Hepatitis B composition

Abstract

There is provided a composition for treating chronic hepatitis B infection comprising an mRNA encoding a hepatitis B virus antigen wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

Description

Technical Field

[0001] The present invention relates to compositions for treating chronic hepatitis B, wherein these compositions comprise mRNA encoding one or more hepatitis B antigens, and to related aspects. Background Art

[0002] Hepatitis B virus (HBV) infection is a major public health problem. The World Health Organization estimates that globally, 296 million people were living with chronic hepatitis B infection in 2019, with 1.5 million new infections each year (WHO, 2021). The clinical course and outcome of HBV infection are largely driven by the age at which infection is acquired and the complex interplay between the virus and the individual's immune response. Consequently, exposure to HBV can result in acute hepatitis that resolves spontaneously, or it can progress to multiple forms of chronic infection, including an inactive hepatitis B surface antigen (HBsAg) carrier state, chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). It is thought that 15%–40% of chronically infected individuals (defined as detectable serum HBsAg for more than 6 months) will develop hepatic sequelae, with cirrhosis (LC), hepatic decompensation, and hepatocellular carcinoma (HCC) being the major complications.

[0003] Although universal hepatitis B vaccination in 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 produced a substantial reduction in the prevalence of chronic hepatitis B (CHB) in adolescents and adults, and an impact on HBV-related deaths is not expected until decades after its introduction. The World Health Organization estimates that 820,000 people died from HBV-related causes in 2019, primarily from 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, consequently, 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. However, the loss (detectability) of hepatitis B e antigen (HBeAg) is a valuable biomarker. HBsAg loss, regardless of anti-HBs seroconversion, is generally considered the optimal endpoint representing a "functional cure" because it demonstrates significant suppression of HBV replication and viral protein expression (Revill, 2019; Block, 2017; Cornberg, 2017). Currently, patients with chronic hepatitis B have two main treatment options: treatment with pegylated interferon α (PegIFNα) or nucleos(t)ide analogs (NAs) (EASL, 2017). PegIFNα, designed to induce long-term immune control with a limited course of treatment, can achieve sustained off-treatment control, but durable virological responses and hepatitis B surface antigen (HBsAg) loss are limited to a small subset of patients. Furthermore, a significant number of patients are not suitable for this treatment due to poor tolerability and long-term safety concerns.

[0005] NAs inhibit DNA replication by inhibiting HBV polymerase reverse transcriptase activity. NAs approved for HBV treatment in Europe include entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF), which are associated with a high barrier to HBV resistance, and lamivudine (LAM), adefovir dipivoxil (ADV), and telbivudine (TBV), which are associated with a low barrier to HBV resistance. The main advantages of treatment with potent NAs with a high barrier to resistance are their predictable, long-term high antiviral efficacy, resulting in HBV DNA suppression in the vast majority of compliant patients, and their favorable safety profile. The disadvantages of NA treatment are that it is a long-term treatment regimen, as NAs generally do not achieve HBV eradication, and discontinuation of NAs may lead to HBV recurrence.

[0006] Due to the low HBsAg seroclearance rate and the high risk of non-NA viral relapse, most patients maintain long-term or even indefinite NA treatment, which may be associated with reduced patient compliance with therapy, increased financial costs, and an increased risk of drug toxicity and resistance mutations after long-term exposure. Therefore, new strategies are needed to achieve "functional cure" with limited-duration regimens.

[0007] Messenger RNA (mRNA) is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by the ribosome during the process of protein production. mRNA-based vaccines offer an alternative vaccination approach to traditional strategies involving live attenuated / inactivated pathogens or subunit vaccines (Zhang, 2019). mRNA vaccines can 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 target antigen 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, enabling intracellular RNA amplification (Pardi, 2018).

[0008] The present invention is intended to help address the need for HBV treatments that can clear HBsAg, allowing patients to safely discontinue NA therapy 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 virus core antigen (HBc), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs). In an embodiment, HBc is fused to a human invariant chain (hli).

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

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

[0012] In other aspects, the composition comprising mRNA is administered sequentially or simultaneously with one or more recombinant hepatitis B polypeptides. In embodiments, the recombinant hepatitis B polypeptides include recombinant hepatitis B core protein (HBc) and recombinant hepatitis B surface protein (HBs). In further embodiments, the one or more recombinant hepatitis B polypeptides are administered with an adjuvant. The adjuvant may be AS01.

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

[0014] Also described herein is a method for treating chronic hepatitis B infection in a human, comprising the steps of: (a) administering to the human an adenoviral vector comprising a polynucleotide encoding a hepatitis B polypeptide; (b) administering to the human an mRNA encoding a hepatitis B viral antigen; and (c) administering to the human at least one recombinant hepatitis B polypeptide. This method can be a heterologous prime-boost regimen comprising (a) administering an adenoviral vector as a prime dose; (b) administering an mRNA as a boost 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-defective chimpanzee adenovirus (ChAd) vector.

[0015] The present invention also provides a composition 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 may be AS01.

[0016] In one aspect, there is a method comprising administering a combination of mRNA and at least one polypeptide to a human. The components (i.e., mRNA and polypeptide) can be administered sequentially in a heterologous prime-boost regimen. If a heterologous prime-boost regimen is used, mRNA can be administered as a primary dose, and at least one polypeptide is administered as a booster dose. In another aspect, at least one polypeptide is administered as a primary dose, and mRNA is administered as a booster dose. At least one polypeptide may be administered with or without an adjuvant. In a specific embodiment, the polypeptide is administered together with an adjuvant. In one embodiment, mRNA and an adjuvanted polypeptide are administered sequentially. In another embodiment, mRNA and an adjuvanted polypeptide are administered concomitantly (e.g., simultaneously at different sites). The adjuvant is preferably AS01.

[0017] Sequence Description

[0018] SEQ ID NO: 1: Amino acid sequence of HBs

[0019] SEQ ID NO: 2: Amino acid sequence of HBc truncate

[0020] SEQ ID NO: 3: Amino acid sequence of the spacer region into which the foot-and-mouth disease virus 2A cleavage region is incorporated

[0021] SEQ ID NO: 4: Nucleotide sequence encoding a spacer region incorporated into the foot-and-mouth disease virus 2A cleavage region

[0022] SEQ ID NO: 5: Amino acid sequence of HBc-2A-HBs

[0023] SEQ ID NO: 6: Nucleotide sequence encoding HBc-2A-HBs

[0024] SEQ ID NO: 7: Amino acid sequence of hIi

[0025] SEQ ID NO: 8: Nucleotide sequence encoding hIi

[0026] SEQ ID NO:9: Amino acid sequence of hIi-HBc-2A-HBs

[0027] SEQ ID NO: 10: Nucleotide sequence encoding hIi-HBc-2A-HBs

[0028] SEQ ID NO: 11: Amino acid sequence of HBc

[0029] SEQ ID NO: 12: Amino acid sequence of hIi substitution variant

[0030] SEQ ID NO: 13: Nucleotide sequence encoding hIi substitution variant

[0031] SEQ ID NO: 14: Alternative nucleic acid sequence of hIi-HBc-2A-HBs

[0032] SEQ ID NO: 15: Alternative amino acid sequence of hIi-HBc-2A-HBs

[0033] SEQ ID NO: 16: Nucleic acid sequence of empty SAM vector

[0034] SEQ ID NO: 17: Human codon-optimized (Genewiz) nucleic acid sequence encoding the hIi_HBc_2A_HBs SAM transgene

[0035] SEQ ID NO: 18: hli_HBc_2A_HBs SAM plasmid sequence

[0036] SEQ ID NO: 19: Human codon-optimized (Genewiz) nucleic acid sequence encoding the HBc_2A_HBs SAM transgene

[0037] SEQ ID NO: 20: HBc_2A_HBs in SAM plasmid sequence

[0038] SEQ ID NO: 21: Amino acid sequence of hli-HBc

[0039] SEQ ID NO: 22: Nucleotide sequence encoding hli-HBc

[0040] SEQ ID NO: 23: hli-HBc mRNA plasmid sequence (UTR4)

[0041] SEQ ID NO: 24: Nucleotide sequence encoding HBs

[0042] SEQ ID NO: 25: HBs mRNA plasmid sequence (UTR4)

[0043] SEQ ID NO: 26: Amino acid sequence of hli-HBs

[0044] SEQ ID NO: 27: Nucleotide sequence encoding hli-HBs

[0045] SEQ ID NO: 28: hli-HBs mRNA plasmid sequence (UTR4)

[0046] SEQ ID NO: 29: IRES nucleotide sequence

[0047] SEQ ID NO:30: Nucleic acid sequence encoding human codon-optimized hIi_HBc mRNA transgene (CodeRNA2)

[0048] SEQ ID NO:31: Nucleic acid sequence encoding human codon-optimized HBs mRNA transgene (CodeRNA2)

[0049] SEQ ID NO:32: Nucleic acid sequence encoding human codon-optimized hIi_HBs mRNA transgene (CodeRNA2) BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A : Shows HBV core antigen (HBc)-specific CD4+ T cell responses in the spleen after priming with ChAd155-hli-HBV and boosting with SAM-HBV (±hli). Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) to assess HB core (HBc)-specific CD4+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0051] Figure 1B: Shows HBV surface (HBs)-specific CD4+ T cell responses in the spleen after priming with ChAd155-hli-HBV and boosting with SAM-HBV (±hli). Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) to assess HBs surface (HBs)-specific CD4+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0052] Figure 2A : Shows HBV core antigen (HBc)-specific CD8+ T cell responses in the spleen after priming with ChAd155-hli-HBV and boosting with SAM-HBV (±hli). Spleens were collected 12 / 13 days after the second immunization (12 / 13 dpII) to assess HB core (HBc)-specific CD8+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0053] Figure 2B : Shows HBV surface (HBs)-specific CD8+ T cell responses in the spleen after priming with ChAd155-hli-HBV and boosting with SAM-HBV (±hli). Spleens were harvested 12 / 13 days after the second immunization (12 / 13 dpII) to assess HBs surface (HBs)-specific CD8+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0054] Figure 3A Figure 3: HBV core antigen (HBc)-specific antibody responses following priming with ChAd155-hli-HBV and boosting with SAM-HBV (±hli). 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 the geometric mean (GM) is represented by a bar with a 95% confidence interval (CI).

