence encoding an IRES may be located between a first coding region A (e.g., encoding hIi-HBc) and a second coding region B (e.g., encoding HBs).

[0073] In one embodiment, the self-replicating RNA has the following configuration: 5' cap / 5' UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hIi / HBc / 2A / HBs / 3' UTR / polyA. In one embodiment, the self-replicating RNA has the following configuration: 5' cap / 5' UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hIi / HBc / IRES / HBs / 3' UTR / polyA.

[0074] In one embodiment, the self-replicating RNA has the following configuration: 5' cap / 5' UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hIi / HBc / 3' UTR / polyA. In one embodiment, the self-replicating RNA has the following configuration: 5' cap / 5' UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / HBs / 3' UTR / polyA.

[0075] In one embodiment, the self-replicating RNA has the following configuration: 5' cap / 5' UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hIi / HBs / 3' UTR / polyA. In one embodiment, the mRNA is non-replicating mRNA. In a second embodiment, the mRNA is replicative mRNA.

[0076] Lipid nanoparticles (LNPs) Lipid nanoparticles (LNPs) are non-virion liposomal particles that can encapsulate mRNA. RNA alone and unprotected can be degraded by the subject's RNases. LNPs provide a means of protecting mRNA by encapsulating a certain amount of mRNA in the overall composition within the LNP. LNP delivery systems and methods for their preparation are known in the art. While LNPs can contain external mRNA (e.g., on the surface of the LNP), it is desirable that at least half (preferably at least 85%, particularly at least 95%, e.g., all) of the mRNA be encapsulated.

[0077] The first lipid In some embodiments, the LNP comprises lipids, including a first lipid (i.e., a cation-ionizable lipid), an optional sterol (e.g., cholesterol), an optional polymer-conjugated lipid, and an optional second lipid (i.e., any neutral lipid, including any anionic lipid or zwitterionic lipid). In some embodiments, the optional neutral lipid comprises a neutral lipid / zwitterionic lipid. In some embodiments, the polymer-conjugated lipid comprises a polyethylene glycol-conjugated lipid.In some embodiments, the LNP is selected from the group consisting of WO2012 / 006376, WO2012 / 030901, WO2012 / 031046, WO2012 / 031043, WO2012 / 006378, WO2011 / 076807, WO2013 / 033563, WO2013 / 006825, WO2014 / 136086, WO2015 / 095340, WO2015 / 095346, WO2016 / 037053, WO2017 / 075531, WO2018 / 0814 80, WO2015 / 074085, WO2018 / 1703322, U.S. Patent Application Publication Numbers: 20220081392, 20220072155, 20220040285, 20210395188, 20210251898, 20210128488, 20210122703, 20210122702, 20210107861, 20200283372, 20200172472, 20200163878, 20200121809, 20200046838, 20190359556, 20190314524, 20190274968, 20190270697, 20190022247, 20180185516, 20170283367, 20170157268, 20170119904, 20160376224, 20160317676, or 20150376115, U.S. Application Nos. 61 / 905,724 or 15 / 614,499, or U.S. Patent Nos. 8,802,863, 9,458,090, and 9,593,077 , 9,567,296, 9,604,908, 9,643,916, 9,669,097, 9,670,487, 9,737,619, 9,738,593, 9,725,720, 9,796,977, 10,106,490, 10,166,298, 10,221,127, 10,723,692, 11,040,112, 11,168,051, or 11,285,222 (including the ionizable lipids and PEG-lipids referred to herein).

[0078] In some embodiments, the cationizable lipid comprises amine, which may be tertiary amine, which can be charged depending on the pH of the solution in which the cationizable lipid is present, when compared with the pKa of the cationizable lipid.In some embodiments, when the pH of the solvent containing the cationizable lipid is higher than pKa, at least half of the cationizable lipid is neutrally charged, and the amine is a tertiary amine.Also, when the pH of the solvent containing the cationizable lipid is lower than pKa, at least half of the cationizable lipid is positively charged.In this regard, without being limited to a particular theory, it is believed that in some embodiments, the positive charge of the ionizable lipid is distributed on the amine, and as a result, when the pH of the solvent containing the cationizable lipid is lower than pKa, the amine is positively charged.Without being limited to a particular theory, the amine is an ionizable amine, because the amine can fluctuate between neutral and positive charge depending on the pH of the solution in relation to the pKa of the cationizable lipid.

[0079] Cationizable lipids are further described when the amine is a tertiary amine and when the cationizable lipid is neutrally charged, but the description is not limited to the lack of ability of the cationizable lipid to be positively charged. That is, lipids in the tertiary amine state and neutrally charged are described herein without the need to describe cationizable lipids when the tertiary amine is charged. In some embodiments, the cationizable lipid comprises, in addition to the ionizable amine described above, a head group (R H ) and fatty acid tail (R FA1 or R FA2 In some embodiments, the cationizable lipid further comprises a head group and at least two fatty acid tails (R ) (in addition to the ionizable amine described above), e.g., as shown in Formula I FA1 and R FA2 ) further includes. [ka]

[0080] In some embodiments, the amine provides a branch point between the head group and the fatty acid tail. FA ) or at least two fatty acid tails (i.e., R FA1 , R FA2 ...) is immediately separated from the ionizable amine. In some embodiments, the fatty acid tail, or at least two fatty acid tails, are linked to a biodegradable group (i.e., R BD1 or R BD2 In some embodiments, the at least two fatty acid tails each comprise a biodegradable group as shown in Formula II, wherein the biodegradable groups are the same or independent of one another. In some embodiments, the biodegradable groups are selected from the group consisting of, in order from ionizable amine, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(═O)O—, wherein X is 0, 1, or 2; and R a is hydrogen or C1-C 12 In some embodiments, the fatty acid, or at least two fatty acids, have a C1-C6 alkyl group between the amine branch point and the biodegradable group, as shown in Formula II. 12 Alkyl, C1-C 12 Alkylene, or C1-C 12 Alkenylene (i.e., R FC1 and R FC2 In some embodiments, the fatty acid or two or more fatty acids may include a C6-C amine group distal to the ionizable amine and biodegradable group, such as shown in Formula II: 24 Alkyl, C6-C 24 Alkylene, C7-C 23 Alkyl, C7-C 23 Alkylene, C8-C 22 Alkyl, C8-C 22Alkylene, C9-C 21 Alkyl, C9-C 21 Alkylene, C 10 -C 20 Alkyl, C 10 -C 20 Alkylene, C 11 -C 19 Alkyl, C 11 -C 19 Alkylene, C 12 -C 18 Alkyl, C 12 -C 18 Alkylene, C 13 -C 17 Alkyl or C 13 -C 17 Alkylene (i.e., R FC3 and R FC4 ) is included. [ka] [In the formula, R FC1 and R FC2 are independently C1-C 12 Alkyl, C1-C 12 Alkylene, or C1-C 12 is alkenylene, R FC3 and R FC4 are independently C6-C 24 Alkyl, C6-C 24 Alkylene, C7-C 23 Alkyl, C7-C 23 Alkylene, C8-C 22 Alkyl, C8-C 22 Alkylene, C9-C 21 Alkyl, C9-C 21 Alkylene, C 10 -C 20 Alkyl, C 10 -C 20 Alkylene, C 11 -C 19 Alkyl, C 11 -C 19 Alkylene, C 12 -C 18 Alkyl, C 12 -C 18 Alkylene, C13 -C 17 Alkyl, C 13 -C 17 is alkylene, R BD1 and R BD2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, or -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(═O)O—, X is 0, 1, or 2, and R a is hydrogen or C1-C 12 It is alkyl.

[0081] In some embodiments, C6-C 24 Alkyl or C6-C 24 Alkylene is a C6-C 12 , C7-C 11 , C8-C 10 or C9 of each of the at least two fatty acid termini. 24 Alkyl or C6-C 24 Alkylene independently includes: [ka]

[0082] In some embodiments, the head group comprises a first group (i.e., R H1 ) and a second group (i.e., R H2 The first group comprises, consists of, is or has a C1-C 24 Alkyl, C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene, and the second group is H, —OH, CN, —C(═O)OR 4, -OC(=O)OR 4 , -NR 5 C(=O)OR 4 OR 5 and the above R 4 is C1-C 12 alkyl, and R 5 is H or C1-C6 alkyl. In some embodiments, the head group comprises a linear or branched form of -(CH2)6OH, -(CH2)5OH, -(CH2)4OH, -(CH2)3OH, -(CH2)2OH, or -CH2OH. In some embodiments, the cationizable lipid comprises, consists of, or is [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) or 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate.

[0083] In some embodiments, the cationizable lipid is: [ka]

[0084] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV28, which has the following structure: [ka]

[0085] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV31, which has the following structure: [ka]

[0086] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV33, which has the following structure: [ka]

[0087] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV37, which has the following structure: [ka]

[0088] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV39, i.e., 2,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl 4-(dimethylamino)butanoate. [ka]

[0089] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV42, which has the following structure: [ka]

[0090] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV44, which has the following structure: [ka]

[0091] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV73 having the following structure: [ka]

[0092] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV75 having the following structure: [ka]

[0093] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV81, which has the following structure: [ka]

[0094] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV84, which has the following structure: [ka]

[0095] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV85 having the following structure: [ka]

[0096] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV86, which has the following structure: [ka]

[0097] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV88, which has the following structure: [ka]

[0098] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV91, which has the following structure: [ka]

[0099] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV92, which has the following structure: [ka]

[0100] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV93 having the following structure: [ka]

[0101] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is 2-(5-((4-((1,4-dimethylpiperidine-4-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate (RV94), having the following structure: [ka]

[0102] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV95, which has the following structure: [ka]

[0103] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV96, which has the following structure: [ka]

[0104] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV97, which has the following structure: [ka]

[0105] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV99, which has the following structure: [ka]

[0106] In one embodiment, the cationizable lipid comprises, consists of, consists essentially of, or is RV101, which has the following structure: [ka]

[0107] In some embodiments, the cationizable lipid comprises, consists of, consists essentially of, or is a lipid having the structure of formula III. [ka] wherein n is an integer from 1 to 3; (i) R1 is CH3, R2 and R3 are both H, and Y is C; or (ii) R1 and R2 together are CH2-CH2 and together with the nitrogen form a 5-, 6-, or 7-membered heterocycloalkyl, R3 is CH3, and Y is C; or (iii) R1 is CH3, R2 and R3 are both absent, and Y is O; o is 0 or 1, X is (i) [ka] wherein R4 and R5 are independently a C alkene group having one or two cis alkene groups at either or both of the omega 6 and omega 9 positions. 10 - 20 a hydrocarbon chain, or (ii) —CH(—R 6 )—R 7 , wherein (1) R6 is -(CH2) p -OC(O)-R8 or -C p -R8; (2) R7 is -(CH2) p’ -OC(O)-R8' or -C p’ -R8'; (3) p and p' are independently 0, 1, 2, 3, or 4; and (4) R8 and R8' are independently (A) -C having one or two cis alkene groups at either or both of the omega 6 and 9 positions 8-20 hydrocarbon chains; (B)-C 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chains; (C)-C 6-16 saturated hydrocarbon chains; (D)-C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chains; (E)-C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 a saturated or unsaturated hydrocarbon chain; and (F)-C 6-16 saturated or unsaturated hydrocarbon chain ]

[0108] In one embodiment, R1 is CH3, R2 and R3 are both H, and Y is C. In some embodiments, R1 and R2 together are CH2-CH2 and together with the nitrogen atom form a 5-, 6-, or 7-membered heterocycloalkyl, R3 is CH3, and Y is C. In some embodiments, R1 is CH3, R2 and R3 are both absent, and Y is O.

[0109] In one embodiment, X is [ka] and R4 and R5 are independently a C 16 having one or two cis alkene groups at either or both of the omega 6 and 9 positions. 10-20 It is a hydrocarbon chain.

[0110] In one embodiment, X is —CH(—R)—R, where R is —(CH)OC(O)—R, and R is —(CH) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 R is a hydrocarbon chain, and R has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 It is a hydrocarbon chain.

[0111] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 R8' is a hydrocarbon chain; 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0112] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0113] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 is a hydrocarbon chain, and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0114] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 R8' is a hydrocarbon chain and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0115] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions.8-20 hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0116] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0117] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0118] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain; 6-16 It is a saturated hydrocarbon chain.

[0119] In one embodiment, X is —CH(—R)—R, where R is —(CH)p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0120] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0121] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0122] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16saturated hydrocarbon chain, R8' has one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0123] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0124] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0125] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0126] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16is a saturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0127] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0128] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0129] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0130] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’-OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0131] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0132] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0133] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0134] In one embodiment, X is —CH(—R)—R, where R is —(CH)p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at one or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0135] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0136] In one embodiment, X is —CH(—R)—R, where R is —(CH)OC(O)—R, and R is —(CH) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0137] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12R8' is a saturated or unsaturated hydrocarbon chain; R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0138] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0139] In one embodiment, X is —CH(—R)—R, where R is —(CH)OC(O)—R, and R is —(CH) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0140] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0141] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2)p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0142] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0143] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0144] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0145] In one embodiment, X is —CH(—R)—R, where R is —(CH) p-OC(O)-R8, where R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0146] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 R is a hydrocarbon chain, and R has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 It is a hydrocarbon chain.

[0147] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0148] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0149] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 R8' is a hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0150] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 is a hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0151] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at one or both of the omega 6 and 9 positions. 8-20 hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0152] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0153] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0154] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3 or 4. R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain; 6-16 It is a saturated hydrocarbon chain.

[0155] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0156] In one embodiment, X is —CH(—R)—R, where R is —(CH) p-OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0157] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain; 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0158] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' has one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0159] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0160] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0161] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0162] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated hydrocarbon chain. 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0163] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0164] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’-R8', where p and p' are independently 0, 1, 2, 3 or 4. R8 is -C(-C 6-16 )-C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0165] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0166] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0167] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0168] In one embodiment, X is —CH(—R)—R, where R is —(CH) p-OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0169] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0170] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 It is a hydrocarbon chain.

[0171] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12It may be a saturated or unsaturated hydrocarbon chain.

[0172] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0173] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain; R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0174] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain. R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0175] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0176] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0177] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chains

[0178] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0179] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0180] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0181] In one embodiment, X is —CH(—R)—R, where R is —(CH) p -OC(O)-R8, where R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0182] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 R8' is a hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0183] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’-OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0184] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0185] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 is a hydrocarbon chain, and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0186] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 is a hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0187] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0188] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 It is a hydrocarbon chain.

[0189] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0190] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0191] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain. 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0192] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0193] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0194] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’-OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0195] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0196] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0197] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0198] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16R8' is a saturated hydrocarbon chain. 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0199] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0200] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C(-C 6-16 )-C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0201] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain; 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0202] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0203] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0204] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0205] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0206] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’-OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 It is a hydrocarbon chain.