[0055] Figure 3B Figure 3: HBV surface antigen (HBs)-specific antibody responses following priming with ChAd155-hli-HBV and boosting with SAM-HBV (±hli). 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 the geometric mean (GM) is represented by a bar with a 95% confidence interval (CI).

[0056] Figure 4A: Shown are HBV core antigen (HBc)-specific CD8+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) 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 to assess HB core (HBc)-specific CD8+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0057] Figure 4B : Shown are HBV core (HBc)-specific CD8+ T cell responses in the spleen at 22 dp IV (i.e., 22 days after the fourth dose) for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 22 days after the fourth immunization (22 dp IV), spleens were harvested to assess HB core (HBc)-specific CD8+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0058] Figure 5A : Shown are HBV surface (HBs)-specific CD8+ T cell responses in the spleen at 14 dp II (i.e., 14 days after the second dose) 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 dp IV), spleens were harvested to assess HB surface (HBs)-specific CD8+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0059] Figure 5B : Shows HBV surface antigen (HBs)-specific CD8+ T cell responses in the spleen at 22 dp IV (i.e., 22 days after the fourth dose) for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 22 days after the fourth immunization (22 dp IV), spleens were harvested to assess HB surface (HBs)-specific CD8+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0060] Figure 6A: Shown are HBV core antigen (HBc)-specific CD4+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) 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 14 days after the second immunization (14 dpII) to assess HB core (HBc)-specific CD4+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0061] Figure 6B : Shown are HBV core antigen (HBc)-specific CD4+ T cell responses in the spleen at 22 days post-IV administration (i.e., 22 days after the fourth dose) 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 post-fourth immunization (22 days post-IV) to assess HB core (HBc)-specific CD4+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0062] Figure 7A : Shown are HBV surface antigen (HBs)-specific CD4+ T cell responses in the spleen at 14 dpII (i.e., 14 days after the second dose) 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 14 days after the second immunization (14 dpII) to assess HB surface (HBs)-specific CD4+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0063] Figure 7B : Shown are HBV surface antigen (HBs)-specific CD4+ T cell responses in the spleen at 22 days post-IV (i.e., 22 days after the fourth dose) for all groups detailed in Table 2 (where C = ChAd155-hIi-HBV, M = MVA-HBV, S = SAM-hIi-HBV, and P = HBc-HBs / AS01). 22 days post-fourth immunization (22 days post-IV), spleens were harvested for assessment of HBs surface (HBs)-specific CD4+ T cells by intracellular staining. Each point represents a single animal, and the geometric mean (GM) is represented by the bar.

[0064] Figure 8AFigure 2 shows anti-HBc binding antibody titers measured on 13 / 14 dpII (i.e., 13 / 14 days after the second dose) 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 a single animal, and the geometric mean (GM) is represented by a bar with a 95% confidence interval (CI).

[0065] Figure 8B Figure 2 shows anti-HBc binding antibody titers measured IV at 22 days post-dose (i.e., 22 days after the fourth dose) 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-HBc IgG antibody titers by ELISA. For each group, each point represents the anti-HBc IgG antibody titer of a single animal, and the geometric mean (GM) is represented by a bar with a 95% confidence interval (CI).

[0066] Figure 9A : Shown are anti-HBs binding antibody titers measured on 13 / 14 dpII (i.e., 13 / 14 days after the second dose) 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 a single animal, and the geometric mean (GM) is represented by a bar with a 95% confidence interval (CI).

[0067] Figure 9B Figure 2 shows anti-HBs binding antibody titers measured 22 days post-IV (i.e., 22 days after the fourth dose) 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 a single animal, and the geometric mean (GM) is represented by a bar with a 95% confidence interval (CI).

[0068] Figure 10: shows the kinetics of circulating HBs antigen titers detected in different groups. The geometric means (GMs) of circulating HBs antigen titers are represented by squares with 95% confidence intervals.

[0069] Figure 11 : Shows circulating HBs antigen titers after the second and fourth immunizations compared to pre-immunization titers in different groups. The geometric mean ratios are represented by squares with 90% confidence intervals.

[0070] Figure 12 : shows the kinetics of AST and ALT levels detected in the different groups. The geometric mean ratios (GMRs) of AST and ALT titers compared to pre-immunization titers are represented by triangles with 95% confidence intervals.

[0071] Figure 13A : Shows the cytokine co-expression profile of HBc-specific CD8+ T cells. The frequency of HBc-specific CD8+ T cells expressing at least one, two, or three cytokines (IL-2, IFN-γ, and TNF-α) was assessed by intracellular staining 14 days after the second immunization. The median of each group is plotted.

[0072] Figure 13B Figure 3: Cytokine co-expression profiles of HBs-specific T cells. The frequency of HBs-specific CD8+ T cells expressing at least one, two, or three cytokines (IL-2, IFN-γ, and TNF-α) was assessed by intracellular staining 14 days after the second immunization. The median values of each group are plotted.

[0073] Figure 14 : The SAM-HBV construct used in the examples is shown. The SAM construct contains the genetic elements of VEEV TC-83 (non-structural protein sequences, nsP1-4) necessary for RNA amplification. The sequences encoding the structural proteins have been replaced by transgenes encoding HBV polypeptides, which are under the control of a subgenomic promoter. The empty SAM plasmid is shown as 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 constant chain (hli) is fused to the HBc.

[0074] Figure 15 : The hli_HBc_2A_HBs SAM plasmid map showing the SAM plasmid sequence of SEQ ID NO: 18.

[0075] Figure 16: HBc_2A_HBs SAM plasmid map showing the SAM plasmid sequence of SEQ ID NO:20.

[0076] Figure 17 : Shows HBV-specific CD8+ and CD4+ T cell responses after co-administration of three different mRNAs.

[0077] Figure 18 : Shows the HBc-specific and HBs-specific CD8+ T cell responses observed in Example 3.

[0078] Figure 19 : Shows Figure 18 Comparison of the geometric mean ratio (GMR) of CD8+ Tell responses of the combination of (hIi-HBc+hIi-HBs) mRNA constructs with hIi-HBc and hIi-HBs alone.

[0079] Figure 20 : Shows the HBc-specific and HBs-specific CD4+ T cell responses observed in Example 3.

[0080] Figure 21 : Shows the HBc-specific IgG response observed in Example 3. DETAILED DESCRIPTION

[0081] HBV antigen

[0082] At least ten HBV genotypes (A to J) have been identified (Liu, 2021). Within a given HBV genotype, multiple subgenotypes have also been identified. For example, genotypes A, B, C, D, and F have been further divided into subgenotypes. The antigens used 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) that encode (i) viral polymerase (Pol), (ii) viral surface proteins (L-HBs, M-HBs, and HBs), (iii) pre-core / core proteins (HBe and HBc), and (iiii) X protein (HBx).

[0083] The hepatitis B virus surface protein (HBsAg) is composed of three related but distinct proteins: the large (L), middle (M), and small (S) surface proteins. The HBV surface proteins (L, M, and S) are derived from alternate translations of the same ORF. The large surface protein consists of three domains: preS1 (108 / 118 / 119 amino acids, depending on the genotype; the genotype ApreS1 domain 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.

[0084] Hepatitis B core protein antigen (HBc) is highly conserved across genotypes and subtypes, and the hepatitis B small surface protein antigen (HBs) sequence is selected to include key B cell epitopes that are conserved across genotypes, which allows for the induction of a broad neutralizing response. Suitably, the sequences of HBc and HBs used in the disclosed methods and compositions are based on those from genotype / subtype A2.

[0085] Suitably, the HBV surface protein antigen used in the disclosed methods and compositions is derived from the small (S) surface antigen protein. In particular, the HBV surface antigen used herein may be derived from HBs.

[0086] In particular, suitable HBV surface protein antigens include the small (S) protein (HBs) of HBV adw2 strain genotype A. For example, a suitable HBs antigen has at least 90%, 95%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 1. In a preferred embodiment, a suitable HBs antigen has 226 amino acids of the amino acid sequence SEQ ID NO: 1. In one aspect, the HBs antigen can be fused with hli. In an embodiment, the hli-HBs antigen has at least 90%, 95%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 26. In a preferred embodiment, hli-HBs has the amino acid sequence of SEQ ID NO: 26.

[0087] Hepatitis B core protein (HBc) is the main component of the nucleocapsid that packages the viral genome. This protein (183-185 aa in length) 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. The HBc antigen used in the disclosed methods and compositions can be full-length or can contain a C-terminally truncated protein (lacking the RNA binding C-terminus), for example, including amino acids 1-145 of the wild-type core antigen protein, such as amino acids 1-145, 1-146, 1-147, 1-148 or amino acids 1-149 of the wild-type hepatitis B core antigen protein. The truncated protein retains the ability to assemble into nucleocapsid particles. Suitable HBc antigens for use in the disclosed methods and compositions have an amino acid sequence from genotype A of the HBV adw2 strain. When used as a recombinant polypeptide, the recombinant HBc protein is appropriately truncated from the wild type at the 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 preferred embodiments, the recombinant HBc protein has the amino acid sequence of SEQ ID NO:2. When expressed in mRNA or from a viral vector, the HBc antigen is suitably a 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 embodiments, the HBc antigen has the amino acid sequence of SEQ ID NO:11. In one aspect, the HBc antigen may be fused to hli. In embodiments, hli-HBc has at least 90%, 95%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:21. In preferred embodiments, hli-HBc has the amino acid sequence of SEQ ID NO:21.

[0088] invariant chain

[0089] Antigens are substances that induce an immune response in the body, especially the production of antibodies. Antigens can be foreign, i.e. pathogenic, originating from or derived from the organism itself, the latter being referred to as self-antigens or autoantigens. Antigens can be presented on the surface of antigen-presenting cells by MHC molecules. There are two types of MHC molecules, MHC class I (MHC-I) and MHC class II (MHC-II). MHC-II molecules are membrane-bound receptors that are synthesized in the endoplasmic reticulum and leave the endoplasmic reticulum to enter the MHC class II compartment. In order to prevent endogenous peptides (i.e., self-antigens) from binding to MHC-II molecules and being presented to produce an immune response, nascent MHC-II molecules interact with another protein (invariant chain), which blocks the peptide binding groove of the MHC-II molecule.

[0090] Human invariant chain (hIi, also known as CD74 when expressed at the plasma membrane) is an evolutionarily conserved type II membrane protein that plays a variety of roles within cells and throughout the immune system (Borghese, 2011). When the MHC class II compartment fuses with the late endosome containing the foreign protein to be engulfed and degraded, the invariant chain is cleaved, leaving only the CLIP region bound to the MHC-II molecule. In a second step, CLIP is removed by HLA-DM molecules, leaving the MHC-II molecule free to bind to the fragments of the foreign protein. Once the MHC class II compartment fuses with the plasma membrane, the fragments are presented on the surface of antigen-presenting cells, thereby presenting the foreign antigen to other cells, primarily T helper cells.

[0091] It is known that when the adenovirus expression system encoding the fusion of constant chain and the antigen is used for vaccination, the immune response to the antigen increases (see WO2007 / 062656, which is also disclosed as US2011 / 0293704 and is incorporated by reference for the purpose of disclosing constant chain sequences), that is, constant chain enhances the immunogenicity of the antigen. In addition, the adenovirus construct has been shown to be useful in eliciting an immune response (see WO2014 / 141176, which is also disclosed as US2016 / 0000904 in the context of a primary immunity-boosting vaccination regimen; and WO2010 / 057501, which is also disclosed as US2010 / 0278904 and is incorporated by reference for the purpose of disclosing constant chain sequences and adenovirus vectors encoding constant chain sequences).

[0092] In the present invention, the mRNA encoding the hepatitis B virus antigen includes a nucleotide sequence encoding the invariant chain (Ii), preferably the human invariant chain (hIi). The 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 to the N-terminus of the nucleotide sequence encoding the HBc antigen and / or the HBs antigen.

[0093] In the present invention, a composition for treating chronic hepatitis B infection is provided, which comprises mRNA encoding at least hepatitis B virus core antigen (HBc), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs), wherein the N-terminus of the nucleotide sequence encoding HBc is fused to human invariant chain (hli).

[0094] The present invention also provides a composition for treating chronic hepatitis B infection, which comprises 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 wherein the N-termini of the nucleotide sequences encoding HBc and HBs are fused to human invariant chain (hli).

[0095] In one embodiment, the mRNA encodes the amino acid sequence of SEQ ID NO: 9 and SEQ ID NO: 15 (preferably SEQ ID NO: 15). SEQ ID NO: 15 is a fusion of hli shown in SEQ ID NO: 12, HBc shown in SEQ ID NO: 11, 2A shown in SEQ ID NO: 3, and HBs shown in SEQ ID NO: 1.

[0096] In certain embodiments, adenoviral vectors (Ad), such as chimpanzee adenoviral vectors (ChAd), used in the methods and compositions disclosed herein may include a nucleotide sequence encoding hIi. The two amino acid sequences of hIi contained in the disclosed adenoviral vectors are shown in SEQ ID NO:7 and SEQ ID NO:12, and the nucleotide sequences encoding these amino acid sequences are shown 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 hIi is fused to the N-terminus of a nucleotide sequence encoding the HBc antigen.

[0097] messenger RNA (mRNA)

[0098] Non-replicating mRNA

[0099] The present disclosure provides a composition comprising a 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 target polypeptide and can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded target polypeptide. The mRNA typically contains a segment encoding the target polypeptide (i.e., a segment encoding a heterologous polypeptide (e.g., 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 target polypeptide; however, the 3'UTR is downstream (i.e., 3') of the target polypeptide. The 5'UTR starts at the transcription start site and ends at one nucleotide before the translation start sequence (i.e., 5'-adenosine, uridine, guanosine-3' (5'-AUG-3') sequence) of the coding region of the target polypeptide. The 3'UTR follows the translation stop codon of the target polypeptide coding region.

[0100] In an embodiment, the mRNA of the present disclosure can be structurally modified or chemically modified. As used herein, "structural" modification is wherein inserting, deleting, duplicating, reversing or randomizing two or more connected nucleosides in a polynucleotide without the modification of significant chemical modification to the nucleotide itself. Because chemical bonds must be broken and reorganized to achieve structural modification, structural modification has chemical properties and is therefore chemically modified. However, structural modification will result in different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified to "ATCCCG" from "ATCG". Here, dinucleotide "CC" has been inserted, resulting in structural modification to the polynucleotide. In one embodiment, the mRNA of the present disclosure can have the consistent chemical modification of all or any identical nucleoside types, or have all or any identical nucleoside types but the measured percentage of the chemical modification randomly incorporated, for example, wherein all uridines are replaced by uridine analogs (such as pseudouridine). In another embodiment, the mRNA can have two, three, or four identical nucleoside types consistently modified throughout the polynucleotide (e.g., all uridines and all cysteines are modified in the same manner). When the polynucleotides of the mRNA disclosed herein are chemically or structurally modified, the polynucleotides can be referred to as "modified polynucleotides."

[0101] In an embodiment, the mRNA has the following configuration: 5' cap / 5' UTR / hli / HBc / 3' UTR / poly A.

[0102] In an embodiment, the mRNA has the following configuration: 5' cap / 5' UTR / HBc / 3' UTR / poly A.

[0103] In an embodiment, the mRNA has the following configuration: 5' cap / 5' UTR / hli / HBs / 3' UTR / poly A.

[0104] In an embodiment, the mRNA has the following configuration: 5' cap / 5' UTR / HBs / 3' UTR / poly A.

[0105] In some embodiments, mRNA includes a 5' cap. In some embodiments, mRNA also includes 7-methylguanosine, 5' first ribonucleoside, optional 5' second ribonucleoside and optional triphosphate bridge. In some embodiments, 7-methylguanosine is directly or indirectly 5' to 5' connected to 5' first ribonucleoside. In some embodiments, 7-methylguanosine is connected to 5' first ribonucleoside 5' to 5' by triphosphate bridge. In some embodiments, 5' first ribonucleoside includes 2'-methylated ribose (2'-O-Me) (i.e., cap-1 or cap-2). In some embodiments, 5' second ribonucleoside is combined with the 3' end of 5' first ribonucleoside. In some embodiments, 5' second ribonucleoside includes 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 connected to the RNA via a 5' to 5' triphosphate connection by an mRNA guanylyltransferase, and wherein the guanine of the guanosine is methylated at its 7 position. In this context and in some embodiments, a 5' to 5' triphosphate connection occurs when the 5' end of the ribose of the guanosine is connected to the 5' end of the ribose of the mRNA via a triphosphate group by an mRNA guanylyltransferase. In some embodiments, thereafter, the guanine of the guanosine is methylated at its 7 position by a (guanine-N7-)-methyltransferase. In some embodiments, the 7-methylguanosine is added to the 5' first ribonucleoside immediately (i.e., without the addition of 7-guanosine and its further methylation to obtain 7-methylguanosine) via a 5' to 5' connection. In some embodiments, 7-methylguanosine is added to the 5' first ribonucleoside via a 5' to 5' addition and the addition of the 5' first ribonucleoside comprising a 2'-methylated ribose or the 5' second ribonucleoside comprising a 2'-methylated ribose occurs immediately (i.e., In some embodiments, a cap structure is preformed (i.e., as cap-1, cap-2, or cap-0, with or without the addition of a 7-methyl group to the 5' guanosine / 7-methylguanosine) and added to the recombinant RNA molecule (i.e., by ligation). In some embodiments, the preformed cap structure has a 5'-AG-3' start sequence added, such as AG product insert (Trilink Corporation Catalog No. N-7113), which is incorporated by reference).