[0207] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0208] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0209] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain; R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0210] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain. R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0211] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0212] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0213] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 1-3-C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0214] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0215] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0216] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0217] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -(CH2) p’ -OC(O)-R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0218] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 R8' is a hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0219] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 is a hydrocarbon chain; R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0220] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 is a hydrocarbon chain; R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0221] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C 8-20 is a hydrocarbon chain, and R8' is -C(-C 6-16 )-C6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0222] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 is a hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0223] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 has one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0224] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain having one or two cis alkene groups at either or both of the omega 6 and 9 positions. 8-20 It is a hydrocarbon chain.

[0225] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC6-12 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0226] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3 or 4. R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain; 6-16 It is a saturated hydrocarbon chain.

[0227] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain; R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0228] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0229] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’-R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 1-3 -C(-OC 6-12 )-OC 6-12 R8' is a saturated or unsaturated hydrocarbon chain. 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0230] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0231] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated hydrocarbon chain. 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0232] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0233] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C(-C 6-16)-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0234] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 is a saturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0235] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0236] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0237] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0238] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0239] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0240] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0241] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C(-C 6-16 )-C 6-16 R8' is a saturated or unsaturated hydrocarbon chain. 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0242] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0243] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain; 1-3 -C(-OC 6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0244] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0245] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0246] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', where p and p' are independently 0, 1, 2, 3, or 4. R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 R8' is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0247] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0248] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 A saturated or unsaturated hydrocarbon chain, R8' having one or two cis alkene groups at either or both of the omega 6 and 9 positions -C 8-20 It is a hydrocarbon chain.

[0249] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 1-3 -C(-OC6-12 )-OC 6-12 It may be a saturated or unsaturated hydrocarbon chain.

[0250] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It is a saturated hydrocarbon chain.

[0251] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 R8' is a saturated or unsaturated hydrocarbon chain and R8' is -C(-C 6-16 )-C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0252] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 is a saturated or unsaturated hydrocarbon chain, and R8' is -C[-COC(O)-C 4-12 ]-COC(O)-C 4-12 It may be a saturated or unsaturated hydrocarbon chain.

[0253] In one embodiment, X is —CH(—R 6 )—R 7 , and R 6 is —C p -R8 and R7 is -C p’ -R8', p and p' are independently 0, 1, 2, 3 or 4, and R8 is -C 6-16 saturated or unsaturated hydrocarbon chain, R8' is -C 6-16 It may be a saturated or unsaturated hydrocarbon chain.

[0254] In some embodiments, the cationizable lipid is selected from the group consisting of WO2012 / 006376, WO2012 / 030901, WO2012 / 031046, WO2012 / 031043, WO2012 / 006378, WO2011 / 076807, WO2013 / 033563, WO2013 / 006825, WO2014 / 136086, WO2015 / 095340, WO2015 / 09534 6, WO2016 / 037053, WO2017 / 075531, WO2018 / 081480, WO2015 / 074085, WO2018 / 1703322, U.S. Patent Application Publication Numbers: 20220081392, 20220072155, 20220040285, 20210395188, 20210251898, 20210128488, 20210122703, 2021012 2702, 20210107861, 20200283372, 20200172472, 20200163878, 20200121809, 20200046838, 20190359556, 20190314524, 20190274968, 20190270697, 20190022247, 20180185516, 20170283367, 20170157268, 2 0170119904, 20160376224, 20160317676, or 20150376115, U.S. Application Nos. 61 / 905,724 or 15 / 614,499, or U.S. Patents Nos. 8,802,863, 9,458,090, 9,593,077, 9,567,296, 9,604,908, 9,643,916, 9,669,097, 9,670,487, and 9,737,619; 9,738,593, 9,725,720, 9,796,977, 10,106,490, 10,166,298, 10,221,127, 10,723,692, 11,040,112, 11,168,051, 11,246,933, or 11,285,222 (referred to in the literature as ionizable lipids).

[0255] In some embodiments, the cationizable lipid comprises a first group and two biodegradable hydrophobic tails. In some embodiments, the first group comprises a central moiety and a head group, and the first group can be positively charged. In some embodiments, the central moiety is directly bonded to each of the two biodegradable groups. In some embodiments, the central moiety is directly bonded to the head group. In some embodiments, the central moiety is selected from a central carbon atom, a central nitrogen atom, a central heteroaryl group, and a central heterocyclic group. In some embodiments, one of the two biodegradable hydrophobic tails, or each of the two biodegradable hydrophobic tails, has the formula: -(C1-C 12 Alkyl, C1-C 12 Alkylene, or C1-C 12 Alkenylene)-(biodegradable group)-(C6-C 24 Alkyl, C6-C 24 Alkylene, C7-C 23 Alkyl, C7-C 23 Alkylene, C8-C 22 Alkyl, C8-C 22 Alkylene, C9-C 21 Alkyl, C9-C 21 Alkylene, C 10 -C 20 Alkyl, C 10 -C 20 Alkylene, C 11 -C 19 Alkyl, C 11 -C 19 Alkylene, C 12 -C 18 Alkyl, C 12 -C 18 Alkylene, C 13 -C 17 Alkyl, C 13 -C 17 In some embodiments, each biodegradable group in the two biodegradable hydrophobic tails is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a-, -OC(=O)NR a - or -NR a C(═O)O—; where X is 0, 1, or 2; and R a is hydrogen or C1-C 12 In some embodiments, one of the two biodegradable tails, or each of the two biodegradable tails, has 1) a terminal hydrophobic chain and a terminus that is a branched alkyl group, 2) the branch of the branched alkyl group is alpha to the biodegradable group, and 3) 6 to 12 carbon atoms of the biodegradable hydrophobic tail separate the terminus from the biodegradable group.

[0256] In some embodiments, the cationizable lipid is selected from the group consisting of bis(2-methacryloyl)oxyethyl disulfide (DSDMA, CAS number 36837-97-5), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA), 98N12-5, 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3- Dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), ICE (imidazole-based), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-dilinoleoyl- 4-Dimethylaminoethyl-[1,3]-dioxolane) HGT4003, 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or their analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), ALNY-100(( 3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-o-3aH-cyclopenta[d][1,3]dioxol-5-amine)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3 ]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLi n-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN® (DOTMA and 1, from GIBCO / BRL, Grand Island, NY)The liposomes may comprise a commercially available cationic liposome containing 2-dioleoyl-sn-3-phosphoethanolamine (DOPE), LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetic acid (DOSPA) (DOPE) manufactured by GIBCO / BRL), or TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol manufactured by Promega Corp., Madison, Wisconsin), or any combination thereof. Further suitable cationic compounds include those described in International Patent Publication WO2010 / 053572 (particularly CI2-200 described in paragraph

[0225] ) and WO2012 / 170930 (both of which are incorporated herein by reference), HGT4003, HGT5000, HGTS001, HGT5001, and HGT5002 (see U.S. Patent Application Publication No. 20150140070A1).

[0257] Representative cationizable lipids include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine) These include, but are not limited to, 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and MC3 (U.S. Patent Publication No. 20100324120).

[0258] Various amphipathic lipids can form bilayers in an aqueous environment and encase an aqueous core containing RNA as an LNP. These lipids can have anionic, cationic, or zwitterionic hydrophilic head groups. Phospholipids can be anionic, zwitterionic, or cationic. Suitable phospholipid classes include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. Some useful phospholipids are listed in Table 1. Useful cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and dodecylphosphocholine. These lipids can be saturated or unsaturated. It is preferred to use at least one unsaturated lipid in preparing the liposomes, and if the unsaturated lipid has two tails, both tails may be unsaturated or one saturated and one unsaturated tail.

[0259] Other useful LNPs are described in the following documents: WO2012 / 006376, WO2012 / 030901, WO2012 / 031046, WO2012 / 031043, WO2012 / 006378, WO2011 / 076807, WO2013 / 033563, WO2013 / 006825, WO2014 / 136086, WO2015 / 095340, WO2015 / 095346, WO2016 / 037053. In some embodiments, the LNP is an RV01 liposome, see WO2012 / 006376 and Geall et al. (2012) PNAS USA. September 4;109(36):14604-9.

[0260] Polyethylene glycol conjugated lipids In some embodiments, the LNPs comprise polyethylene glycol-conjugated (PEG-conjugated) lipids. In some embodiments, the PEG-conjugated lipids comprise polyethylene glycols (PEGs) of various lengths and molecular weights.

[0261] In some embodiments, the median molecular weight of PEG in the PEG-conjugated lipid is 0.5 kDa, 0.6 kDa, 0.7 kDa, 0.8 kDa, 0.9 kDa, 1.0 kDa, 1.1 kDa, 1.2 kDa, 1.3 kDa, 1.4 kDa, 1.5 kDa, 1.6 kDa, 1.7 kDa, 1.8 kDa, 1.9 kDa, 2.0 kDa, 2.1 kDa, 2.2 kDa, 2.3 kDa, 2.4 kDa, 2.5 kDa, 2.6 kDa, 2.7 kDa, 2.8 kDa, 2.9 kDa, 3.0 kDa, or 4.0 kDa. a, 3.1kDa, 3.2kDa, 3.3kDa, 3.4kDa, 3.5kDa, 3.6kDa, 3.7kDa, 3.8kDa, 3.9kDa, 4.0kDa, 4.1kDa, 4.2kDa, 4.3kDa, 4.4kDa, 4.5kDa, 4 .6kDa, 4.7kDa, 4.8kDa, 4.9kDa, 5.0kDa, 5.1kDa, 5.2kDa, 5.3kDa, 5.4kDa, 5.5kDa, 5.6kDa, 5.7kDa, 5.8kDa, 5.9kDa, or 6.0kDa.

[0262] In some embodiments, the PEG-conjugated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, "2000" represents the median molecular weight of PEG in Daltons. In some embodiments, the PEG-conjugated lipid comprises 1,2-dimyristoyl-sn-glycero-2-phosphoethanolamine-N-[methoxy(polyethylene glycol)]. In some embodiments, the PEG-conjugated lipid comprises 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

[0263] Secondary lipids In some embodiments, the LNP further comprises a second lipid comprising an anionic lipid, a neutral lipid, or a zwitterionic lipid. In some embodiments, the neutral lipid comprises a neutral zwitterionic lipid. In some embodiments, the anionic lipid, neutral lipid, or zwitterionic lipid comprises a phosphate group (i.e., a phospholipid), choline, or a sphingolipid.

[0264] In some embodiments, the second lipid is 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (17:0 PE), 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (06:0 PE), 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (08:0 PE), 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (10:0 PE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0 PE), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (15:0 PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (15:0 PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (10:0 PE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0 PE), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (15:0 PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (16:0 PE), 1,2-dipentadecanoyl-sn-glycero-3-phospho PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (18:0 PE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (14:0 PE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (16:1 PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (18:1 (Δ9-trans) PE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (18:2 PE), 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (18:3 PE), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6 PE), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (20:4 PE), 1-pentadecanoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (15:0-18:1 PE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2 PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (16:0-20:4PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6 PE), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2 PE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4 PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6 PE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 lyso-PE), 1-hydroxy-2-oleoyl-sn-glycero-3-phosphoethanolamine (2-18:1 lysoPE), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0 lysoPE), 1-tridecanoyl-sn-glycero-3-phosphoethanolamine (13:0 lysoPE), 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphoethanolamine (17:1 lysoPE), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:0 lysoPE), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (14:0 lysoPE), L-α-phosphatidylethanolamine, 1,2-dibutyryl-sn-glycero-3-phosphocholine (4:0 lysoPE), 1-hydroxy-sn-glycero-3-phosphoethanolamine (1 ... PC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (7:0 PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC), 1,2-dinonanoyl-sn-glycero-3-phosphocholine (9:0 PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (11:0 PC), 1,3-dipalmitoyl-rac-glycero-2-phosphocholine (16:0 PC) 2-PC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (13:0PC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15:0 PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17:0 PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (19:0 PC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (20:0 PC), 1,2-diheneicosanoyl-sn-glycero-3-phosphocholine (21:0 PC) PC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (22:0 PC), 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (23:0 PC), 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (24:0 PC), 1,2-dibacenoyl-sn-glycero-3-phosphocholine (18:1 (11-cis) PC), 1,2-di[(8Z)octadecenoyl]-sn-glycero-3-phosphocholine (18:1 (8-cis) PC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (14:1 (Δ9-cis) PC), 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18:1 (Δ6-cis) PC) PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dielaidoyl-sn-glycero-3-phosphocholine (18:1 (Δ9-trans)PC), 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-didecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2- Dilauroyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipentadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-distearoyl-s n-Glycero-3-phospho-(1'-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dielaidoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilinolenoyl-sn-glycero-3-phospho phospho-(1'-rac-glycerol), 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], 1,2-didocosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 (cis) PC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (20:1 (cis) PC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (20:4 (cis) PC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (22:1 (cis) PC), 1,2-dinervonoyl-sn-glycero-3-phosphocholine (24:1 (cis) PC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6 (cis) PC), 1-pentadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine (15:0-18:1PC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0 PC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC) PC) 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (16:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC) PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC), 1-(8Z-octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine (18:1(n10)-16:0 PC). PC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0PC), 1-palmitoyl-2-[12'-(palmitoyloxy)octadecanoyl]-sn-glycero-3-phosphocholine (16:0-(12-PAHSA) PC), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-hexanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-heptanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-octanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-nonanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-decanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1 -lauroyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-tridecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-(10Z-heptadecenoyl)-2-hydroxy-sn-glycero-3-phosphocholine, 1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine, 2-stearoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-ligno 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0), 1-hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0), diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lyso-PC), 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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), N-lignoceroyl-D-erythro-sphingosylphosphoethanolamine, or sphingomyelin.