[0106] In the absence of further methylation, a 7-methylguanosine attached to the 5' first ribonucleoside via a 5' to 5' sequence is called cap-0 and is expressed as 5'(m7Gp)(ppN)[pN] N , where the first "N" represents the first (5') base of the mRNA, "pN" represents another nucleotide in the RNA, and "[pN] N "middle"[..] N The addition of " represents the repeating polymer structure of RNA, thereby collectively representing each sequentially adjacent nucleotide in the RNA.

[0107] Additional oxygen-linked methylation of the 2' carbon of the ribose of the nucleoside of the mRNA immediately adjacent to the 7-methylguanosine (i.e., the 5' first ribonucleoside) by a 2'-O-methyltransferase generates a cap-1 structure denoted as 5'(m7Gp)(ppm2N)[pN] N , wherein the addition of "m2" represents the methylation of the oxygen-linked 2' carbon of the ribose of the nucleoside adjacent to (via triphosphate connection) the 7-methylguanosine. And further, the 5' second ribonucleoside (i.e., the next (3') nucleoside adjacent to the 5' first nucleoside methylated in cap-1) is further methylated to produce a cap-2 structure, which is represented by 5'(m7Gp)(ppm2N)(m2pN)[pN]n, wherein the addition of the following "m2" represents the methylation of the nucleotide adjacent to the nucleotide methylated in cap-1. This cap-2 methylation is also directed to the 2' carbon (i.e., 2'-O-Me) of the ribose of the adjacent nucleotide. 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.

[0108] 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 native to the mRNA (i.e., if the native sequence is 5'-UUAAT-3', then when a triphosphate bridge is present, addition of m7Gp will produce 5'-m7Gp(ppUUAAT-3'; if the native sequences are identical, the cap-1 structure will produce 5'-(m7Gp)(ppm2U)UAAT-3', and the cap-2 structure will produce 5'-(m7Gp)(ppm2U)(m2U)AAT-3').

[0109] Kits that provide all the materials for a 5' cap, either Cap-1 or Cap-2, are available, as well as supplemental kits that add Cap-1 and Cap-2 capabilities to the Cap-0 kit. 5' capping can be performed according to the manufacturer's instructions.

[0110] 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 located at the 3' end of the mRNA.

[0111] The mRNA disclosed herein can be modified. As used herein, the term "modified mRNA" or "RNA modification" can refer to chemical modifications, including backbone modifications and sugar modifications or base modifications. In this case, the modified RNA molecule as defined herein can contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications. The backbone modification associated with the present invention is a modification wherein the phosphates of the backbone of the nucleotides contained in the RNA molecule defined herein are chemically modified. The sugar modification associated with the present invention is a chemical modification of the sugar of the nucleotides of the RNA molecule as defined herein. In addition, the base modification associated with the present invention is a chemical modification of the base moiety of the nucleotides of the RNA molecule. In this article, nucleotide analogs or modifications are preferably selected from nucleotide analogs suitable for transcription and / or translation.

[0112] In one aspect, the modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules as described herein can be modified in the sugar moiety. For example, the 2' hydroxyl group (OH) can be modified or replaced by many different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, such as R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl group is connected to the 4' carbon of the same ribose, for example, by a methylene bridge; and amino groups (-O-amino, where the amino group, such as NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be linked to the sugar via a linker, wherein the linker comprises one or more of the atoms C, N, and O. The sugar group can also contain one or more carbons with the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, the modified RNA molecule can include nucleotides containing, for example, arabinose as the sugar.

[0113] In another aspect, the phosphate backbone can be further modified in modified nucleosides and nucleotides, and the modified nucleosides and nucleotides can be incorporated into modified RNA molecules as described herein. The phosphate group of main chain can be modified by replacing one or more oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include fully replacing unmodified phosphate moieties with modified phosphoric acid as described herein. The limiting examples of modified phosphate group include but are not limited to phosphorothioate, selenophosphate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester. Two non-connected oxygens of dithiophosphate are all substituted by sulfur. It is also possible to modify the phosphate joint by replacing connection oxygen with nitrogen (phosphoramidate of bridging), sulfur (phosphorothioate of bridging) and carbon (methylene phosphonate of bridging).

[0114] The modified nucleosides and nucleotides that can be used in the present invention can be further modified in the core base portion. Examples of core bases found in RNA include but are not limited to adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the 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.

[0115] Modified mRNA can comprise 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 following references: U.S. Patent Nos. 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.