[0265] sterols In some embodiments, the lipid nanoparticles further comprise a sterol. In some embodiments, the sterol is cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, cymosterol, lathosteriol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydrolanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone. Desmosterol, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9), 14-dien-3β-ol (dihydro-FF-MAS-d6), 4,4-dimethyl(d6)-cholest-8(9)-en-3β-ol (dihydro-T-MAS-d6), zymostenol, sitostanol, campestanol, camperstanol, 7-dehydrodesmosterol, pregnenolone, 4,4-dimethyl-cholest-8(9)-en-3β-ol (dihydro-T-MAS), Δ5-avengerstenol, buccal Lasicasterol, dihydro FF-MAS, 24-methylenecholesterol, oxysterols, deuterated sterols, fluorinated sterols, sulfonated sterols, phosphorylated sterols, A-ring substituted sterols, cholest-5-ene-3β,4β-diol, 5α-cholestan-3β-ol, 4-cholesten-3-one, cholesta-8(9),24-dien-3-one, cholesta-8(9),24-dien-3-one, 2,2,3,4,4-pentadeuterio-5a-cholestan-3β-ol, cholesterylphosphocholine Phosphorus, cholesteryl-d7 pentadecanoate, cholesteryl-d7 palmitoylate, B-ring substituted sterols, cholestanol, 5β,6β-epoxy-d7, 3β-hydroxy-5-cholesten-7-one, 6α-hydroxy-5α-cholestane, cholestanol, 5α,6α-epoxycholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, cholestanol, 5α,6α-epoxy-d7Δ5,7-cholesterol, cholesta-5,8(9)-dien-3β-ol, cholesta-5,8(14)-dien-3β-ol, 7α-hydroxy-4-cholesten-3-one, zymostenol-d7, zymostenol, 7-dehydrodesmosterol, 3b,5a-dihydroxy-cholestan-6-one, D-ring substituted sterols, 3β-hydroxy-5α-cholest-8(14)-en-15-one, 3β-hydroxy-5α-cholestan-15-one, 5α-cholest-8(14)-ene-3β,15α-diol, 5α-cholest-8(14)-ene-3β,15β-diol, lanosterol-95, 5α-7,24-cholestadi ene, 14-dehydrozymostenol, ergosta-5,7,9(11),22-tetraen-3β-ol, cholest-5-ene-3β,25-diol, cholest-(25R)-5-ene-3β,27-diol, 24(R / S)-25-epoxycholesterol, 24(S)-25-epoxycholesterol, 24(R / S),25-epoxycholesterol-d6, cholest-5-ene-3β,22(S)-diol, cholest-5-ene-3β,22(R)-diol, cholest-5-ene-3β,2 4(S)-diol, cholest-5-ene-3β,24(R)-diol, 27-hydroxy-4-cholesten-3-one, campestanol, N,N-dimethyl-3β-hydroxycholenamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3β-hydroxycholenamide, 25,27-dihydroxycholesterol, 5-cholesten-3β,20α-diol, 24S,25-epoxy-5α-cholest-8(9)-en-3β-ol, 24(S / R),25-epoxylanostachys-8(9) -en-3β-ol, 7-keto-27-hydroxycholesterol, 7α,27-dihydroxy-4-cholesten-3-one, 7α,27-dihydroxycholesterol, 7β,27-dihydroxycholesterol, 5α,6β-dihydroxycholestanol, 7α,25-dihydroxycholesterol, 7β,25-dihydroxycholesterol, 7α,24(S)-dihydroxycholesterol, 7α,24(S)-dihydroxy-4-cholesten-3-one, 7-keto-25-hydroxycholesterol, 7α,24S,27-trihydroxycholesterol, dihydrotestosterone, testosterone, estrone, estrogen, estradiol, corticosterone, cortisol, or 24S,27-dihydroxycholesterol.

[0266] Fabrication of LNPs encapsulating mRNA As described above, in some embodiments, mRNA molecules are encapsulated within LNPs. In some embodiments, the mRNA contained within or encapsulated within the LNPs can be obtained by mixing and / or purifying the mRNA and lipids of the LNPs. In some embodiments, the mRNA encapsulated within the LNPs can be obtained at the above-mentioned percentages by mixing and / or purifying the mRNA and lipids of the LNPs.

[0267] In some aspects, a method for obtaining a composition comprising mRNA and LNPs, wherein the mRNA is encapsulated within the LNPs in the above-described ratio, and the LNPs comprise the lipids, is provided, the method comprising mixing a first solution comprising the recombinant RNA molecule with a second solution comprising the lipids. In some embodiments, the mixing is performed using at least a T-mixer, a microfluidics device, or an impinging jet mixer. In some embodiments, the first solution further comprises a citrate buffer (e.g., sodium citrate) or an acetate buffer (e.g., sodium acetate).

[0268] In some embodiments, the second solution further comprises an organic solvent. In some embodiments, the organic solvent comprises chloroform, dichloromethane, diethyl ether, cyclohexane, cyclopentane, benzene, toluene, methanol, benzyl alcohol, and an aliphatic alcohol (e.g., a C1-C8 alcohol). In some embodiments, the aliphatic alcohol comprises ethanol, propanol, isopropanol, butanol, tert-butanol, isobutanol, pentanol, benzyl alcohol, and hexanol. In some embodiments, the organic solvent comprises an alcohol solution. In some embodiments, the organic alcohol solution comprises 70% to 100% ethanol by volume.

[0269] In some embodiments, the method includes mixing a first solution containing recombinant RNA molecules and lipids of the LNPs described above with a second solution that is an aqueous solution. In some embodiments, the RNA and lipids of the LNPs are mixed in an organic solvent. In some embodiments, the organic solvent includes chloroform, dichloromethane, diethyl ether, cyclohexane, cyclopentane, benzene, toluene, methanol, benzyl alcohol, and aliphatic alcohols (e.g., C1-C8 alcohols). In some embodiments, aliphatic alcohols include ethanol, propanol, isopropanol, butanol, tert-butanol, isobutanol, pentanol, benzyl alcohol, and hexanol. In some embodiments, the organic solvent comprises an alcohol solution. In some embodiments, the organic alcohol solution includes 70% to 100% ethanol by volume. In some embodiments, the organic alcohol solution includes 70% to 100% ethanol by volume and 30% to 0% benzyl alcohol by volume. In some embodiments, the aqueous solution includes a citrate buffer (e.g., sodium citrate) or an acetate buffer (e.g., sodium acetate). In some embodiments, the first and second solutions are mixed in a ratio of 1:1 to 5:1, 2:1 to 4:1, 2.5:1 to 3.5:1, or 3:1.

[0270] In some embodiments, the mixing of the first solution and the second solution (i.e., either of the above two methods) is performed at a pH ranging from 4.5 to the pKa of the first lipid (e.g., a cationizable lipid), thereby obtaining a first mixture. In some embodiments, the mixing of the first solution and the second solution is performed at a pH ranging from 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, or 6.0 to the pKa of the first lipid (e.g., a cationizable lipid), thereby obtaining a first mixture. In some embodiments, the method further includes a first increasing step of increasing the pH of the first mixture to above the pKa of the first lipid, thereby obtaining a pH-adjusted first mixture. In some embodiments, the first increasing step results in a pH-adjusted first mixture having a pH from the pKa of the first lipid (e.g., a cationizable lipid) to 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, or 7.0.

[0271] In some embodiments, the first enrichment or purification step comprises cross-flow filtration or tangential flow filtration. In some embodiments, the first enrichment or purification further comprises transferring the composition comprising the LNP and the recombinant RNA molecule to a third solution different from the first solution. In some embodiments, the third solution comprises phosphate buffered saline. In some embodiments, the transfer comprises dialysis. In some embodiments, the tangential flow filtration comprises the use of hollow fiber filters. In some embodiments, the hollow fibers comprise polyethersulfone hollow fiber filters or polysulfone hollow fiber filters.

[0272] In some embodiments, the first enrichment or purification step comprises passing the LNP / RNA mixture through an ion exchange solid support prior to the filtration step described above. In some embodiments, the ion exchange solid support comprises an anion exchange column or a cation exchange column.

[0273] In some embodiments, the lipids of the LNPs are mixed with an organic solvent to obtain a concentrated stock (e.g., a stock lipid / organic solvent mixture) prior to mixing the mRNA with the lipids of the LNPs. In some embodiments, mixing is performed (e.g., by stirring, rocking, vortexing, sonicating, or agitating the stock lipid / organic solvent mixture at 25°C to 37°C) for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 minutes to form a homogenous stock lipid / organic solvent mixture. In some embodiments, mixing occurs within 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes, 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.4 hours, or 1.5 hours (e.g., stirring, shaking, vortexing, sonicating, or agitating the stock lipid / organic solvent mixture at 25°C-37°C) to form a homogenous stock lipid / organic solvent mixture. In light of the above embodiments, it is contemplated and supported that any of the above "at least" and "within" times can be combined to provide a closed range (i.e., stirring, shaking, vortexing, sonicating, or agitating the stock lipid / organic solvent mixture at 25°C-37°C for 5 minutes to 19 minutes).

[0274] Regimen The present invention encompasses a method for treating chronic hepatitis B infection (CHB) by administering to a human a first mRNA encoding a first hepatitis B virus antigen in combination with a second mRNA encoding a second hepatitis B virus antigen. In one embodiment, the first and second mRNAs are administered simultaneously. In a first embodiment, the first and second mRNAs are in separate LNP formulations that are mixed into a single composition prior to administration. This can be performed at the bedside immediately prior to administration. In a second embodiment, the first and second mRNAs can be formulated together in a single LNP. In a third embodiment, the first and second mRNAs are packaged in a single vial.

[0275] Thus, the present invention encompasses an immunogenic combination comprising a first mRNA encoding a first Hepatitis B virus antigen and a second mRNA encoding a second Hepatitis B virus antigen, wherein the first and second mRNAs are in separate LNP formulations. The present invention also encompasses the resulting composition formed by mixing the separate LNP formulations.

[0276] The present invention further encompasses immunogenic compositions comprising a first mRNA encoding a first Hepatitis B virus antigen and a second mRNA encoding a second Hepatitis B virus antigen, in which the first and second mRNAs may be encapsulated by separate LNPs or the first and second mRNAs may be formulated in the same LNP.

[0277] The present invention encompasses methods for treating chronic hepatitis B infection (CHB) by administering to a human an mRNA encoding at least one hepatitis B virus antigen in combination with at least one recombinant hepatitis B polypeptide. The components (e.g., mRNA and recombinant hepatitis B polypeptide) can be administered sequentially in a heterologous prime-boost regimen. When a heterologous prime-boost regimen is used, preferably, the mRNA is administered as a priming dose and at least one recombinant hepatitis B polypeptide is administered as a booster dose. In this regimen, the method comprises administering the mRNA first and then the recombinant hepatitis B polypeptide. In another embodiment, at least one recombinant hepatitis B polypeptide is administered as a priming dose and the mRNA is administered as a booster dose. In this regimen, the method comprises administering the recombinant hepatitis B polypeptide and then the mRNA. In a further embodiment, at least one recombinant hepatitis B polypeptide is administered as a priming dose and the mRNA, together with an adjuvanted recombinant protein, is administered as a booster dose. In this regimen, the method involves administering a recombinant Hepatitis B polypeptide and then administering mRNA in conjunction with the recombinant protein.

[0278] In some embodiments, the at least one hepatitis B virus polypeptide is at least one recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc), or a combination thereof. The at least one recombinant hepatitis B polypeptide may be administered with or without an adjuvant. In a preferred embodiment, the mRNA is administered sequentially with an adjuvanted recombinant hepatitis B polypeptide, wherein the recombinant hepatitis B polypeptide comprises both hepatitis B small surface antigen (HBs) and hepatitis B virus core antigen (HBc). In this embodiment, the adjuvant is preferably AS01.

[0279] The above regimens may include multiple priming and / or booster administrations. In one embodiment, there is a single mRNA priming and multiple subsequent administrations of at least one recombinant hepatitis B polypeptide. For example, there may be two administrations of recombinant hepatitis B polypeptide. In another embodiment, there are multiple priming administrations of mRNA followed by multiple administrations of recombinant hepatitis B polypeptide. For example, two administrations of mRNA followed by two administrations of recombinant hepatitis B polypeptide. In a further embodiment, there is a single priming with at least one recombinant hepatitis B polypeptide followed by multiple administrations of mRNA. In yet a further embodiment, there are multiple priming administrations of recombinant hepatitis B polypeptide and multiple subsequent administrations of mRNA.

[0280] In other embodiments, the mRNA is administered simultaneously with at least one recombinant hepatitis B polypeptide. Additional doses of these components may be administered at subsequent times. In some embodiments, the mRNA is administered simultaneously with at least one recombinant hepatitis B polypeptide. The at least one recombinant hepatitis B polypeptide is at least one recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B core antigen (HBc), or a combination thereof. The recombinant HBc may be full-length or truncated, preferably truncated. The at least one recombinant hepatitis B polypeptide may be administered with or without an adjuvant. In one embodiment, the mRNA is administered simultaneously with an adjuvanted recombinant hepatitis B polypeptide, wherein the recombinant hepatitis B polypeptide comprises both hepatitis B small surface (HBs) and hepatitis B core (HBc) antigens. In this case, the adjuvant is preferably AS01.

[0281] In these above embodiments, the recombinant hepatitis B surface antigen (HBs) may have the amino acid sequence of SEQ ID NO: 1. In such above embodiments, the recombinant hepatitis B core antigen (HBc) may have the amino acid sequence of SEQ ID NO: 2 or 11. Preferably, the HBc has the amino acid sequence of SEQ ID NO: 2.

[0282] In all of these regimens, at least one recombinant hepatitis B polypeptide may be administered with a suitable adjuvant. A suitable adjuvant is one that can enhance the immune response in chronically ill and immunocompromised subjects. CHB patients are characterized by an inability to mount efficient innate and adaptive immune responses against the virus, making the development of an effective vaccine challenging. In these patients, one of the important functions of adjuvanted vaccine formulations is to target the cellular immune response toward a T helper 1 (Th1) profile, which is recognized as essential for the elimination of intracellular pathogens.

[0283] Examples of suitable adjuvants include inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic non-peptide adjuvants (e.g., saponins such as QS21, or squalene), oil-based adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immune stimulating complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A (MPL), such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL), or muramyl peptides), synthetic adjuvants (non-ionic block copolymers, muramyl peptide analogs, synthetic lipid A, etc.), synthetic polynucleotide adjuvants (polyarginine, polylysine, etc.), and immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides ("CpG"). In particular, the adjuvant can be an organic non-peptide adjuvant (e.g., a saponin such as QS21, squalene, etc.) and / or a bacterially derived adjuvant (e.g., monophosphoryl lipid A (MPL), such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL)).

[0284] One suitable adjuvant is monophosphoryl lipid A (MPL), particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Chemically, it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with four, five, or six acylated chains. It can be purified and prepared by the methods disclosed in GB 2122204B, which also discloses the preparation of diphosphoryl lipid A and its 3-O-deacylated variant. Other purified and synthetic lipopolysaccharides have also been described (U.S. Patent No. 6,005,099 and EP 0 729 473 B1; Hilgers, 1986; Hilgers, 1987; and EP 0 549 074 B1).

[0285] Saponins are also suitable adjuvants (Lacaille-Dubois, 1996). For example, the saponin Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina) and its fractions are described in U.S. Patent No. 5,057,540 and Kensil, 1996, as well as EP 0 362 279 B1. Purified fractions of Quil A are also known as immunostimulants, such as QS21 and QS17, the preparation of which is disclosed in U.S. Patent No. 5,057,540 and EP 0 362 279 B1. The use of QS21 is further described in Kensil, 1991. Combinations of QS21 with polysorbates or cyclodextrins are also known (WO 99 / 10008). Particulate adjuvant systems comprising fractions of QuilA such as QS21 and QS7 are described in WO 96 / 33739 and WO 96 / 11711.

[0286] Such adjuvants may be formulated with carriers such as liposomes, oil-in-water emulsions, and / or metal salts (including aluminum salts such as aluminum hydroxide). For example, 3D-MPL may be formulated with aluminum hydroxide (EP 0 689 454) or an oil-in-water emulsion (WO 95 / 17210). QS21 may be formulated with cholesterol-containing liposomes (WO 96 / 33739), an oil-in-water emulsion (WO 95 / 17210), or alum (WO 98 / 15287).

[0287] The disclosed compositions may utilize combinations of adjuvants, particularly combinations of monophosphoryl lipid A and saponin derivatives (see, e.g., WO 94 / 00153, WO 95 / 17210, WO 96 / 33739, WO 98 / 56414, WO 99 / 12565, WO 99 / 11241), more particularly the combination of QS21 and 3D-MPL disclosed in WO 94 / 00153, or compositions in which QS21 is quenched in cholesterol-containing liposomes (DQ) disclosed in WO 96 / 33739. A potent adjuvant formulation comprising QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion is described in WO 95 / 17210, and is another formulation that may be used in the disclosed compositions. Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, and an aluminum salt (e.g., as described in WO 00 / 23105). Further exemplary adjuvants include QS21 and / or MPL and / or CpG. QS21 can be quenched in cholesterol-containing liposomes, as disclosed in WO 96 / 33739.

[0288] Thus, a suitable adjuvant for use with at least one recombinant Hepatitis B polypeptide is AS01 (sometimes referred to as "AS-01"), a liposome-based adjuvant containing MPL and QS-21. The liposome vehicle for the MPL and QS-21 immunopotentiators is composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in phosphate-buffered saline. AS01 B-4 is a particularly preferred variant of the AS01 adjuvant, consisting of the immunopotentiators QS-21 (a triterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A), and the vehicle for these immunopotentiators, DOPC / cholesterol liposomes, and sorbitol in PBS solution. B-4 A single human dose (0.5 mL) of AS01 contains 50 μg of QS-21 and 50 μg of MPL. E-4 is AS01 B-4 This corresponds to a two-fold dilution of QS-21 and MPL, i.e., 25 μg of QS-21 and 25 μg of MPL per human dose.

[0289] In all of these regimens, in a preferred embodiment, the mRNA encodes a hepatitis B core (HBc) polypeptide, with or without an hIi fusion. The HBc encoded by the mRNA can be full-length or truncated, preferably full-length. In a preferred embodiment, the HBc encoded by the mRNA is full-length and fused to an hIi. In another preferred embodiment, the mRNA encodes full-length hepatitis B core (HBc) antigen, with or without an hIi fusion, and hepatitis B surface protein (HBsAg), with or without an hIi fusion. Preferably, the HBsAg is hepatitis B small surface protein (HBs), with or without an hIi fusion. In another preferred embodiment, the hepatitis B small surface protein (HBs) is fused to an hIi. The mRNA-encoded HBc and / or mRNA-encoded HBs are preferably fused to hIi.

[0290] The present invention also encompasses treating chronic hepatitis B infection (CHB) by administering to a human an adenoviral vector comprising a polynucleotide encoding a hepatitis B polypeptide in combination with mRNA encoding at least one hepatitis B virus antigen. These components may be administered in a heterologous prime-boost regimen. When using a prime-boost regimen, the adenoviral vector is preferably administered as a priming dose and the mRNA as a first booster dose. In such a regimen, multiple priming and / or booster doses may be administered. In one embodiment, a single priming dose of an adenoviral vector (e.g., a replication-deficient chimpanzee adenovirus (ChAd) vector) is administered, followed by multiple booster doses containing mRNA and / or recombinant HBV polypeptides.

[0291] In an alternative embodiment of a heterologous prime-boost regimen, mRNA is used as the first priming administration and an adenoviral vector (eg, a replication-deficient chimpanzee adenoviral (ChAd) vector) is used as the first boost administration.

[0292] The present invention also encompasses treating chronic hepatitis B infection (CHB) in humans by administering to them (i) an adenoviral vector comprising a polynucleotide encoding a hepatitis B polypeptide, and (ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B core antigen (HBc), and an adjuvant, in combination with mRNA encoding at least one hepatitis B viral antigen. The present invention may also include multiple sequential administrations of mRNA. In such embodiments, the mRNA used in the priming and booster administrations is preferably the same.

[0293] subject The present invention contemplates human subjects. Subjects treated using the methods of the present invention can be of any age.

[0294] The methods of the present invention are suitable for use in treating HBV, i.e., for administration to subjects infected with hepatitis B. The subject may be infected with hepatitis B alone, or with both hepatitis B and hepatitis D.

[0295] Formulation and Administration The mRNA can be administered by a variety of suitable routes of administration, including parenteral administration, such as intramuscular or subcutaneous administration. Preferably, the mRNA is administered intramuscularly.

[0296] The mRNA may be provided in liquid or dried (e.g., lyophilized) form. The preferred form will depend on factors such as the exact nature of the mRNA (e.g., whether the mRNA is susceptible to drying) and other components that may be present. Preferably, the mRNA is provided in liquid form.

[0297] Compositions containing mRNA intended to be combined with other compositions need not themselves have a physiologically acceptable pH or physiologically acceptable tonicity prior to administration. Formulations intended for administration should have a physiologically acceptable pH and a physiologically acceptable osmolality.

[0298] The pH of the liquid formulation is adjusted taking into account the components of the composition and the compatibility required for administration to a human subject.

[0299] For parenteral administration, the solution should have a physiologically acceptable osmolality to avoid excessive cell deformation or lysis. Physiologically acceptable osmolality generally means that the osmolality of the solution is approximately isotonic or slightly hypertonic. Osmolality can be measured according to techniques known in the art, such as using a commercially available osmometer (e.g., Advanced® Model 2020 manufactured by Advanced Instruments Inc., USA).

[0300] The liquid used for reconstitution is substantially aqueous, such as water for injection, phosphate buffered saline, etc. As noted above, the need for a buffer and / or tonicity adjuster depends on both the contents of the reconstitution container and the subsequent use of the contents after reconstitution. The buffer may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate, and TRIS. The buffer may be a phosphate buffer, such as Na / Na2PO4, Na / K2PO4, or K / K2PO4. The mRNA may be provided in a variety of containers, such as vials or pre-filled syringes.

[0301] In some embodiments, the mRNA is provided in a single dose form, while in other embodiments, the mRNA is provided in multiple doses, including 2 doses, 5 doses, or 10 doses.

[0302] When transferring liquids between containers, such as from a vial to a syringe, it is common to provide an "overage" to facilitate the transfer of the entire required volume. The required overage level varies depending on the situation, but excessive overage should be avoided to reduce waste, and insufficient overage can cause practical problems. The overage can be approximately 20-100 μL per dose, e.g., 30 μL or 50 μL.

[0303] Stabilizers may be added, which may be particularly important when multi-dose containers are provided, as doses of the final formulation may be administered to a subject over a period of time. The formulation is preferably sterile.

[0304] Approaches to establishing strong and durable immunity often involve repeated immunization, i.e., boosting the immune response with one or more booster doses. Such booster doses can be administered with the same immunogenic composition (homologous boost) or with different immunogenic compositions (heterologous boost). The present invention can be applied as either a priming immunization or a booster immunization, as part of a homologous or heterologous prime / boost regimen.

[0305] Therefore, the administration of mRNA can be part of a multiple-dose regimen.For example, mRNA can be administered as a priming dose in a multiple-dose regimen, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more doses, particularly 6 doses over 6 months.MRNA can be administered as a booster dose in a multiple-dose regimen, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more doses, for example 6 doses over 6 months.In a specific example, mRNA is administered as a 4-dose regimen.

[0306] The priming and boosting doses can be homologous or heterologous. Thus, mRNA can be administered as a priming dose and a boosting dose in a homologous multiple dose regimen, specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more doses, particularly a 6-dose regimen over a 6-month period. In one embodiment, the mRNA is administered as a 4-dose regimen. Alternatively, mRNA can be administered as a priming or boosting dose in a heterologous multiple dose regimen, specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more doses, particularly a 6-dose regimen over a 6-month period. The boosting dose can be different (e.g., mRNA with or without an adjuvant, such as AS01 or a squalene emulsion adjuvant, or an alternative antigen presentation, such as a protein or viral vector antigen). In one embodiment, the mRNA is administered as a four dose regimen.

[0307] The interval between doses can be 2 weeks to 6 months, for example, 3 weeks to 3 months. Preferably, two doses, one priming dose and one boosting dose, are administered simultaneously monthly for 6 months. Periodic booster doses over longer periods, such as every 2 to 10 years, can also be administered.

[0308] The present invention encompasses immunogenic combinations or compositions comprising a first mRNA encoding a first Hepatitis B virus antigen and a second mRNA encoding a second Hepatitis B virus antigen. In one embodiment, the immunogenic combination comprises a first mRNA encoding a first Hepatitis B virus antigen and a second mRNA encoding a second Hepatitis B virus antigen, wherein the first and second mRNAs are present in separate LNP formulations.

[0309] In another example, the immunogenic composition comprises a first mRNA encoding a first Hepatitis B virus antigen and a second mRNA encoding a second Hepatitis B virus antigen. The first and second mRNAs can be encapsulated in separate LNPs, or the first and second mRNAs can be co-formulated in the same LNP.

[0310] In some examples, the combination or composition includes the first and second mRNAs in equal amounts by weight. However, in other examples, the combination or composition may include the first and second mRNAs in unequal amounts by weight. In some embodiments, the first hepatitis B virus antigen is HBc and the second hepatitis B virus antigen is HBs. In such embodiments, the combination or composition includes more of the first mRNA than the second mRNA by weight.

[0311] In an example of a formulation containing both HBc mRNA and HBs mRNA, the composition contains equal amounts (by weight) of HBc mRNA and HBs mRNA. However, in one embodiment, the composition contains more mRNA encoding HBc ("HBc-mRNA") than mRNA encoding HBs ("HBs-mRNA"), by weight. In one embodiment, the composition contains 1.25 to 2 times, e.g., 1.5 to 2 times, the amount of HBc-mRNA compared to HBs-mRNA. In a specific embodiment, the composition contains 1.5 times as much HBc-mRNA as HBs-mRNA, by weight.

[0312] In another embodiment, - a first composition comprising mRNA encoding hepatitis B virus core antigen (HBc) encapsulated in lipid nanoparticles (LNPs) and mRNA encoding hepatitis B small surface protein (HBs) encapsulated in lipid nanoparticles (LNPs); and - a second composition comprising recombinant hepatitis B core protein (HBc), recombinant hepatitis B small surface protein (HBs) and AS01. There is an immunogenic combination comprising:

[0313] This combination may be used in a method of treating chronic hepatitis B (CHB) by sequentially or simultaneously administering the first and second compositions. The first composition may comprise mRNAs encoding HBc and HBs co-formulated in a single LNP. Alternatively, the mRNAs encoding HBc and HBs may be formulated in separate LNPs, and the LNPs may be co-packaged in a single vial.

[0314] definition Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0315] Throughout this specification, including the claims, where the context permits, the term "comprising" and variations thereof, such as "comprises," should be construed to include the recited element (e.g., an integer) or elements (e.g., integers), without necessarily excluding other elements (e.g., integers). Thus, a composition "comprising" X may consist solely of X, or it may include additional elements, such as X+Y.

[0316] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the term "substantially" may be omitted from the definition of the invention.

[0317] The term "about" or "approximately" in reference to a numerical value x is arbitrary and means, for example, x ±10% of the given numerical value, such as x ±5% of the given numerical value.

[0318] As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the content clearly dictates otherwise.

[0319] Unless otherwise specified, processes that involve mixing two or more components do not require a specific mixing order. Thus, the components may be mixed in any order. If there are three components, two components may be mixed together, and then that combination may be mixed with the third component, etc.

[0320] The terms "protein," "polypeptide," "antigen," and "peptide" are used interchangeably herein to refer to a peptide-linked chain of amino acids, regardless of length, co-translational, or post-translational modification. A fusion protein (or "chimeric protein") is a recombinant protein containing two or more peptide-linked proteins. Fusion proteins are produced by joining two or more genes that originally encoded separate proteins. Translation of the fusion genes produces a single fusion protein.

[0321] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymer composed of nucleotide monomers. Preferably, the polynucleotides of the present invention are recombinant. By recombinant, we mean that the polynucleotide has been produced by at least one of cloning, restriction enzyme digestion, ligation, or other method that results in a polynucleotide that differs from naturally occurring polynucleotides.

[0322] A heterologous nucleic acid sequence refers to a nucleic acid sequence that is not isolated from, derived from, or based on a naturally occurring nucleic acid sequence found in a host organism. "Naturally occurring" means a sequence that occurs in nature and is not synthetic or modified. A sequence is "derived" from an original sequence if it has been separated from the original sequence but has been suitably modified (e.g., deleted, substituted (mutated), inserted, or otherwise modified) so as not to interfere with the normal function of the original gene.

[0323] Preferably, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is one that has been separated from its native environment. For example, a naturally occurring polynucleotide is isolated if it has been separated from some or all of the materials with which it coexists in nature. A polynucleotide is considered to be isolated, for example, if it is cloned into a vector that is not part of its natural environment or if it is contained within a cDNA.

[0324] As used herein, "concurrent" administration refers to administration while the same immune response is ongoing. Preferably, both components are administered simultaneously (e.g., co-administration of a vector-containing composition and a protein-containing composition), although one component may be administered within minutes (e.g., during the same medical or physician visit) or within hours. Such administration is also referred to as co-administration. In some embodiments, co-administration may refer to administration of an adenoviral vector and a protein component. In other embodiments, co-administration refers to administration of an adenoviral vector and another viral vector, such as a poxvirus, e.g., MVA. In other embodiments, co-administration refers to administration of an adenoviral vector and an adjuvanted protein component.

[0325] "Sequential" administration refers to the administration of a first composition followed by a second composition at a significant time interval, e.g., when the immune response elicited by the first administration is not ongoing. Thus, sequential administration includes the first administration and subsequent administrations in the case of prime-boost. The period between two sequential administrations can be, for example, 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks. More specifically, it can be 4 weeks or 8 weeks.

[0326] The term "adjuvant" refers to an agent that auguments, stimulates, activates, potentiates, or modulates the immune response to an antigen of the composition at either the cellular or humoral level. For example, an immunogenic adjuvant stimulates the immune system's response to an antigen but has no immunological effect by itself. The immunogenic compositions disclosed herein may include an adjuvant as a separate component in the formulation, regardless of whether a vector included in the composition (or another component of the composition) encodes a "genetic adjuvant" such as a hIi. [Example]

[0327] Preclinical data were obtained comparing the immunogenicity of SAM-HBV with and without the human invariant chain in mice (Example 1, below). Additionally, preclinical data were obtained comparing the immunogenicity of vaccination regimens using one or more of MVA-HBV and ChAd155-hIi-HBV with vaccination regimens using at least one SAM-hIi-HBV construct (Example 2, below). Preclinical data were also obtained examining the immunogenicity of co-administered LNP-mRNA in HLA-A2 / DRB1 naive mice (Example 3, below).

[0328] These experiments demonstrate that LNP-formulated SAM-HBV containing a human invariant chain (hIi) fused to hepatitis B core antigen (HBc) elicits a higher frequency of HBc-specific CD8+ T cell responses (i.e., a higher percentage of HBc-specific CD8+ cells were found to respond) compared to constructs lacking the human invariant chain (Example 1, below). Furthermore, the use of SAM-hIi-HBV demonstrated the ability to enhance the frequency of HBc- and HBs-specific CD8+ T cell responses in a mouse model of chronic HBV infection (Example 2, below). Furthermore, these experiments demonstrate a favorable ratio for co-administration of LNP-formulated hIi-HBc and hIi-HBs mRNA (Example 3, below).