[0116] Modified nucleobases that can 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-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxy Norvalylcarbamoyladenosine; 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-methyl-adenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; α-thioadenosine; 2( 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(thio)adenine 8-(haloalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)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'-α-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine TP;2-Amino-ATP; 2'-α-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2'-b-ethynyladenosine TP; 2-bromoadenosine TP; 2'-b-trifluoromethyladenosine 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-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'-homo-adenosine TP; 8-aza-ATP; 8-bromoadenosine TP; 8-trifluoromethyladenosine TP; 9-deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-dihydroadenosine Aminopurine; 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-dimethylcytidine; 5-formyl-2'-O-methylcytidine; lysine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-Aza-cytidine; pseudo-iso-cytidine; 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(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(propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine;5-(Propynyl)cytosine; 5-(Trifluoromethyl)cytosine: 5-bromo-cytidine; 5-iodo-cytidine; 5-propynylcytosine; 6-(Azo)cytosine; 6-Azacytidine; Azacytosine; Deazacytosine; N4(Acetyl)cytosine; 1-Methyl-1-deaza-pseudoisocytidine; 1-Methyl-pseudoisocytidine; 2-Methoxy-5-methyl-cytidine: 2-Methoxycytidine; 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-Zebullin; 5-Methyl-Zebullin; Pyrrolopseudoisocytidine Zebulin; (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-thiocytidine TP; 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-thiocytidine TP; 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)arabinoside TP; 5-(2-chloro-phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl-C TP; 5-cyanocytidine TP; 5-ethynylarabinoside TP; 5-ethynylcytidine TP; 5'-homo-cytidine TP; 5-methoxycytidine TP; 5-trifluoromethyl-cytidine TP; N4-amino-cytidine TP; N4-benzoyl-cytidine 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; purine; methyl 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 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-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thio)guanine; azaguanine; deazaguanine; N(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-azaguanosine; 6-thio-7-deazaguanosine; 6-thio-7-deazaguanosine; Methylguanosine; 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-isobutylguanosine TP; 2'O-methyl-N2-isobutylguanosine 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'-α-mercaptoguanosine TP; 2'-deoxy-2'-a-thiomethoxyguanosine TP; 2'-deoxy-2'-b-aminoguanosine TP; 2'-deoxy-2'-b-azidoguanosine guanosine 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-thioguanosine 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; epoxy-braided glycosides; galactosyl-braided glycosides;Mannosyl braided thymidine; braided thymidine; allylamino-thymidine; azathymidine; deazathymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-tauromethyl-2-thiouridine; 5-tauromethyluridine; dihydrouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseudouridine; 1-methyl-pseudouridine; 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-methyl-pseudouridine 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 ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine TP; 5-methoxycinnamylmethyl- 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-oxyacetic acid-uridine TP; 5-oxyacetic acid-methyl ester-uridine TP; N1-methyl-pseudouridine; N1-ethyl-pseudouridine; uridine 5-oxyacetic 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)- 1-pentenylaminomethyl)uridine TP; 5-propynyl uracil; α-thiouridine; 1(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouridine; 1(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-4(thio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-pseudouridine; 1(aminocarbonylvinyl)-2(thio)-pseudouridine; 1(aminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminocarbonylvinyl)-4(thio)pseudouridine; 1(aminocarbonylvinyl)-pseudouridine; 1-substituted 2(thio)-pseudouridine; 1-substituted 2,4-(dithio)pseudouridine; 1-substituted 4(thio)pseudouridine;1-substituted pseudouridine; 1-(aminoalkylaminocarbonylvinyl)-2-(thio)-pseudouridine; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-pseudouridine TP; 2-(thio)pseudouridine; 2'-deoxyuridine; 2'-fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouridine; 2'methyl; 2'amino; 2'azido; 2'fluoro-guanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine TP; 2'-O-methyl pseudouridine; 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-oxadiazol-1-yl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidinyl)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(methylaminomethyl)-2( thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5(propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4(dithio)pseudouracil; 5-(alkyl)-4(thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidinyl)uracil; 5-(halogenated)uracil; 5-( 1,3-oxadiazole-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)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(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-(propargyl)uracil;5-(Trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylamino-uracil; aza-uracil; deaza-uracil; N3(methyl)uracil; pseudo-UTP-1-2-acetic 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-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; -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-pseudouridine TP; (E)-5-(2-bromo-vinyl)pseudouridine TP; (Z)-5-(2-bromo-vinyl)ara- Pseudouridine TP; (Z)-5-(2-bromo-vinyl)pseudouridine 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-trimethyl-benzyl)pseudouridine TP; 1-(2,4,6-trimethyl-phenyl)pseudouridine; 1-(2-amino-2-carboxyethyl)pseudouridine TP; 1-(2-amino-ethyl)pseudouridine TP; 1-(2-hydroxyethyl)pseudouridine TP; 1-(2-methoxyethyl)pseudouridine TP; 1-(3,4-bis(trifluoromethoxybenzyl)pseudouridine TP; 1-(3,4-dimethoxybenzyl)pseudouridine TP; 1-(3-amino-3-carboxypropyl)pseudouridine TP; 1-(3-aminopropyl)pseudouridine TP; 1-(3-cyclopropyl-alkyn-2-yl)pseudouridine TP; 1-(4-amino-4-carboxybutyl)pseudouridine TP; 1-(4-amino-benzyl)pseudouridine TP; 1-(4-amino-butyl)pseudouridine TP; 1-(4-amino-phenyl)pseudouridine TP; 1-(4-azidobenzyl)pseudouridine TP; 1-(4-bromobenzyl)pseudouridine TP; 1-(4-chlorobenzyl)pseudouridine TP; 1-(4-fluorobenzyl)pseudouridine TP; 1-(4-iodobenzyl)pseudouridine TP; 1-(4-methylsulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-methoxybenzyl)pseudouridine TP; 1-(4-methoxyphenyl)pseudouridine TP; 1-(4-methylbenzyl)pseudouridine TP; 1-(4-methyl-benzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitro-phenyl)pseudouridine TP; 1-(4-thiomethoxybenzyl)pseudouridine TP; 1-(4-trifluoromethoxybenzyl)pseudouridine TP; 1-(4-trifluoromethylbenzyl)pseudouridine TP; 1-(5-amino-pentyl)pseudouridine TP; 1-(6-amino-hexyl)pseudouridine TP; 1,6-dimethyl-pseudouridine TP; 1-[3-(2-{2- [2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl] pseudouridine TP; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-acetyl pseudouridine 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-allyl pseudouridine TP; 1-aminomethyl-pseudo-UTP; 1-benzoyl pseudouridine TP; 1-benzyloxymethyl pseudouridine TP; 1-benzyl-pseudo- UTP; 1-biotinyl-PEG2-pseudouridine TP; 1-biotinyl-pseudouridine TP; 1-butyl-pseudouridine TP; 1-cyanomethyl-pseudouridine TP; 1-cyclobutylmethyl-pseudouridine TP; 1-cyclobutyl-pseudouridine TP; 1-cycloheptylmethyl-pseudouridine TP; 1-cycloheptyl-pseudouridine TP; 1-cyclohexylmethyl-pseudouridine TP; 1-cyclohexyl-pseudouridine TP; 1-cyclooctylmethyl-pseudouridine TP; 1-cyclooctyl-pseudouridine TP; 1-cyclopentylmethyl-pseudouridine TP; 1-cyclopentyl-pseudouridine TP; 1-cyclopropylmethyl-pseudouridine TP; 1-cyclopropyl-pseudouridine TP; 1-ethyl-pseudouridine TP; 1-hexyl-pseudouridine TP; 1-homoallyl-pseudouridine TP; 1- Hydroxymethyl pseudouridine TP; 1-isopropyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-α-thio-pseudo-UTP; 1-methylsulfonylmethyl pseudouridine TP; 1-methoxymethyl pseudouridine TP; 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP; 1-methyl-6-(4-morpholino)-pseudo-UTP; 1-methyl-6-(4-thiomorpholino)-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-ethylcarboxylic acid-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-morpholinomethyl-pseudo-UTP; 1-pentyl-pseudo-UTP; 1-phenyl-pseudo-UTP; 1-pivaloyl-pseudo-UTP; 1-propargyl-pseudo-UTP; 1-propyl-pseudo-UTP; 1-propargyl-pseudo-UTP; 1-p-tolyl-pseudo-UTP; 1-tert-butyl-pseudo-UTP; 1-thiomethoxymethyl-pseudo-UTP; 1-thiomorpholinomethyl-pseudo-UTP; 1-trifluoroacetyl-pseudo-UTP; 1-trifluoromethyl-pseudo-UTP; 1-vinyl-pseudo-UTP; 2,2'-deoxyuridine Aqua-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-ethynyluridine TP; 2'-a-trifluoromethyluridine TP; 2'-b-ethynyluridine TP; 2'-b-trifluoromethyluridine TP; 2'-deoxy-2',2'-difluorouridine 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-thiomethoxyuridine TP; 2-methoxy-4-thiouridine; 2-methoxyuridine; 2'-O-methyl-5-(1-propargyl)uridine TP; 3-alkyl-pseudo-UTP; 4'-azidouridine TP; 4'-carbocyclic uridine TP; 4'-ethynyluridine TP; 5-(1-propynyl)uridine TP; 5-(2-furyl)uridine TP; 5-cyanouridine TP; 5-dimethylaminouridine TP; 5'-homo-uridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5-phenylethynyluridine TP;5-trideuteriomethyl-6-deuterouridine TP; 5-trifluoromethyl-uridine TP; 5-vinyluridine 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-UTP; 6-cyano-pseudo-UTP; 6-dimethylamino-pseudo-UTP; 6-ethoxy-pseudo-UTP; 6-ethylcarboxylic acid-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; α-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 TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-methylphosphonic acid; pseudouridine TP Diethyl 1-methylphosphonate; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric 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; wyobutosine; hydroxywyobutosine; isowyobutosine; peroxywyobutosine; undermodified hydroxywyobutosine; 4-demethylwyobutosine; 2,6-(diamino)purine; 1-(aza)-2 -(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenothiazin-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-chloro-purine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'-fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isocarboxystyryl; 3-(methyl)isocarboxystyryl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl) )isocarboxystyryl; 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)-phenothiazin-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-(aza)indolyl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; -yl; 7-(propynyl)isocarboxystyryl; 7-(propynyl)isocarboxystyryl; propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(aza)-phenoxazin-1-yl; 9-(methyl)-imidazopyridinyl; aminoindolyl; anthracenyl; bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isocarboxystyryl; isoguanosine; N2-substituted purine;N6-Methyl-2-amino-purine; N6-substituted purine; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purine; O-alkylated derivatives; o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentaphenyl yl); phenanthrenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrazinyl; stilbenebenzyl; substituted 1,2,4-triazole; tetracenyl; tuberculin; xanthine; xanthine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridine-4-one ribonucleoside; 2-aminoriboside-TP; formycin ATP; formycin B TP; pyrrolosine TP; 2'-OH-arabinoadenosine TP; 2'-OH-arabinocytidine TP; 2'-OH-arabinouridine TP; 2'-OH-arabinoguanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; or N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0117] In