[0329] In all these experiments, HLA.A2 / DRB1 mice (human HLA-A2 and HLA-DRB1 transgenic mice) were used to evaluate the ability of HBV mRNA vaccines to induce HBc-specific CD8+ T cell responses. HBV-specific CD4+ T cell and antibody responses were evaluated in the same HLA.A2 / DRB1 mice.

[0330] ChAd155-hIi-HBV Drug Substance Manufacturing: To manufacture ChAd155-hIi-HBV drug substance, Procell-92.S cells are grown to a defined cell density. The cells are then infected with ChAd155-hIi-HBV master virus seed (MVS) at a defined multiplicity of infection. The resulting ChAd155-hIi-HBV virus is purified by a multistep process based on anion exchange chromatography.

[0331] Formulation and filling of ChAd155-hIi-HBV vaccine: The purified ChAd155-hIi-HBV drug substance is processed in the following steps. - Dilution of purified ChAd155-hIi-HBV drug substance in formulation buffer. - Sterile filtration. - Filling into final containers. The ChAd155-hIi-HBV vaccine is a liquid formulation filled in vials.

[0332] MVA-HBV Drug Substance Manufacturing: The MVA-HBV drug substance is manufactured by primary culture of chicken embryo fibroblasts (CEF) to a defined cell density and then infection with MVA-HBV master virus seed (MVS) at a defined multiplicity of infection. The resulting MVA-HBV virus is purified by a multistep process based on differential gradient centrifugation.

[0333] Preparation and filling of MVA-HBV vaccine: The purified MVA-HBV drug substance is processed in the following steps: - Dilution of purified MVA-HBV DS in preparation buffer. - Filling into final containers. The MVA-HBV vaccine is a liquid formulation packaged in vials with an extraction volume of 0.5 mL.

[0334] HBc drug substance manufacturing: The manufacturing process for HBc DS involves inoculating a pre-culture flask with recombinant E. coli working seeds, followed by a fermentation process and a multi-step purification process including harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0335] HBs drug substance manufacturing: The manufacturing process of HBs DS consists of inoculating a pre-culture flask with recombinant S. cerevisiae working seeds, followed by a fermentation process and a multi-step purification process including harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0336] HBc and HBs vaccine formulation and filling: The purified HBs and HBc DS are diluted with a formulation buffer containing sucrose as a cryoprotectant and poloxamer as a surfactant, filled into 4 mL clear glass vials, and lyophilized.

[0337] Dosage of AS01 adjuvant system: The AS01B-4 adjuvant system consists of the immunopotentiators QS-21 (a triterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A), along with liposomes and sorbitol as vehicles for these immunopotentiators. Specifically, one clinical dose container (0.5 mL) of AS01B-4 contains 50 μg of QS-21 and 50 μg of MPL. One-tenth the human dose, or 50 μl, is the volume injected into mice (equivalent to 5 μg of QS-21 and MPL).

[0338] Preparation of mRNA constructs: The plasmid was linearized with BspQI restriction enzyme to prepare a DNA template for in vitro transcription. mRNA was produced by in vitro transcription using the capping analog, TRILINK CLEANCAP A / G, and 100% N1-methylpseudouridine, followed by DNase I and phosphatase treatment and silica column purification. The newly synthesized mRNA was verified by capillary gel electrophoresis and denaturing agarose gel.

[0339] Preparation of SAM-HBV and SAM-hIi-HBV constructs: The HBV and hIi-HBV sequences were codon-optimized for human protein expression and synthesized by GENEWIZ and cloned into the SAM plasmid. RNA was synthesized by in vitro transcription. Briefly, the DNA plasmid encoding the SAM replicon was linearized at the 3' end of the poly(A) tail by restriction enzyme digestion with BspQI and purified by phenol-chloroform extraction. The linearized DNA was used as a template for in vitro transcription using T7 RNA polymerase. After in vitro transcription, the RNA was capped using a vaccinia capping kit, purified by LiCl precipitation, and resuspended in nuclease-free water.

[0340] The preparation of LNPs containing SAMs followed established methods for LNP preparation using microfluidic mixing. Lipids (cationic lipids, zwitterionic lipids, cholesterol, and PEG-lipid conjugates) were dissolved in an ethanol solution, and SAMs were dissolved in an aqueous buffer solution. The ethanol and aqueous solutions were rapidly mixed using a microfluidic mixing chamber. SAM-entrapped lipid nanoparticles formed spontaneously through the nucleation of supersaturated lipids in the mixture. The lipids condensed and precipitated, trapping the SAMs and forming lipid nanoparticles. After a short maturation period, the SAM-LNP buffer was exchanged with a storage buffer. The SAM-LNP solution was characterized for size, lipid content, RNA capture, and in vitro efficacy.

[0341] The SAM vector VEE TC-83 was used as a background construct for cloning in the examples. The background empty construct has the nucleic acid sequence of SEQ ID NO: 16.

[0342] The design of the HBV-SAM construct in Figure 14 involves cloning the HBV antigen-encoding sequence under the subgenomic promoter of the SAM vector. The SAM HBV construct was modified, including codon optimization of the antigen-encoding sequence.

[0343] The SAM constructs were evaluated for antigenicity and antigenicity, and further tested for immunogenicity and efficacy using in vivo models.

[0344] SAM constructs having the sequences of SEQ ID NOs: 17 and 19 were designed and obtained for further characterization and testing in the following examples.

[0345] Characterization of SAM-HBV and SAM-hIi-HBV constructs: RNA pattern uniformity assessment To examine the uniformity of the RNA patterns, RNA samples were analyzed in a 1% agarose gel. RNA samples were prepared as follows: 100–250 ng of RNA was mixed with 3 μL of loading buffer (50 mM EDTA pH 8, 30% w / v sucrose, 0.05% bromophenol blue) and water to a final volume of 10 μL. Samples were denatured at 50°C for 20 minutes. Agarose gels were run at 130 V for 45 minutes using NorthernMax-Gly Gel Running Buffer (Invitrogen™). No significant RNA degradation was observed, and similar patterns were obtained between both constructs.

[0346] Protein expression assessment by Western blot The ability of cells to express antigens derived from various HBV SAM constructs was assessed according to the following method.

[0347] On day 0, baby hamster kidney (BHK) cells were plated in a T225 flask at 1 × 10 7 Cells were seeded in growth medium (DMEM high glucose (Gibco™), 1% L-glutamine, 1% Pen-Strep (Corning®), 5% FBS (Gibco™)). For trypsinization, the medium was removed and the cells were washed with 5 mL of PBS. The PBS wash was removed, and 5 mL of pre-warmed trypsin was added and thoroughly coated over the entire plate. Trypsin was removed, and the plate was incubated at 37°C for 1-2 minutes. The cells were then resuspended in 10 mL of growth medium. The cells were counted and seeded into new flasks at the required concentration. The cells were incubated at 37°C, 5% CO2 for approximately 20 hours.

[0348] On day 1, 2 mL of growth medium (DMEM high glucose, 1% L-glutamine, 1% Pen-Strep, 1% FBS) was added to each well of a 6-well plate (one well per electroporation). The plate was incubated in a 37°C incubator. The electroporator was set to output 120 V, 25 ms pulses, 0.0 pulse intervals, and 1 pulse per 2 mm cuvette. The cuvettes were labeled and stored on ice. Cells in the proliferating phase were harvested in BHK growth medium and counted using a cell counter. The cells were trypsinized using the same trypsinization protocol described above. The cells were then centrifuged at 462 x g for 3 minutes. The medium was aspirated, and the cells were washed once with 20 mL of cold Opti-MEM medium (Gibco™). The cells were again centrifuged at 462 x g for 5 minutes. Aspirate the medium and resuspend the cells in Opti-MEM medium at 1 x 10 per electroporation. 6 The volume was 0.25 mL per cell. Standard solutions and negative controls were also prepared.

[0349] For each sample, 2 μg of RNA was mixed with 250 μL of cells, and the mixture was gently pipetted 4–5 times. The cell and RNA mixture was transferred to a 2 mm cuvette and electroporated using the parameters described above. The cells were allowed to stand at room temperature for 10 minutes. Cells were removed from one cuvette and added to one well of a pre-warmed 6-well plate. The plate was tilted back and forth and then at a 45° angle to distribute the cells evenly. On day 2 (17 hours after electroporation), cell culture supernatants were collected and analyzed by Western blotting at different concentrations. The cell monolayer was detached and resuspended in 1 mL of 20 mM HEPES, 150 mM NaCl, 5% glycerol (pH 7.6) buffer supplemented with cOmplete™ protease inhibitor cocktail (Roche, catalog no. 11697498001), followed by sonication to lyse the cells. After cell lysis, the intracellular fractions were analyzed by Western blotting. Mouse anti-HBc monoclonal antibody and rabbit anti-HBs polyclonal serum (prepared in-house) were used as primary antibodies.

[0350] In vitro potency of SAMs after LNP preparation After LNP preparation, in vitro efficacy testing was also performed.

[0351] Cellular Immune Response - Intracellular Cytokine Staining (ICS): Fresh pools of peripheral blood leukocytes (PBLs), spleen cells, or liver-infiltrating lymphocytes collected at different time points were stimulated ex vivo for 6 hours with pools of 15 bases containing 11 amino acid overlaps covering the HBc or HBs sequence. HBc- and HBs-specific cellular responses were assessed by ICS, which measures the amount of CD4+ or CD8+ T cells expressing IFN-γ, IL-2, and / or tumor necrosis factor (TNF)-α. Technical acceptance criteria for considering ICS results included a minimum number of acquired CD8+ or CD4+ T cells exceeding 3,000 events.

[0352] Humoral immune response - enzyme-linked immunosorbent assay (ELISA): HBc- and HBs-specific antibody responses were measured by ELISA using sera collected from immunized mice at different time points. Briefly, purified hepatitis B core antigen (HBc) or purified hepatitis B surface antigen (HBs) was coated onto a 96-well ELISA plate. Vaccinated mouse sera were serially diluted and incubated. Serial dilutions of standard and control substances were used to calculate the standard titers of anti-HBc or anti-HBs antibodies in the test sera, confirming the validity of the test. After each incubation step, the plate was washed with 0.1% PBS and Tween 20 buffer. Horseradish peroxidase-infused goat anti-mouse IgG (H+L) antibody was then added, and the antibody complex was detected by incubation with tetramethylbenzidine liquid substrate (TMB). Optical density (OD) was recorded between 450 and 620 nm. Anti-HBc or HBs antibody titers in each mouse serum were determined by a regression model using the ELISA standard curve. Geometric mean titers (GMTs) were then calculated for each group of mice. For each time point and each antigen (HBc, HBs), an analysis of variance (ANOVA) model was applied to the log10 titers using a heterogeneous variance model (identical variances between groups are not assumed), with group, test, and interaction as fixed effects. This model was used to estimate the geometric mean (and its 95% confidence interval) and the geometric mean ratio and its 95% confidence interval. Because no predefined criteria were set, this analysis was descriptive, and the 95% confidence intervals for the ratios between groups were calculated without adjusting for multiplicity.

[0353] ALT / AST measurement: ALT and AST levels in mouse serum were quantified using the following commercially available kits. Alanine Aminotransferase Activity Assay Kit (Sigma Aldrich, Catalog Number: MAK052) Aspartate Aminotransferase Activity Assay Kit (Sigma Aldrich, Catalog Number: MAK055)

[0354] Serum HBs antigen quantification Circulating HBs antigen in mouse serum was quantified using the Monolisa Anti-HBs PLUS kit (BIO-RAD, catalog number: 72566) and an international standard (Abbott Diagnostics).

[0355] Example 1: SAM-HBV with and without human invariant chain In this experiment, male and female HLA.A2 / DR1 naive mice were injected intramuscularly on days 0 and 28. Details of the compositions administered to each group on days 0 and 28 are listed in Table 1 below. [Table 1]

[0356] Fourteen mice (N = 14) were used in each group, except for the control group 7. At 14 days after the first inoculation (14 dpI), two mice from each group were sacrificed, and spleen and serum samples were collected to measure T cell responses at this time point. Serum samples were also collected from all mice. At 12 and 13 days after the second inoculation (12 / 13 dpII), all remaining mice were sacrificed, and spleen, liver, and serum samples were collected.

[0357] In groups 1 to 6, all mice were primed with ChAd155-hIi-HBV and boosted with SAM-HBV (±hIi). ChAd155-hIi-HBV was administered at a dose of 10 per mouse. 8 All patients were administered the same dose of HBV vp, except for SAM-HBV (± hIi) at three different doses: 2.5 μg, 1 μg, and 0.1 μg. The specific doses used in each group are listed in Table 1 above.

[0358] The HBc- and HBs-specific CD4+ and CD8+ T cell responses, as well as the HBc- and HBs-specific antibody responses obtained in Example 1, are shown in Figures 1, 2, and 3. Figure 1 shows the CD4+ response, Figure 2 shows the CD8+ response, and Figure 3 shows the antibody response. In all of these figures, panel "A" shows the hepatitis B core antigen response, and panel "B" shows the hepatitis B surface antigen response.

[0359] As shown in Figures 2A and 2B, the SAM construct containing the invariant chain (SAM-hIi-HBV) induced a significantly higher frequency of HBc-specific CD8+ T cell responses to HBc compared with the construct lacking the invariant chain (SAM-HBV) (geometric mean ratio, GMR = 4.1, 95% CI [1.96-8.40]). First, the geometric means of the HBc-specific CD8+ T cell responses of mice immunized with SAM-hIi-HBV and mice immunized with SAM-HBV were calculated. Next, the ratio of these two geometric means was calculated. In this case, it was observed that SAM-hIi-HBV induced a four-fold higher HBc-specific CD8+ T cell response.

[0360] Efficient control of HBV infection is associated with the induction and persistence of CD4+ and CD8+ T cells that specifically target HBV core and surface antigens, which play a key role in the control and cure of HBV infection.

[0361] Several studies comparing HBV antigen-specific T cells in various subgroups of HBV-infected patients (post-acute infection, convalescent, active chronic infection, and inactive carriers) have emphasized the need to induce strong polyspecific T cell responses to HBV antigens, particularly HBc antigen, to promote clearance of HBV infection. In line with this, comparison of T cells from patients with chronic HBV infection who are on the path to recovery from chronic HBV infection and those who are not on the path to recovery has revealed a higher prevalence of HBc antigen-specific CD4+ and CD8+ T cells in patients on the path to recovery [Boni, 2012; Li, 2011; Liang, 2011].

[0362] Furthermore, the role of functional CD8+ T cells appears to be crucial. In chimpanzees, depletion of CD8+ T cells during acute HBV infection results in persistent viremia [Thimme, 2003]. In humans, clearance of HBV in acute hepatitis B is associated with strong, polyclonal, and multispecific CD8+ T cell responses against the viral nucleocapsid, envelope, and polymerase proteins, which persist for decades after clinical recovery. In contrast, patients with chronic hepatitis B (CHB) typically do not mount strong CD8+ T cell responses to the virus. CHB patients who experience spontaneous or interferon-induced remission exhibit CD8+ T cell responses to HBV that are comparable in strength and specificity to those of patients who have recovered from acute hepatitis [Rehermann, 1996].

[0363] SAM constructs containing the invariant chain (SAM-hIi-HBV) were chosen for use in Example 2 because they were shown to induce stronger CD8+ T cell responses to HBcAg and HBsAg.