some embodiments, modified nucleotides that can be substituted for adenosine include: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 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). phosphate); isopentenyl adenosine; 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-methyl-adenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; 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 ) adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)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'-α-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine TP; 2-amino-ATP; 2'-α-trifluoromethyladenosine TP; 2-azidoadenosine TP;2'-b-ethynyladenosine TP; 2-bromoadenosine TP; 2'-b-trifluoromethyladenosine 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-fluoro Adenosine 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-aza-ATP; 8-bromoadenosine TP; 8-trifluoromethyladenosine TP; 9-deazaadenosine TP; wyobutosine; hydroxywyobutosine; isowyobutosine; peroxywyobutosine; undermodified hydroxywyobutosine; 4-demethylwyobutosine; 2,6-(dimethoxy)adenosine (amino) purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenothiazin-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-chloro-purine; 2-aza-inosyl; 2'-azido-2'-deoxyuridine Oxyribose; 2'-fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isocarboxystyryl; 3-(methyl)isocarboxystyryl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isocarboxystyryl; 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)-phenothiazin-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)-phenothiazin-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-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarboxystyryl; 7-(propynyl)isocarboxystyryl; propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(Aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(aza)-phenoxazin-1-yl; 9-(methyl)-imidazopyridinyl; aminoindolyl; anthracenyl; bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isocarboxystyryl; isoguanosine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; gouache Nubularine; O6-substituted purine; O-alkylated derivatives; o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentaphenyl; phenanthrenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl;Pyrrolo-pyrimidin-2-one-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrazinyl; Stilbenzyl; Substituted 1,2,4-triazole; Tetracenyl; Tubercidine; Xanthine; Xanthine-5'-TP; 2-thio-zebulin; 5-aza-2-thio-zebulin; 7-deaza-2-amino-purine; Pyridine-4-one ribonucleoside; 2-aminoriboside-TP; Formycin A TP; Formycin B TP; pyrrolosine TP; 2'-OH-arabinoadenosine TP; 2'-OH-arabinocytidine TP; 2'-OH-arabinouridine TP; 2'-OH-arabinoguanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; or N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0118] In some embodiments, the modified nucleotides substituted for uridine or thymidine include pseudouridine; N1-methyl pseudouridine; N1-ethyl pseudouridine; inosine; 1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2'-O-methylinosine; epoxy braids; galactosyl-braids; mannosyl braids; braids; allylamino-thymidine; azathymidine; deazathymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-tauromethyl-2-thiouridine; 5-tauromethyluridine; dihydrouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseudouridine; 1-methyl-pseudouridine; 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-methyl-pseudouridine 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- Carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine TP; 5-methoxycinnamylmethyl-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-oxyacetic acid-uridine TP; 5-oxyacetic acid Acid-methyl ester-uridine TP; N1-methyl-pseudouridine; N1-ethyl-pseudouridine; uridine 5-oxoacetic 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(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouridine; 1(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-4(thio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-pseudouridine;1(aminocarbonylvinyl)-2(thio)-pseudouridine; 1(aminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminocarbonylvinyl)-4(thio)pseudouridine; 1(aminocarbonylvinyl)-pseudouridine; 1-substituted 2(thio)-pseudouridine; 1-substituted 2,4-(dithio)pseudouridine; 1-substituted 4(thio)pseudouridine; 1-substituted pseudouridine; 1-(aminoalkylaminocarbonylvinyl)-2-(thio)-pseudouridine; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine-UTP; 1-methyl-pseudouridine; 2(thio)pseudouridine; 2'-deoxyuridine; 2'-fluorouridine; 2-(Thio)uracil; 2,4-(dithio)pseudouridine; 2'methyl; 2'amino; 2'azido; 2'fluoro-guanosine; 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-oxadiazol-1-yl)uracil; 5(2-aminopropyl) Uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidinyl)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(methylaminomethyl)-2(thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5(propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4(dithio)uracil )pseudouracil; 5-(alkyl)-4(thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidinyl)uracil; 5-(halo)uracil; 5-(1,3-oxadiazol-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)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(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-(Propargyl)uracil; 5-(Trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylamino-uracil; azauracil; deazauracil; N3(methyl)uracil; pseudo-UTP-1- 2-acetic 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-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; dihydro Pseudouridine; (.+-.)1-(2-hydroxypropyl)pseudouridine TP; (2R)-1-(2-hydroxypropyl)pseudouridine TP; (2S)-1-(2-hydroxypropyl)pseudouridine TP; (E)-5-(2-bromo-vinyl)ara-pseudouridine TP; (E)-5-(2-bromo-vinyl)pseudouridine TP; (Z)-5-(2-bromo-vinyl)ara-pseudouridine TP; (Z)-5-(2-bromo-vinyl)pseudouridine TP; 1-(2,2,2-trifluoroethyl)-pseudouridine-UTP; 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-trimethyl-benzyl) pseudo-UTP; 1-(2,4,6-trimethyl-phenyl) pseudo-UTP; 1-(2-amino-2-carboxyethyl) pseudo-UTP; 1-(2-amino-ethyl) pseudo-UTP; 1-(2-hydroxyethyl) pseudo-UTP; 1-(2-methoxyethyl) pseudo-UTP; 1-(3,4-bis(trifluoromethoxybenzyl) pseudo-UTP; 1-(3,4-dimethoxybenzyl) pseudo-UTP; 1-(3-amino-3-carboxypropyl) pseudo-UTP; 1-(3-aminopropyl) pseudo-UTP; 1-(3-cyclopropyl-alkyn-2-yl) pseudo-UTP; 1-(4-amino-4-carboxybutyl) pseudo-UTP; 1-(4-amino-benzyl) pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-amino-phenyl)pseudo-UTP; 1-(4-azidobenzyl)pseudouridine TP; 1-(4-bromobenzyl)pseudouridine TP; 1-(4-chlorobenzyl)pseudouridine TP; 1-(4-fluorobenzyl)pseudouridine TP; 1-(4-iodobenzyl)pseudouridine TP; 1-(4-methylsulfonylbenzyl)pseudouridine TP; 1-(4-methoxybenzyl)pseudouridine TP; 1-(4-methoxybenzyl)pseudouridine TP; 1-(4-methoxyphenyl)pseudouridine TP; 1-(4-methylbenzyl)pseudouridine TP; 1-(4-methyl-benzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitro-benzyl)pseudouridine TP; 1-(4 -nitro-phenyl) pseudo-UTP; 1-(4-thiomethoxybenzyl) pseudo-UTP; 1-(4-trifluoromethoxybenzyl) pseudo-UTP; 1-(4-trifluoromethylbenzyl) pseudo-UTP; 1-(5-amino-pentyl) pseudo-UTP; 1-(6-amino-hexyl) pseudo-UTP; 1,6-dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl] pseudo-UTP; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl} pseudo-UTP; 1-acetyl pseudo-UTP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-UTP P; 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-allyl pseudouridine TP; 1-aminomethyl-pseudo-UTP; 1-benzoyl pseudouridine TP; 1-benzyloxymethyl pseudouridine TP; 1-benzyl-pseudo-UTP; 1-biotinyl-PEG2-pseudouridine TP; 1-biotinyl pseudouridine TP; 1-butyl-pseudo-UTP; 1-cyanomethyl pseudouridine TP; 1-cyclobutylmethyl-pseudo-UTP; 1-cyclobutyl-pseudo-UTP; 1-cycloheptylmethyl-pseudo-UTP; 1-cycloheptyl-pseudo-UTP; 1-cyclohexylmethyl-pseudo-UTP P; 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 pseudouridine TP; 1-hydroxymethyl pseudouridine TP; 1-isopropyl pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-α-thio-pseudo-UTP; 1-methylsulfonylmethyl pseudouridine TP; 1-methoxymethyl pseudouridine TP; 1-methyl-6-(2,2,2-trifluoroethyl) pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP; 1-methyl-6-(4-thiomorpholino)-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-ethylcarboxylic acid-pseudo-UTP; 1-methyl-6-ethyl-pseudo-UTP; 1-methyl-6-fluoro-pseudo-UTP; 1-methyl 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-morpholinomethyl-pseudouridine TP; 1-pentyl-pseudouridine TP; 1-phenyl-pseudouridine TP; 1-pivaloyl-pseudouridine TP; 1 -Propargyl-pseudouridine TP; 1-propyl-pseudouridine; 1-propargyl-pseudouridine; 1-p-tolyl-pseudouridine; 1-tert-butyl-pseudouridine; 1-thiomethoxymethyl-pseudouridine TP; 1-thiomorpholinomethyl-pseudouridine TP; 1-trifluoroacetyl-pseudouridine TP; 1-trifluoromethyl-pseudouridine; 1-vinyl-pseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudouridine; 2'-a-ethynyl-uridine TP; 2'-a-trifluoromethyl-uridine TP; 2'-b-ethynyl-uridine TP; 2'-b-trifluoromethyl-uridine TP; 2'-deoxy-2',2'-difluorouridine 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-thiomethoxyuridine TP; 2-methoxy-4-thiouridine; 2-methoxyuridine; 2'-O-methyl-5-(1-propargyl)uridine TP;3-Alkyl-pseudo-UTP; 4'-azidouridine TP; 4'-carbocyclic uridine TP; 4'-ethynyl uridine TP; 5-(1-propynyl)uridine TP; 5-(2-furyl)uridine TP; 5-cyanouridine TP; 5-dimethylaminouridine TP; 5'-homo-uridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5-phenylethynyl uridine TP; 5-trideuteriomethyl-6-deuterouridine TP ; 5-trifluoromethyl-uridine TP; 5-vinyluridine 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-UTP P; 6-cyano-pseudo-UTP; 6-dimethylamino-pseudo-UTP; 6-ethoxy-pseudo-UTP; 6-ethylcarboxylic acid-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; α-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 TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-methylphosphonic acid; pseudouridine TP Diethyl 1-methylphosphonate; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric 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; wyoside; hydroxywyoside; isowyoside; peroxywyoside; undermodified hydroxywyoside; 4-demethylwyoside; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxo)-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-chloro-purine; 2-aza-inosyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo- Pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isocarboxystyryl; 3-(methyl)isocarboxystyryl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isocarboxystyryl; 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)-phenothiazin-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)-phenothiazin-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)-phenoxazin-1-yl; 7-(guanidiniumhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidiniumhydroxy)-1-(aza)- 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarboxystyryl; 7-(propynyl)isocarboxystyryl; propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(aza)-phenoxazin-1-yl;9-(Methyl)-imidazopyridinyl; aminoindolyl; anthracenyl; bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isocarboxystyryl; N2-substituted purine; N6-methyl-2-amino-purine; N6-substituted purine; N-alkylated Derivatives; Naphthyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; O6-substituted purines; O-alkylated derivatives; o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Oxoformycin TP; p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl alkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentacenyl; phenanthrenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolo Pyrimidinyl; pyrrolopyrazinyl; stilbenzyl; substituted 1,2,4-triazole; tetracenyl; tuberculin; xanthine; xanthine-5'-TP; 2-thio-zebulin; 5-aza-2-thio-zebulin; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-aminoriboside-TP; formycin A TP; formycin B TP; pyrrolosine TP; 2'-OH-arabinocytidine TP; 2'-OH-arabinouridine TP; or 5-(2-carbomethoxyvinyl)uridine TP.