[0364] Example 2: Evaluation of replacement of MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV with SAM-hIi-HBV in HLA.A2 / DR1 transgenic mice Due to limitations on the number of animals per experiment, two independent experiments were conducted, both of which included animals from all groups shown in Table 2.

[0365] In this study, we used male and female HLA.A2 / DR1 transgenic mice. The AAV2 / 8-HBV-transduced HLA.A2 / DR1 mouse model recapitulates the virological and immunological characteristics of chronic HBV infection. It was selected to evaluate the immunogenicity of different vaccine regimens, the influence of liver-infiltrating HBc-specific CD8+ T cells, the potential for targeting HBcAg-expressing hepatocytes, and the potential for vaccine-associated liver inflammation as measured by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities.

[0366] Therefore, in these experiments, male and female HLA.A2 / DR1 mice (groups 1–6 and group 8) were injected with 10 10 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 40 46 47 48 49 50 51 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 99 100 101 10 The viral genome (vg) was inoculated intravenously.

[0367] Before immunization, HLA.A2 / DR1 mice were randomly assigned to seven different groups (groups 1–6 and group 8) based on the circulating HBs antigen levels detected in serum on days 21 and 22, age, and gender distribution.

[0368] Mice in group 7 were not transduced with the AAV2 / 8-HBV viral vector but were immunized intramuscularly (IM) with the co-administered vaccine regimen, and this group served as a positive control for immunological testing.

[0369] HLA.A2 / DR1-transduced mice were injected intramuscularly (gastrocnemius) with various formulations containing HBcAg and HBsAg (listed in Table 2) on days 31 or 33 (first immunization), 59 or 61 (second immunization), 73 or 75 (third immunization), and 86 or 88 (fourth immunization). The results of the two separate experiments were combined for graphical presentation and statistical analysis of the results.

[0370] In all cases, the same dose of each composition was used. - ChAd155-hIi-HBV was administered at 10 8 was administered at a dose of vp. - MVA-HBV-inoculated mice received 10 7 pfu doses were administered. - SAM-hIi-HBV was administered at a dose of 1 μg per mouse, and - The group receiving adjuvanted proteins received AS01 containing 4 μg of HBc, 1 μg of HBs, and 5 μg of MPL and 5 μg of QS21 per mouse.

[0371] The objective of this study was to evaluate whether SAM-hIi-HBV could replace MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV in sequential or simultaneous vaccine regimens by inducing at least the same level of HBc-specific CD8+ T cell responses compared to vaccine regimens using MVA-HBV. [Table 2]

[0372] Mice in groups 1-6 and 8 were transduced with AAV2 / 8-HBV 31 or 33 days before the first injection. As mentioned above, for practical reasons in experimental conduct, each group was divided into two groups and two separate experiments were performed. All results shown are a combination of results from both experiments.

[0373] At 13 and 14 days after the second injection (13 / 14 dpII), 15 mice each from groups 1 to 6 (from each experiment), 6 and 4 mice from groups 7 and 8 in experiment number 20200719, and 4 and 5 mice from groups 7 and 8 in experiment number 20200720 were sacrificed to collect spleen samples and serum samples from all mice. At 22 days after the fourth injection (22 dPIV), all remaining animals were sacrificed, and spleen, liver, and serum samples were collected.

[0374] The CD8+ T cell and antibody responses generated in the various groups in Example 2 are shown in Figures 4A and 4B. The results in Figure 4 indicate that 14 days after the second immunization, priming with ChAd155-hIi-HBV and boosting with SAM-hIi-HBV induced a 7.36-fold higher HBc-specific CD8+ T cell response (GMR = 7.36, 90% CI [3.96-13.70]) compared with priming with ChAd155-hIi-HBV and boosting with MVA-HBV. Similarly, priming with SAM-hIi-HBV and boosting with SAM-hIi-HBV induced a 9-fold higher HBc-specific CD8+ T cell response (GMR = 9.07, 90% CI [4.87-16.87]) compared with priming with ChAd155-hIi-HBV and boosting with MVA-HBV.

[0375] Similar CD8+ T cell response results are shown in Figures 5A and 5B. In this experiment, priming with ChAd155-hIi-HBV and boosting with SAM-hIi-HBV induced a 3.6-fold higher HBs-specific CD8+ T cell response (GMR = 3.64, 90% CI [2.34-5.67]) compared with priming with ChAd155-hIi-HBV and boosting with MVA-HBV 14 days after the second immunization. Priming with SAM-hIi-HBV and boosting with SAM-hIi-HBV also induced a 7.78-fold higher HBs-specific CD8+ T cell response (GMR = 7.78, 90% CI [5-12.12]) compared with priming with ChAd155-hIi-HBV and boosting with MVA-HBV.

[0376] Interestingly, replacing MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV with SAM-hIi-HBV vaccine induced greater numbers of polyfunctional HBV-specific CD8+ T cells, as indicated by the cytokine coexpression profile (Figures 13A and 13B). The majority of HBV-specific CD8+ T cells primarily expressed IFN-γ and TNFα, and this population was further expanded by allogeneic prime-boost with SAM-hIi-HBV.

[0377] Figures 6 and 7 show CD4+ responses measured in the spleen. 14 days after the second immunization, all vaccine regimens induced very low or undetectable HBc-specific CD4+ T cells (<0.1%) in AAV2 / 8-HBV-transduced HLA-A2 / DR1 mice. Only HBc-HBs / AS01 alone or in combination with both vectors induced slightly higher HBc-specific CD4+ T cells.

[0378] Regarding anti-HBs-specific CD4+ T cell responses 14 days after the second immunization, priming with ChAd155-hIi-HBV and boosting with SAM-hIi-HBV induced 7.12-fold higher HBs-specific CD4+ T cell responses (geometric mean ratio (GMR) = 7.12, 90% CI [4.61-11]) than priming with ChAd155-hIi-HBV and boosting with MVA-HBV. Although replacing both ChAd and MVA with SAM-hIi-HBV did not induce higher responses, priming with SAM-hIi-HBV and boosting with SAM-hIi-HBV induced 2.56-fold higher HBs-specific CD4+ T cell responses (GMR = 2.56, 90% CI [1.66-3.95]) compared with priming with ChAd155-hIi-HBV and boosting with MVA-HBV. As previously observed for HBc-specific CD4+ T cell responses, strong HBs-specific CD4+ T cell responses were induced only when HBc-HBs / AS01 was administered alone or in combination with both vectors.

[0379] Regarding humoral immune responses, replacement of MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV with SAM-hIi-HBV did not affect the levels of anti-HBc and anti-HBs antibody responses.

[0380] Interestingly, immunization with the HBV vaccine resulted in a ±1.5-fold decrease in serum HBsAg in all groups, regardless of the regimen used (sequential or simultaneous administration), with no significant differences between groups (Figures 10 and 11). Finally, as liver-related inflammation parameters, AST and ALT activities were measured in mouse serum after the second and fourth immunizations. In each group, ALT levels remained stable throughout the study period, and replacement of MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV with SAM-hIi-HBV had no significant effect. Slightly elevated AST levels were measured in all groups, but no significant differences were observed between groups (Figure 12).

[0381] Summary: Substitution of SAM-hIi-HBV for MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV significantly increased HBc- and HBs-specific CD8+ T-cell responses (SAM / SAM > ChAd / SAM > ChAd / MVA), had a small positive effect on HBs-specific CD4+ T-cell responses, and had no significant effect on the levels of anti-HBc and anti-HBs IgG antibody responses.

[0382] Regarding circulating HBsAg, a trend toward a ±1.5-fold decrease in circulating HBsAg was observed in all groups, with no intergroup differences. Furthermore, we assessed the possibility of vaccine-associated liver inflammation by measuring serum activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), but no elevation of liver enzymes was detected in the vaccinated group compared to the unvaccinated group.

[0383] [Example 3: Immunogenicity evaluation of LNP-mRNA co-administration in HLA-A2 / DRB1 naive mice] Details of this experiment are shown in Table 3. The LNP-mRNA construct contains the UTR4 backbone and RV39 LNP. The formulation further contains 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), polyethylene glycol-conjugated (PEG-conjugated) lipid, and cholesterol.

[0384] In this experiment, 8- to 12-week-old HLA-A2 / DRB1 naive mice (54 males and 47 females) were used. The administration schedule consisted of intramuscular immunization on days 0, 21, 42, and 63. The doses used were as follows: - mRNA: as listed in the table - ChAd155-hIi-HBV: 10 per mouse 8 vp - MVA-HBV: 10 per mouse 7 pfu - HBc-HBs: 4-1 μg / AS01, i.e. 4 μg of HBc and 1 μg of HBs (in the case of adjuvanted proteins administered simultaneously with mRNA in group 7 of Table 3).

[0385] The ChAd155-hIi-HBV vector encodes the hIi-HBc-2A-HBs amino acid sequence of SEQ ID NO:15, and the MVA-HBV vector encodes the HBc-2A-HBs amino acid sequence of SEQ ID NO:5.

[0386] The primary objective of this study was to investigate the immune interference caused by the coadministration of hIi-HBc mRNA and hIi-HBs mRNA (i.e., "hIi-HBc + hIi-HBs"). The coadministration of three mRNAs (including hIi-HBc and hIi-HBs) was compared with a formulation containing only a single mRNA type. In this previous study, it was observed that coadministration adversely affected HBc- and HBs-specific CD8+ T cell responses (see Figure 17). Notably, coadministration of these mRNAs resulted in a 6.7-fold decrease in HBc-specific responses and a 2-fold decrease in HBs-specific responses.

[0387] The first change was simply to co-administer HBc mRNA and HBs mRNA (i.e., no third mRNA). Additionally, the amount of HBs mRNA was reduced relative to HBc mRNA, as this had the greatest effect on the HBc-specific CD8+ response. This resulted in different HBc mRNA to HBs mRNA ratios observed in groups 2–5 in Table 3.

[0388] - Mice in group 2 were administered a composition containing 7 μg of HBc mRNA and 7 μg of HBs mRNA. - In group 3, HBs mRNA was diluted 1.5 times and 4.6 μg (rounded down to one decimal place) of HBs mRNA was administered to mice. Therefore, the ratio of mRNA used in this composition was 1.5 HBc mRNA:1 HBs mRNA. - In group 4, 4.6 μg of the HBs mRNA composition was diluted 1.5 times and the composition containing 3.1 μg (rounded down to one decimal place) was administered to mice. Therefore, the ratio of mRNA used in this composition was 2.3 HBc mRNA:1 HBs mRNA. - In group 5, 3.1 μg of the HBs mRNA composition was further diluted 1.5 times, and a composition containing 2.0 μg (rounded down to one decimal place) was administered to mice. Therefore, the ratio of mRNA used in this composition was 3.5 HBc mRNA:1 HBs mRNA.

[0389] To select the co-administration (from groups 2, 3, 4, and 5) containing the LNP-mRNA ratio with the lowest level of immune interference compared to the administration of LNP-mRNA alone (i.e., groups 8, 9, 10, 11, and 12). - Potential adverse effects on HBc-specific CD8+ T cell responses were assessed. - Potential adverse effects on HBs-specific CD8+ T cell responses were assessed.

[0390] To assess immune interference, HBc- and HBs-specific CD8+ T cell responses in the spleen were measured on days 75 and 77 (see Figures 18, 19, 20).

[0391] The success criteria were defined as follows: "Evaluate the non-inferiority of simultaneous administration of different LNP-mRNA ratios to a single LNP-mRNA formulation. Non-inferiority is demonstrated if the lower limit of the 90% confidence interval of the geometric mean ratio exceeds 0.33. If non-inferiority is demonstrated statistically, biological relevance will be evaluated by scientists."

[0392] A sample size of 8 mice with an SD of less than 0.36 would indicate at least 80% 3-fold non-inferiority at an α level of 5%. Eight mice were assigned to each group and five to the NaCl group, for a total of 101 mice.

[0393] The results showed that reducing the amount of hIi-HBs mRNA relative to hIi-HBc mRNA improved the HBc-specific CD8+ response compared to the control group (mice immunized with 7 μg of hIi-HBc mRNA alone) (see Figure 19). The highest HBc-specific CD8+ response was observed when 4.6 μg of hIi-HBs mRNA was used.

[0394] Interestingly, a composition containing 7 μg of hIi-HBc and 4.6 μg of hIi-HBs was found to elicit a similar level of HBs-specific CD8+ T cell response as a composition containing 7 μg of hIi-HBc and 7 μg of hIi-HBs, and this response was similar to that detected in mice immunized with 4.6 μg of HBs hIi-HBs mRNA alone. Although the 7 μg-3.1 μg and 7 μg-2 μg compositions induced higher HBc-specific T cell responses than the 7 μg-7 μg composition, the HBs-specific CD8+ T cell responses were significantly reduced with these two formulations.

[0395] Therefore, co-administration of 7 μg hIi-HBc-mRNA and 4.6 μg hIi-HBs-mRNA (i.e., a ratio of 1.5 hIi-HBc-mRNA:1 hIi-HBs-mRNA) was found to be a preferred composition, as the immune response induced by the co-administered mRNAs was similar to that induced by each mRNA administered alone.

[0396] The results of this experiment also showed that: - there is no adverse effect of coadministration on HBc-specific CD4+ T cell responses; and - No adverse effects of coadministration (hIi-HBc + hIi-HBs) on HBc-specific IgG responses were observed.

[0397] This experiment was also designed to: (i) To directly compare prime-boost immunization with co-administration of mRNA (hIi-HBc + hIi-HBs) with co-administration of ChAd / MVA (compare groups 1 with 2, 3, 4, and 5 in Table 3). (ii) To evaluate the immunogenicity of four doses of simultaneous administration of mRNA (hIi-HBc + hIi-HBs) versus two doses (compare groups 4 and 6 in Table 3 ). (iii) To investigate the immune interference between co-administered mRNA (hIi-HBc + hIi-HBs) and AS01-adjuvanted protein (compare groups 6 and 7 in Table 3). In group 7, the "hIi-HBc + hIi-HBs" and "HBc-HBs / AS01" compositions were administered simultaneously, but in different injections into different limbs of the mice.

[0398] These secondary endpoints were HBc- and HBs-specific CD4+ and CD8+ T cell responses in the spleen measured by ICS on days 75 and 77, and HBc- and HBs-specific antibody responses measured by ELISA on days 75 and 77 (see Figures 18, 20, and 21).

[0399] The results are as follows: (i) Comparison of two doses of mRNA and ChAd / MVA prime-boost: Two doses of mRNA were found to induce higher CD8+ T cell responses than ChAd / MVA prime-boost, particularly a three-fold increase in CD8+ T cell responses to HBc and a two-fold increase in CD8+ T cell responses to HBs (see Figure 18). Two doses of mRNA were also found to induce an 8-fold higher HBc-specific CD4+ T cell response. Neither mRNA nor ChAd / MVA induced an HBs-specific CD4+ T cell response (see Figure 19). Two doses of mRNA were also found to induce an 8-fold higher HBc-specific IgG response (see Figure 21). Neither mRNA nor ChAd / MVA induced an HBs-specific IgG response.