[0119] In some embodiments, the modified nucleotides in which cytosine may be substituted include 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; lysine; N4,2'-O-dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-azacytidine; pseudo-iso-cytidine; 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(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(propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine: 5-bromo-cytidine; 5-iodo-cytidine; 5-propynylcytosine; 6-( azo)cytosine; 6-aza-cytidine; azacytosine; deazacytosine; N4(acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxycytidine; 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-zebulin; 5-methyl-zebulin; pyrrolopseudoisocytidine; zebulin; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'-fluoro-N 4-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-thiocytidine TP; 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-thiocytidine TP; 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)arabinocytidine TP; 5-(2-chloro -phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylarabinoside TP; 5-ethynylcytidine TP; 5'-homo-cytidine TP; 5-methoxycytidine TP; 5-trifluoromethyl-cytidine TP; N4-amino-cytidine TP; N4-benzoyl-cytidine TP; pseudoisocytidine; 2'fluoro-cytidine; or 2'-OH-arabinoside TP.

[0120] In some embodiments, the modified nucleotides include: 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; purine; methyl wyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine ; 8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 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-methyl-guanosine; 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-(Hydroxy)guanine; 8-(Thioalkyl)guanine; 8-(Thio)guanine; Azaguanine; Deazaguanine; N(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 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-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2 'Fluoro-N2-isobutylguanosine TP; 2'O-methyl-N2-isobutylguanosine 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'-α-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-thioguanosine 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; epoxy-braided glycoside; galactosyl-braided glycoside; mannosyl-braided glycoside; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinyl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinyl phenoxazin-1-yl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarboxystyryl; 7-(propynyl)isocarboxystyryl; propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(aza)-phenoxazin-1-yl; 9-(methyl)-imidazopyridinyl; aminoindolyl; anthracenyl; bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isocarboxystyryl; isoguanosine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purines; O-alkylated derivatives; o-(Aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; p-(Aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentaphenyl; phenanthrenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrazinyl; stilbenebenzyl;Substituted 1,2,4-triazole; Tetracenyl; Tubercidine; Xanthine; Xanthine-5'-TP; 2-thio-zebulin; 5-aza-2-thio-zebulin; 7-deaza-2-amino-purine; Pyridin-4-one ribonucleoside; 2-aminoriboside-TP; Formycin ATP; Formycin B TP; Pyrrolosine TP; or 2'-OH-arabinoguanosine TP.

[0121] Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers.

[0122] Self-amplifying mRNA (SAM)

[0123] The mRNA disclosed herein can be replicative, also known as self-amplification. Self-amplifying mRNA molecules can be alphavirus-derived mRNA replicons. MRNA amplification can also be achieved by providing a non-replicating mRNA encoding an antigen and a separate mRNA encoding the replication machinery.

[0124] Self-replicating RNA molecules are well known in the art, and can be produced by using the replication elements derived from, for example, alphaviruses, and by replacing the structural viral proteins with the nucleotide sequence encoding the target protein. Self-replicating RNA molecules are normally + chain molecules, which can directly translate after being delivered to the cell, and this translation provides RNA-dependent RNA polymerase, and then this enzyme produces antisense and sense transcripts from the RNA of delivery. Therefore, the RNA of delivery causes the generation of a plurality of sub-RNAs. These sub-RNAs and collinear subgenomic transcripts can self-translate to provide the in situ expression of coded antigens, or can be transcribed to provide the RNA of delivery with the further transcript of identical sense, and described transcript is translated to provide the in situ expression of antigens. The overall result of this transcription sequence is the huge amplification of the replicon RNA quantity of introducing, and therefore the coded antigen becomes the main polypeptide product of cell.

[0125] 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 replicons, see the following reference: WO 2005 / 113782.

[0126] In certain embodiments, the self-replicating RNA molecules described herein encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and (ii) an antigen. The polymerase can be an alphavirus replicase, for example comprising one or more alphavirus proteins nsP1, nsP2, nsP3, and nsP4 (wherein nsP stands for nonstructural protein).

[0127] While the natural alphavirus genome encodes structural virion proteins in addition to the nonstructural replicase polyprotein, self-replicating RNA molecules do not encode alphavirus structural proteins. Thus, self-replicating RNA can lead to the production of its own genomic RNA copies in cells, but does not produce RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, self-replicating RNA molecules cannot persist in an infectious form. The alphavirus structural proteins necessary for permanent survival in wild-type viruses are not present in the self-replicating RNA of the present invention; their positions are replaced by one or more genes encoding the immunogen of interest, such that the subgenomic transcripts encode the immunogen rather than the alphavirus structural virion proteins.

[0128] The self-replicating RNA molecules useful in the present invention may have two open reading frames: the first (5') open reading frame encodes the replicase; the second (3') open reading frame encodes one or more HBV antigens.

[0129] In certain embodiments, the self-replicating RNA molecules disclosed herein have a 5' cap (e.g., 7-methylguanosine). The cap can enhance the 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.

[0130] The self-replicating RNA molecule may have a 3' poly A tail. It may also include a poly A polymerase recognition sequence (eg, AAUAAA) near its 3' end.

[0131] Self-replicating RNA molecules can have different lengths, but they are generally 5000-25000 nucleotides long. Self-replicating RNA molecules are typically single-stranded. Single-stranded RNA can usually start adjuvant action by combining TLR7, TLR8, RNA helicase and / or PKR. The RNA (dsRNA) delivered in double-stranded form can be combined with TLR3, and the receptor can also be triggered by dsRNA, which is formed during the single-stranded RNA replication process or in the secondary structure of the single-stranded RNA.

[0132] 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 comprising an RNA construct for expressing an alphavirus replicase, and one RNA molecule comprising 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.

[0133] Self-replicating RNA can be conveniently prepared by in vitro transcription (IVT). IVT can use a (cDNA) template that is generated and propagated in bacteria as a plasmid or that is synthetically produced (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). For example, a DNA-dependent RNA polymerase (e.g., bacteriophage T7, T3, or SP6 RNA polymerase) can be used to transcribe self-replicating RNA from a DNA template. Appropriate capping and poly A addition reactions can be used as needed (although the poly A of the replicon is typically encoded within the DNA template). These RNA polymerases have strict requirements for the 5' nucleotide of the transcript, and in some embodiments, these requirements must match those of the encoded replicase to ensure that the IVT-transcribed RNA can effectively serve as a substrate for its own encoded replicase.

[0134] The self-replicating RNA may comprise (in addition to any 5' cap structure) one or more nucleotides having modified nucleobases. The RNA used in the present invention ideally comprises only phosphodiester bonds between nucleosides, but in some embodiments, it may contain phosphoramidate bonds and / or methylphosphonate bonds.

[0135] The self-replicating RNA molecule can encode a single heterologous polypeptide antigen (i.e., antigen), or optionally, two or more heterologous polypeptide antigens are linked together in such a way that when expressed as an amino acid sequence, each sequence retains its identity (e.g., in tandem). The heterologous polypeptides generated from the self-replicating RNA can then be generated as fusion polypeptides or engineered in such a way as to generate separate polypeptide or peptide sequences.

[0136] The self-replicating RNA molecules described herein can be engineered to express multiple nucleotide sequences, thereby allowing 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 immunomodulators (which can enhance the generation of an immune response).

[0137] If necessary, various in vitro or in vivo test methods well known to those skilled in the art can be used to screen or analyze self-replicating RNA molecules to confirm their treatment and prevention properties. For example, the vaccine comprising self-replicating RNA molecules can be tested for the proliferation induction or effector function of specific purpose lymphocyte types (such as B cells, T cells, T cell lines and T cell clones). For example, splenocytes from immunized mice can be separated, and cytotoxic T lymphocytes have the ability to lyse autologous target cells, and the autologous target cells include self-replicating RNA molecules encoding antigens. In addition, helper T cell differentiation can be analyzed by ELISA measurement of TH1 (IL-2 and IFN-γ) and / or TH2 (IL-4 and IL-5) cytokine proliferation or production, or directly analyzed in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry.

[0138] The ability of the self-replicating RNA molecules encoding antigens to induce humoral immune responses can also be tested, for example, by inducing B cells to produce antibodies specific for the target antigen. These assays can be performed using, for example, peripheral B lymphocytes from immunized individuals. Such assays are known to those skilled in the art. Other assays that can be used to characterize self-replicating RNA molecules can involve detecting the expression of target cell-encoded antigens. For example, FACS can be used to detect antigen expression on the cell surface or within the cell. Another advantage of FACS selection is that different levels of expression can be classified; sometimes lower expression may be required. Other suitable methods for identifying cells expressing specific antigens include panning with monoclonal antibodies on a plate or capturing with magnetic beads coated with monoclonal antibodies.

[0139] In one embodiment, the self-replicating RNA of the present disclosure can include a sequence encoding a self-cleaving peptide. The self-cleaving peptide can be, but is not limited to, the 2A cleavage region (herein referred to as "2A") of foot-and-mouth disease virus (FMDV). The 2A peptide has the amino acid sequence of SEQ ID NO:3. On the one hand, the 2A peptide cuts between the last glycine and the last proline. The 2A peptide causes the ribosome to skip the 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 peptide downstream. In an embodiment, the 2A peptide can 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 can be between the first coding region A (e.g., encoding hli-HBc) and the second coding region B (e.g., encoding HBs).

[0140] In another embodiment, the self-replicating RNA of the present disclosure can include a sequence encoding an internal ribosome entry site (IRES). The effect of the IRES element is similar to that of another ribosome recruitment site, allowing translation to occur in the internal region of the mRNA, thereby causing the downstream ORF to be translated separately from the upstream ORF. In an embodiment, IRES can be used to separate the coding regions of two or more polypeptides of interest. As a non-limiting example, the nucleotide sequence encoding IRES can be between the first coding region A (e.g., encoding hli-HBc) and the second coding region B (e.g., encoding HBs).