[0400] (ii) Comparison of four doses of mRNA vs. two doses: - Two booster immunizations with co-administration of mRNA resulted in CD8+ T cell responses, specifically a two-fold increase in CD8+ T cell responses to HBc and a three-fold increase in CD8+ T cell responses to HBs (see Figure 18). Four doses of mRNA also induced a 3.5-fold higher HBc-specific CD4+ T cell response. HBs-specific CD4+ T cell responses were not induced after either four or two doses (see Figure 19). Four doses of mRNA also induced a two-fold higher HBc-specific IgG response (see Figure 21). HBs-specific IgG responses were not induced after either four or two doses.

[0401] (iii) Co-administration of mRNA and adjuvanted protein: - Co-administration of mRNA (hIi-HBc + hIi-HBs) co-administered with adjuvanted protein was found to negatively affect HBc-specific CD8+ T cell responses, but this co-administration regimen did not negatively affect HBs-specific CD8+ T cell responses (see Figure 18). This co-administration regimen was found to have a negative impact on HBc-specific CD4+ T cell responses, however, the addition of adjuvanted proteins to the formulation also induced HBs-specific CD4+ T cell responses (see Figure 19). - This co-administration regimen was also confirmed to have a positive impact on HBc-specific IgG responses (see Figure 21). Furthermore, the inclusion of adjuvanted proteins in the formulation induced HBs-specific IgG responses. [Table 3]

[0402] Embodiments of the present invention The embodiments of the present invention are described in the following three groups of embodiments: Features of the three groups can be combined to form individual embodiments, if desired.

[0403] Group 1 of the embodiments describes the following: Embodiment A. A composition for the treatment of chronic hepatitis B infection comprising mRNA encoding at least the hepatitis B virus core antigen (HBc), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

[0404] Embodiment B. The composition of embodiment A, wherein the Hepatitis B virus core antigen (HBc) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11.

[0405] Embodiment C. The composition of any preceding embodiment, wherein the hepatitis B virus core antigen (HBc) is fused to a human invariant chain (hIi).

[0406] Embodiment D. The composition of any preceding embodiment, wherein the composition further comprises mRNA encoding hepatitis B small surface protein (HBs).

[0407] Embodiment E. The composition of embodiment D, wherein the Hepatitis B small surface protein (HBs) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0408] Embodiment F. The composition of any preceding embodiment, wherein the hepatitis B small surface antigen (HBs) is fused to a human invariant chain (hIi).

[0409] Embodiment G. A composition for the treatment of chronic hepatitis B infection, comprising mRNA encoding at least the hepatitis B virus surface protein (HBsAg), wherein the mRNA is encapsulated in a lipid nanoparticle (LNP).

[0410] Embodiment H. The composition of embodiment G, wherein the HBsAg is hepatitis B small surface protein (HBs).

[0411] Embodiment I. The composition of embodiment H, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0412] Embodiment J. The composition of any of embodiments GI, wherein the HBsAg is fused to a human invariant chain (hIi).

[0413] Embodiment K. The composition of any of the previous embodiments, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:12.

[0414] Embodiment L. The composition of embodiment K, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:12.

[0415] Embodiment M. The composition of any preceding embodiment administered sequentially or simultaneously with one or more recombinant Hepatitis B polypeptides.

[0416] Embodiment N. The composition of embodiment M, wherein the recombinant Hepatitis B polypeptide comprises recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

[0417] Embodiment O. The composition of embodiment M or N, wherein the HBc comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:2.

[0418] Embodiment P. The composition of embodiment M or N, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0419] Embodiment Q. The composition of any of Embodiments MP, wherein the recombinant Hepatitis B polypeptide is administered with an adjuvant.

[0420] Embodiment R. The composition of embodiment Q, wherein the adjuvant is AS01.

[0421] Embodiment S. A method of treating chronic hepatitis B infection comprising administering to a human a prime-boost regimen, wherein mRNA encoding at least one hepatitis B viral antigen is administered as a priming dose and one or more recombinant hepatitis B polypeptides are administered as booster doses.

[0422] Embodiment T. The method of embodiment S, wherein the mRNA encodes at least one hepatitis B viral antigen selected from the group consisting of hepatitis B core antigen (HBc) and hepatitis B surface antigen (HBsAg).

[0423] Embodiment U. The method of embodiment T, wherein said hepatitis B surface antigen (HBsAg) is hepatitis B small surface protein (HBs).

[0424] Embodiment V. The method of any of embodiments SU, wherein the Hepatitis B virus antigen is fused to the hIi.

[0425] Embodiment W. The method of any one of embodiments S through U, wherein the recombinant Hepatitis B polypeptide comprises recombinant Hepatitis B virus core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

[0426] Embodiment X. The method of any of embodiments S-W, wherein the recombinant Hepatitis B polypeptide is administered with an adjuvant.

[0427] Embodiment Y. The method of embodiment X wherein the adjuvant is AS01.

[0428] Group 2 of embodiments describes the following: Embodiment i. A composition for treating chronic hepatitis B infection, comprising a first mRNA encoding at least hepatitis B virus core antigen (HBc), wherein the first mRNA is encapsulated in a lipid nanoparticle (LNP).

[0429] Embodiment ii. The composition of embodiment i, wherein the Hepatitis B virus core antigen (HBc) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11.

[0430] Embodiment iii. The composition of any of the preceding embodiments, wherein the Hepatitis B virus core antigen (HBc) is fused to a human invariant chain (hIi).

[0431] Embodiment iv. The composition of any preceding embodiment, wherein the composition further comprises a second mRNA encoding the Hepatitis B small surface protein (HBs).

[0432] Embodiment v. The composition of embodiment iv, wherein a first mRNA encoding HBc ("HBc mRNA") is encapsulated in a different LNP than a second mRNA encoding HBs ("HBs mRNA").

[0433] Embodiment vi. The composition of embodiment iv, wherein a first mRNA encoding HBc ("HBc mRNA") is encapsulated in the same LNP as a second mRNA encoding HBs ("HBs mRNA").

[0434] Embodiment vii. The composition of any of embodiments iv-vi, wherein the Hepatitis B small surface protein (HBs) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0435] Embodiment viii. The composition of any of embodiments iv-vii, wherein the hepatitis B small surface antigen (HBs) is fused to a human invariant chain (hIi).

[0436] Embodiment ix. The composition of any of embodiments iv-viii, comprising a greater amount of the first mRNA by weight than the second mRNA.

[0437] Embodiment x. The composition of any of embodiments iv-ix, wherein the first mRNA and the second mRNA are present in a weight ratio of 1.5:1, respectively.

[0438] Embodiment xi. A composition for the treatment of chronic hepatitis B infection, comprising a first mRNA encoding at least the hepatitis B small surface protein (HBs), wherein the first mRNA is encapsulated in a lipid nanoparticle (LNP).

[0439] Embodiment xii. The composition of embodiment xi, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0440] Embodiment xiii. The composition of embodiment xi or xii, wherein HBs is fused to a human invariant chain (hIi).

[0441] Embodiment xiv. The composition of any of embodiments xi to xiii, further comprising a second mRNA encoding Hepatitis B virus core antigen (HBc).

[0442] Embodiment xv. The composition of embodiment xii, wherein the Hepatitis B virus core antigen (HBc) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11.

[0443] Embodiment xvi. The composition of embodiment xiv or xv, wherein the Hepatitis B virus core antigen (HBc) is fused to a human invariant chain (hIi).

[0444] Embodiment xvii. The composition of any of the preceding embodiments, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:12.

[0445] Embodiment xviii. The composition of embodiment xvii, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:12.

[0446] Embodiment xix. The composition of any preceding embodiment, administered sequentially or simultaneously with one or more recombinant Hepatitis B polypeptides.

[0447] Embodiment xx. The composition of embodiment xix, wherein the recombinant Hepatitis B polypeptide comprises recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

[0448] Embodiment xxi. The composition of embodiment xix or xx, wherein the HBc comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:2.

[0449] Embodiment xxii. The composition of embodiment xix or xx, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0450] Embodiment xxiii. The composition of any of embodiments xix to xxii, wherein the recombinant Hepatitis B polypeptide is administered with an adjuvant.

[0451] Embodiment xxiv. The composition of embodiment xxiii, wherein the adjuvant is AS01.

[0452] Group 3 of embodiments describes the following: Embodiment 1. A composition for treating chronic hepatitis B infection, comprising mRNA encoding at least hepatitis B virus core antigen (HBc), wherein the mRNA is encapsulated in a lipid nanoparticle (LNP).

[0453] Embodiment 2. The mRNA of any preceding embodiment, wherein the Hepatitis B virus core antigen (HBc) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11.

[0454] Embodiment 3. The mRNA of any of the previous embodiments, wherein the hepatitis B virus core antigen (HBc) is fused to a human invariant chain (hIi).

[0455] Embodiment 4. The mRNA of any of the preceding embodiments, wherein the mRNA further encodes a hepatitis B small surface protein (HBs).

[0456] Embodiment 5. The mRNA of any of the preceding embodiments, wherein the Hepatitis B small surface protein (HBs) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0457] Embodiment 6. The mRNA of embodiment 4 or 5, comprising a greater amount by weight of mRNA encoding HBc (HBc mRNA) than of mRNA encoding HBs (HBs mRNA).

[0458] Embodiment 7. The mRNA of any one of embodiments 4 to 6, wherein the HBc mRNA and HBs mRNA are present in a weight ratio of 1.5:1, respectively.

[0459] Embodiment 8. A composition for the treatment of chronic hepatitis B infection, comprising mRNA encoding at least the hepatitis B virus surface protein (HBsAg), said mRNA encapsulated in lipid nanoparticles (LNPs).

[0460] Embodiment 9. The mRNA of embodiment 8, wherein the HBsAg is hepatitis B small surface protein (HBs).

[0461] Embodiment 10. The mRNA of embodiment 9, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0462] Embodiment 11. The mRNA of embodiments 8 to 10, wherein HBsAg is fused to a human invariant chain (hIi).

[0463] Embodiment 12. The mRNA of any of the preceding embodiments, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:12.

[0464] Embodiment 13. The mRNA of any of the previous embodiments, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:12.

[0465] Embodiment 14 The mRNA of any preceding embodiment, wherein the composition is administered sequentially or simultaneously with one or more recombinant Hepatitis B polypeptides.

[0466] Embodiment 15. The mRNA of any preceding embodiment, wherein the recombinant Hepatitis B polypeptide comprises recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

[0467] Embodiment 16 The mRNA of any preceding embodiment, wherein the HBc comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:2.

[0468] Embodiment 17. The mRNA of any preceding embodiment, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0469] Embodiment 18 The mRNA of any preceding embodiment, wherein the recombinant Hepatitis B polypeptide is administered with an adjuvant.

[0470] Embodiment 19 The mRNA of embodiment 18, wherein the adjuvant is AS01.

[0471] Embodiment 20. A method of treating chronic hepatitis B infection comprising administering to a human a prime-boost regimen, in which mRNA encoding at least one hepatitis B viral antigen is administered as a priming dose and mRNA encoding at least one hepatitis B viral antigen is administered as a booster dose.

[0472] Embodiment 21 The method of embodiment 20, comprising administering four consecutive doses of mRNA to the human.

[0473] Embodiment 22. The method of embodiment 20 or 21, wherein another composition comprising adjuvanted recombinant Hepatitis B polypeptides is administered simultaneously with the mRNA, and the recombinant Hepatitis B polypeptides comprise recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

[0474] Embodiment 23. A method of treating chronic hepatitis B infection comprising administering to a human a prime-boost regimen in which mRNA encoding at least one hepatitis B viral antigen is administered as a priming dose and one or more recombinant hepatitis B polypeptides are administered as booster doses.

[0475] Embodiment 24 The method of embodiment 23, wherein the hepatitis B viral antigen is selected from the group consisting of hepatitis B core antigen (HBc) or hepatitis B surface antigen (HBsAg).

[0476] Embodiment 25 The method of embodiment 23, wherein the hepatitis B surface antigen (HBsAg) is hepatitis B small surface protein (HBs).

[0477] Embodiment 26 The method of any one of embodiments 23 to 25, wherein the hepatitis B virus antigen is fused to the hIi.

[0478] Embodiment 27. The method of any one of embodiments 23-26, wherein the recombinant Hepatitis B polypeptide comprises recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

[0479] Embodiment 28 The method of any one of Embodiments 23-27, wherein the recombinant Hepatitis B polypeptide is administered with an adjuvant.

[0480] Embodiment 29. The mRNA of any preceding embodiment, wherein the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and a non-ionizable cationic lipid.

[0481] Embodiment 30. The mRNA of any preceding embodiment, wherein the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable cationic lipid.

[0482] Embodiment 31. The mRNA of any preceding embodiment, wherein the non-cationic lipid is a neutral lipid such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or sphingomyelin (SM).

[0483] Embodiment 32 The mRNA of any preceding embodiment, wherein the sterol is cholesterol.

[0484] Embodiment 33. The mRNA of any preceding embodiment, wherein the LNP comprises about 0.5-15 mol% PEG-modified lipid, about 5-25 mol% non-cationic lipid, about 25-55 mol% sterol, and about 20-60 mol% ionizable cationic lipid.

[0485] Embodiment 34. The mRNA of any of the preceding embodiments, wherein the LNP has a diameter of 50 to 200 μm.

[0486] Embodiment 35 The mRNA of any preceding embodiment, wherein the polydispersity of the LNP is 0.4 or less, such as 0.3 or less.

[0487] Embodiment 36. The mRNA of any preceding embodiment, wherein the ratio of nucleotides (N) to phospholipids (P) is in the range of 1N:1P to 20N:1P, 1N:1P to 10N:1P, 2N:1P to 8N:1P, 2N:1P to 6N:1P, or 3N:1P to 5N:1P.

[0488] Embodiment 37. The mRNA of any preceding embodiment, wherein at least half of the mRNA is encapsulated in the LNPs, preferably at least 85%, particularly at least 95%, for example 100% encapsulated.

[0489] Embodiment 38 The mRNA of any preceding embodiment, wherein the mRNA is non-replicating or self-replicating mRNA (SAM).

[0490] Embodiment 39. The mRNA of any preceding embodiment, wherein the self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) a Hepatitis B polypeptide.

[0491] Embodiment 40. The mRNA of any preceding embodiment, having the following configuration: 5' cap-5' UTR-nonstructural protein (NSP) 1-4-subgenomic promoter-Hepatitis B polypeptide-3' UTR-polyA.

[0492] Embodiment 41 The mRNA of any preceding embodiment for administration to a human subject with chronic hepatitis B infection.

[0493] Embodiment 42 The mRNA of any preceding embodiment, which is a non-replicating mRNA.

[0494] Embodiment 43 The mRNA of any preceding embodiment, wherein the one or more recombinant Hepatitis B polypeptides are administered with an AS01 adjuvant.

[0495] Embodiment 44 The mRNA of any preceding embodiment, wherein the method comprises first administering the mRNA, and then administering the one or more recombinant Hepatitis B polypeptides.

[0496] Embodiment 45 The mRNA of any preceding embodiment, wherein the method comprises a prime-boost regimen, wherein the mRNA is administered as a priming dose and the one or more recombinant Hepatitis B polypeptides are administered as a booster dose.

[0497] Embodiment 46 The mRNA of embodiment 45, wherein the method comprises a single priming administration of the mRNA and multiple subsequent booster administrations of the recombinant Hepatitis B polypeptide.

[0498] Embodiment 47. The mRNA of embodiment 46, wherein the method comprises two or three subsequent booster administrations of the one or more recombinant Hepatitis B polypeptides.

[0499] Embodiment 48 The mRNA of embodiment 45, wherein the method comprises multiple priming administrations of the mRNA and multiple subsequent booster administrations of the recombinant Hepatitis B polypeptide.

[0500] Embodiment 49 The mRNA of embodiment 48, wherein the method comprises two priming administrations of the mRNA followed by two booster administrations of the recombinant Hepatitis B polypeptide.

[0501] Embodiment 50. An immunogenic composition comprising the mRNA of any of the preceding embodiments.

[0502] Embodiment 51. The immunogenic composition of embodiment 50, further comprising said one or more recombinant Hepatitis B polypeptides.

[0503] Embodiment 52. The following: (a) the mRNA of any one of embodiments 1 to 49; and (b) one or more recombinant Hepatitis B polypeptides administered in conjunction with said mRNA; An immunogenic combination comprising:

[0504] Embodiment 53. The immunogenic combination of embodiment 52, wherein the one or more recombinant Hepatitis B polypeptides comprise Hepatitis B surface antigen (HBs), Hepatitis B core antigen (HBc), and an adjuvant.

[0505] Embodiment 54. The immunogenic combination of embodiment 53, wherein one or more recombinant Hepatitis B polypeptides are combined with an AS01 adjuvant.

[0506] Embodiment 55. The immunogenic combination of any of embodiments 52 to 54, further comprising an adenovirus vector, which may be a replication-deficient chimpanzee adenovirus (ChAd) vector encoding a hepatitis B polypeptide.

[0507] Embodiment 56. The immunogenic combination of embodiment 55, wherein the adenoviral vector encodes hepatitis B virus core antigen (HBc) fused to a human invariant chain (hIi).

[0508] Embodiment 57. The immunogenic combination of embodiment 56, wherein the adenoviral vector further encodes hepatitis B virus surface antigen (HBs).

[0509] Embodiment 58. The immunogenic combination of any one of embodiments 55 to 57, wherein the adenoviral vector encodes a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0510] Embodiment 59. The immunogenic combination of any of embodiments 55 to 58, wherein the adenoviral vector encodes a polypeptide consisting of an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0511] Embodiment 60. The immunogenic combination of any one of Embodiments 55 to 59, wherein the adenoviral vector encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15.

[0512] Embodiment 61. The immunogenic combination of any one of Embodiments 55 to 60, wherein the adenoviral vector encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:15.

[0513] Embodiment 62. The following: a first composition comprising an mRNA encoding hepatitis B virus core antigen (HBc) encapsulated in a lipid nanoparticle (LNP) and an mRNA encoding hepatitis B small surface protein (HBs) encapsulated in a lipid nanoparticle; and a second composition comprising a recombinant hepatitis B core protein (HBc) and a recombinant hepatitis B small surface protein (HBs) and an adjuvant; 10. An immunogenic combination comprising:

[0514] Embodiment 63 The immunogenic combination of embodiment 62, wherein the second composition comprises an AS01 adjuvant.

[0515] Embodiment 64. A combination according to embodiment 62 or 63 for use in a method for treating chronic hepatitis B (CHB) by sequential or simultaneous administration of the compositions.

[0516] Embodiment 65. A method of treating chronic hepatitis B (CHB) infection in a human, comprising administering to the human the mRNA of any of embodiments 1 to 49, either sequentially or simultaneously, with one or more recombinant hepatitis B polypeptides.

[0517] Embodiment 66. A method of treating chronic hepatitis B infection (CHB) in a human according to embodiment 62, wherein the one or more recombinant hepatitis B polypeptides is recombinant hepatitis B virus core antigen (HBc).

[0518] Embodiment 67. The method of treating chronic hepatitis B infection (CHB) in a human of embodiment 63, wherein the composition further comprises recombinant hepatitis B surface antigen (HBs) and an adjuvant.

[0519] Embodiment 68. A method of treating chronic hepatitis B infection (CHB) in a human according to embodiment 62 or 63, wherein the composition further comprises an adjuvant.

[0520] Embodiment 69. The method of treating chronic hepatitis B infection (CHB) in a human according to embodiment 65, wherein the adjuvant comprises MPL and QS-21.

[0521] Embodiment 70. The method of treating chronic hepatitis B infection (CHB) in a human of embodiment 63, wherein the recombinant hepatitis B surface antigen (HBs) is a C-terminally truncated recombinant hepatitis B virus core antigen (HBc).

[0522] Embodiment 71. A method of treating a chronic hepatitis B (CHB) infection in a human of any of Embodiments 62 to 67, further comprising administering to the human an adenoviral vector comprising a polynucleotide encoding a hepatitis B polypeptide.

[0523] Embodiment 72. A method of treating chronic hepatitis B infection (CHB) in a human according to embodiment 68, wherein the adenoviral vector is a replication-deficient chimpanzee adenoviral (ChAd) vector.

[0524] Embodiment 73. A method of treating chronic hepatitis B infection (CHB) in a human according to embodiment 68 or 69, wherein the adenoviral vector encodes a hepatitis B polypeptide fused to a human invariant chain (hIi).

[0525] Embodiment 74. A method of treating chronic hepatitis B infection (CHB) in a human according to any of embodiments 68 to 70, wherein the adenoviral vector encodes hepatitis B core antigen (HBc).

[0526] Embodiment 75. A method of treating chronic hepatitis B infection (CHB) in a human according to embodiment 71, wherein the adenoviral vector further encodes hepatitis B surface antigen (HBs).

[0527] Embodiment 76. Use of an mRNA according to any one of embodiments 1 to 49 or an immunogenic combination according to any one of embodiments 50 to 61 in the treatment of HBV.

[0528] Embodiment 77. Use of an mRNA according to any one of embodiments 1 to 49 or an immunogenic combination according to any one of embodiments 50 to 61 for reducing circulating hepatitis B surface antigen (HBs) levels in patients infected with HBV.

[0529] Embodiment 78. Use of an mRNA according to any one of embodiments 1 to 49 or an immunogenic combination according to any one of embodiments 50 to 61 in the manufacture of a medicament.

[0530] Embodiment 79. Use of an mRNA according to any one of embodiments 1 to 49 or an immunogenic combination according to any one of embodiments 50 to 61 in the manufacture of a medicament for the treatment of HBV.

[0531] Embodiment 80: The following components: (a) the mRNA of any one of embodiments 1 to 49; and (b) one or more recombinant Hepatitis B polypeptides administered with the mRNA; Kit including:

[0532] References TIFF2026501211000035.tif245161TIFF2026501211000036.tif50161

[0533] Array List SEQ ID NO: 1: Amino acid sequence of HBs TIFF2026501211000037.tif26165 SEQ ID NO: 2: Amino acid sequence of truncated HBc TIFF2026501211000038.tif23164 SEQ ID NO: 3: Amino acid sequence of the spacer incorporating the 2A cleavage region of foot-and-mouth disease virus TIFF2026501211000039.tif8164 SEQ ID NO: 4: Nucleotide sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus TIFF2026501211000040.tif15165 SEQ ID NO: 5: Amino acid sequence of HBc-2A-HBs TIFF2026501211000041.tif48165 SEQ ID NO: 6: Nucleotide sequence encoding HBc-2A-HBs TIFF2026501211000042.tif28164TIFF2026501211000043.tif128165SEQ ID NO: 7: Amino acid sequence of hIi TIFF2026501211000044.tif27165 SEQ ID NO: 8: Nucleotide sequence encoding hIi TIFF2026501211000045.tif62165TIFF2026501211000046.tif23166SEQ ID NO: 9: Amino acid sequence of hIi-HBc-2A-HBs TIFF2026501211000047.tif74165 SEQ ID NO: 10: Nucleotide sequence encoding hIi-HBc-2A-HBs TIFF2026501211000048.tif121166TIFF2026501211000049.tif116166SEQ ID NO: 11: Amino acid sequence of HBc TIFF2026501211000050.tif22166 SEQ ID NO: 12: Amino acid sequence of hIi alternative variant TIFF2026501211000051.tif27166 SEQ ID NO: 13: Nucleotide sequence encoding the hIi alternative variant TIFF2026501211000052.tif43166TIFF2026501211000053.tif43166SEQ ID NO: 14: Alternative nucleic acid sequence of hIi-HBc-2A-HBs TIFF2026501211000054.tif188167TIFF2026501211000055.tif42167SEQ ID NO: 15: Alternative amino acid sequence of hIi-HBc-2A-HBs TIFF2026501211000056.tif74167 SEQ ID NO: 16: Nucleic acid sequence of empty SAM vector TIFF2026501211000057.tif101167TIFF2026501211000058.tif242168TIFF2026501211000059.tif24216 8TIFF2026501211000060.tif242168TIFF2026501211000061.tif242168TIFF2026501211000062.tif69169 1 The insertion begins here after nucleotide 7561 SEQ ID NO: 17: Human codon-optimized (Genewiz) nucleic acid sequence encoding the hIi_HBc_2A_HBs SAM transgene TIFF2026501211000063.tif147169TIFF2026501211000064.tif81169SEQ ID NO: 18: hIi_HBc_2A_HBs SAM plasmid sequence in AA098 TIFF2026501211000065.tif149169TIFF2026501211000066.tif241169TIFF2026501211000067.tif241169TIFF2026501211000068.tif241169TIFF2026501211000069.tif241169TIFF2026501211000070.tif231169SEQ ID NO: 19: Human codon-optimized (Genewiz) nucleic acid sequence encoding the HBc_2A_HBs SAM transgene. TIFF2026501211000071.tif149169 SEQ ID NO: 20: HBc_2A_HBs SAM plasmid sequence in AA098 TIFF2026501211000072.tif69169TIFF2026501211000073.tif241169TIFF2026501211000074.tif244169TIFF2026501211000075.tif244169TIFF2026501211000076.tif244169TIFF2026501211000077.tif234169SEQ ID NO: 21: Amino acid sequence of hIi-HBc TIFF2026501211000078.tif47169 SEQ ID NO: 22: Nucleotide sequence encoding hIi-HBc TIFF2026501211000079.tif149169 SEQ ID NO: 23: hIi-HBc plasmid sequence (UTR4) TIFF2026501211000080.tif21169TIFF2026501211000081.tif245169TIFF2026501211000082.tif158169SEQ ID NO: 24: Nucleotide sequence encoding HBs TIFF2026501211000083.tif71169TIFF2026501211000084.tif14169SEQ ID NO: 25: HBs plasmid sequence (UTR4) TIFF2026501211000085.tif215168TIFF2026501211000086.tif134168SEQ ID NO: 26: Amino acid sequence of hIi-HBs TIFF2026501211000087.tif60168 SEQ ID NO: 27: Nucleotide sequence encoding hIi-HBs TIFF2026501211000088.tif29168TIFF2026501211000089.tif135168SEQ ID NO: 28: hIi-HBs plasmid sequence (UTR4) TIFF2026501211000090.tif97168TIFF2026501211000091.tif243168TIFF2026501211000092.tif96168SEQ ID NO: 29: IRES nucleotide sequence TIFF2026501211000093.tif88168 SEQ ID NO: 30: Human codon-optimized (CodeRNA2) nucleic acid sequence encoding the hIi_HBc mRNA transgene TIFF2026501211000094.tif35168TIFF2026501211000095.tif116168SEQ ID NO: 31: Human codon-optimized (CodeRNA2) nucleic acid sequence encoding the HBs mRNA transgene TIFF2026501211000096.tif82168 SEQ ID NO: 32: Human codon-optimized (CodeRNA2) nucleic acid sequence encoding the hIi_HBs mRNA transgene TIFF2026501211000097.tif163168

Claims

1. A composition for the treatment of chronic hepatitis B infection, comprising mRNA encoding at least the hepatitis B virus core antigen (HBc), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

2. 2. The composition of claim 1, wherein the hepatitis B virus core antigen (HBc) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

11.

3. 3. The composition of claim 1 or 2, wherein the hepatitis B virus core antigen (HBc) is fused to a human invariant chain (hIi).

4. The composition of any one of claims 1 to 3, further comprising mRNA encoding hepatitis B small surface protein (HBs).

5. 5. The composition of claim 4, wherein the hepatitis B small surface protein (HBs) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

1.

6. The composition of any one of claims 1 to 5, wherein the hepatitis B small surface antigen (HBs) is fused to a human invariant chain (hIi).

7. The composition according to any one of claims 4 to 6, which contains a greater amount by weight of mRNA encoding HBc (HBc-mRNA) than of mRNA encoding HBs (HBs-mRNA).

8. The composition according to any one of claims 4 to 7, wherein the mRNA encoding HBc and the mRNA encoding HBs are present in a weight ratio of 1.5:1, respectively.

9. A composition for the treatment of chronic hepatitis B infection, comprising mRNA encoding hepatitis B small surface protein (HBs), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

10. 10. The composition of claim 9, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

1.

11. 11. The composition of claim 9 or 10, wherein the HBs is fused to a human invariant chain (hIi).

12. The composition of claim 3, 6 or 11, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO:

12.

13. The composition of claim 12, wherein the human invariant chain (hIi) comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

12.

14. The composition of any one of claims 1 to 13, administered sequentially or simultaneously with one or more recombinant Hepatitis B polypeptides.

15. 15. The composition of claim 14, wherein the recombinant hepatitis B polypeptide comprises a recombinant hepatitis B virus core protein (HBc) and a recombinant hepatitis B small surface protein (HBs).

16. 16. The composition of claim 15, wherein the HBc comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

2.

17. 17. The composition of claim 15 or 16, wherein the HBs comprises an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

1.

18. The composition of any one of claims 14 to 17, wherein the recombinant Hepatitis B polypeptide is administered together with an adjuvant.

19. The composition of claim 18, wherein the adjuvant is AS-01.

20. A method for treating chronic hepatitis B infection comprising administering to a human a prime-boost regimen, wherein mRNA encoding at least one hepatitis B viral antigen is administered as a priming dose and one or more recombinant hepatitis B polypeptides are administered as booster doses.

21. 21. The method of claim 20, wherein the mRNA encodes at least one hepatitis B virus antigen selected from the group comprising hepatitis B core antigen (HBc) or hepatitis B small surface protein (HBs).

22. 22. The method of claim 20 or 21, wherein the hepatitis B virus antigen is fused to the hIi.

23. 23. The method of any one of claims 20 to 22, wherein the recombinant Hepatitis B polypeptides comprise recombinant Hepatitis B core protein (HBc) and recombinant Hepatitis B small surface protein (HBs).

24. The method of any one of claims 20 to 22, wherein the recombinant Hepatitis B polypeptide is administered with an adjuvant.

25. The method of claim 24, wherein the adjuvant is AS-01.

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