[0141] In an embodiment, the self-replicating RNA has the following configuration: 5' cap / 5'UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hli / HBc / 2A / HBs / 3'UTR / poly A.

[0142] In an embodiment, the self-replicating RNA has the following configuration: 5' cap / 5'UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hli / HBc / IRES / HBs / 3'UTR / poly A.

[0143] In an embodiment, the self-replicating RNA has the following configuration: 5' cap / 5'UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hli / HBc / 3'UTR / poly A.

[0144] In an embodiment, the self-replicating RNA has the following configuration: 5' cap / 5'UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / HBs / 3'UTR / poly A.

[0145] In an embodiment, the self-replicating RNA has the following configuration: 5' cap / 5'UTR-nonstructural proteins (NSP) 1-4 / subgenomic promoter / hli / HBs / 3'UTR / poly A.

[0146] In one embodiment, the mRNA is a non-replicating mRNA. In a second embodiment, the mRNA is a replicating mRNA.

[0147] Lipid nanoparticles (LNPs)

[0148] Lipid nanoparticles (LNPs) are non-virion liposomal particles in which mRNA can be encapsulated. Unprotected RNA itself may 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. LNP delivery systems and methods for their preparation are known in the art. LNPs may include some external mRNA (e.g., on the surface of the LNP), but it is desired that at least half of the mRNA (and suitably at least 85%, in particular at least 95%, e.g., all) is encapsulated.

[0149] First lipid

[0150] In some embodiments, the LNP comprises a lipid comprising: a first lipid (i.e., a cationically ionizable lipid), an optional sterol (e.g., cholesterol), an optional polymer-conjugated lipid, and an optional second lipid (i.e., an optional anionic lipid or an optional neutral lipid, including a 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 comprises a lipid from 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, W O2018 / 081480, WO2015 / 074085, WO2018 / 1703322, U.S. Patent Application Nos. 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. Patent Application Nos. 61 / 905,724 or 15 / 614,499, or U.S. Patent Nos. 8,802,863, 9, or 11,285,222 (including the ionizable lipids and PEG-lipids mentioned therein).

[0151] In some embodiments, the cationically ionizable lipid comprises an amine, which may be a tertiary amine that becomes charged depending on the pH of the solution in which the cationically ionizable lipid is present, when compared to the pKa of the cationically ionizable lipid. In some embodiments, when the pH of the solvent in which the cationically ionizable lipid is present is above the pKa, at least half of the cationically ionizable lipid is neutrally charged and the amine is a tertiary amine; when the pH of the solvent in which the cationically ionizable lipid is present is below the pKa, at least half of the cationically ionizable lipid is positively charged. In this regard, in some embodiments, but without being limited to a particular theory, it is believed that the positive charge of the ionizable lipid is distributed across the amine, and thus, when the pH of the solvent in which the cationically ionizable lipid is present is below the pKa, the amine is positively charged. Because amines can vary between neutral and positively charged depending on the pH of the solution relative to the pKa of the cationically ionizable lipid, and without being bound by a particular theory, amines are ionizable amines.

[0152] When the amine is a tertiary amine and when the cation ionizable lipid is neutral, the cation ionizable lipid will be further described, but such description should not limit the cation ionizable lipid to lacking the ability to become positively charged. That is, the lipid is described herein in the tertiary amine state and is neutral, and it is not necessary to describe the cation ionizable lipid when the tertiary amine is charged. In some embodiments, in addition to the above-mentioned ionizable amine, the cation ionizable lipid further comprises a head group (R H ) and fatty acid tails (R FA1 or R FA2 In some embodiments, the cationic ionizable lipid further comprises (in addition to the ionizable amine described above) a head group and at least two fatty acid tails (R FA1 and R FA2 ), for example in Formula I.

[0153] Formula I:

[0154]

[0155] In some embodiments, the amine provides a branch point between the head group and the fatty acid tail. In some embodiments, the fatty acid tail (R FA ) or at least two fatty acid tails (ie, R FA1 、R FA2 ...) next to the ionizable amine. In some embodiments, the fatty acid tail comprises or at least two fatty acid tails comprise a biodegradable group (ie, R BD1 or R BD2), and at least two fatty acid tails are the same or independent of each other. In some embodiments, at least two fatty acid tails each contain a biodegradable group, such as in Formula II, and the biodegradable groups are the same or independent of each other. In some embodiments, the biodegradable groups include, 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 wherein R a is hydrogen or C1-C 12 In some embodiments, the fatty acid comprises or the at least two fatty acids comprise a C1-C1 between the amine branch point and the biodegradable group. 12 Alkyl, C1-C 12 Alkylene, or C1-C 12 Alkenylene (i.e., R FC1 and R FC2 In some embodiments, the fatty acid comprises or the two or more fatty acids comprise an ionizable amine and a 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 or C 13 -C 17 Alkylene (ie R FC3 and R FC4 ), for example in Formula II.

[0156] Formula II:

[0157]

[0158] Where: R FC1 and R FC2 Each independently is C1-C 12 Alkyl, C1-C 12 Alkylene or C1-C 12 alkenylene;

[0159] R FC3 and R FC4 Each 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, C 13 -C 17 Alkyl, C 13 -C 17 alkylene;

[0160] R BD1 and R BD2 Each independently represents: -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 wherein R a is hydrogen or C1-C 12 alkyl.

[0161] In some embodiments, C6-C 24 Alkyl or C6-C 24 The alkylene group is attached to the biodegradable group at the following positions: 12 、C7-C 11 、C8-C 10 In some embodiments, the C6-C 24 Alkyl or C6-C 24 Alkylene groups independently include:

[0162]

[0163] In some embodiments, the head group comprises, consists of, is, or has: a first group (ie, R H1 ) and the second group (ie, R H2 ), wherein the first group is 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 ; where R 4 It is C1-C 12 Alkyl and R 5 is H or C1-C6 alkyl. In some embodiments, the head group comprises -(CH2)6OH, -(CH2)5OH, -(CH2)4OH, -(CH2)3OH, -(CH2)2OH, or -CH2OH in linear or branched form. In some embodiments, the cationically ionizable lipid comprises, consists of, or is [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or 9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecanyloxy)hexyl]amino}octanoate.

[0164] In some embodiments, the cationic ionizable lipid is:

[0165]

[0166]

[0167] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV28 having the structure:

[0168]

[0169] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV31 having the structure:

[0170]

[0171] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV33 having the structure:

[0172]

[0173] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV37 having the structure:

[0174]

[0175] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV39, i.e., 2,5-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)benzyl 4-(dimethylamino)butyrate):

[0176]

[0177] RV39

[0178] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV42 having the structure:

[0179]

[0180] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV44 having the structure:

[0181]

[0182] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV73 having the structure:

[0183]

[0184] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV75 having the structure:

[0185]

[0186] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV81 having the structure:

[0187]

[0188] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV84 having the structure:

[0189]

[0190] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV85 having the structure:

[0191]

[0192] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV86 having the structure:

[0193]

[0194] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV88 having the structure:

[0195]

[0196] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV91 having the structure:

[0197]

[0198] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV92 having the structure:

[0199]

[0200] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV93 having the structure:

[0201]

[0202] In embodiments, the cationic ionizable 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-diyl dioctanoate (RV94) having the structure:

[0203]

[0204] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV95 having the structure:

[0205]

[0206] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV96 having the structure:

[0207]

[0208] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV97 having the structure:

[0209]

[0210] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV99 having the structure:

[0211]

[0212] In embodiments, the cationic ionizable lipid comprises, consists of, consists essentially of, or is: RV101 having the structure:

[0213]

[0214] In some embodiments, the cationically ionizable lipid comprises, consists of, consists essentially of, or is a lipid having the structure of Formula III:

[0215]

[0216] wherein n=an integer from 1 to 3 and

[0217] (i) R1 is CH3, R2 and R3 are both H, and Y is C; or

[0218] (ii) R1 and R2 together are CH2-CH2 and together with nitrogen form a five-, six- or seven-membered heterocycloalkyl, R3 is CH3, and Y is C; or

[0219] (iii) R1 is CH3, R2 and R3 are absent, and Y is O;

[0220] Where o is 0 or 1;

[0221] Where X is:

[0222] (i) wherein R4 and R5 are independently C with one or two cis-olefinic groups at one or both of the ω6 and ω9 positions 10-20 hydrocarbon chain; or

[0223] (ii) –CH(–R6)–R7, where

[0224] (1) R6 is –(CH2) p –O–C(O)–R8 or –C p –R8;

[0225] (2) R7 is –(CH2) p' –O–C(O)–R8' or –C p' –R8',

[0226] (3) p and p' are independently 0, 1, 2, 3 or 4; and

[0227] (4) R8 and R 8’ Independently

[0228] (A) a –C with one or two cis-olefinic groups at one or both of the ω6 and ω9 positions 8-20 hydrocarbon chain;

[0229] (B)–C 1-3 –C(–O–C 6-12 )–O–C 6-12 saturated or unsaturated hydrocarbon chains;

[0230] (C)–C 6-16 saturated hydrocarbon chains;

[0231] (D)–C(–C 6-16 )–C 6-16 saturated or unsaturated hydrocarbon chains;

[0232] (E)–C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 saturated or unsaturated hydrocarbon chains; and

[0233] (F)–C 6-16Saturated or unsaturated hydrocarbon chain.

[0234] In embodiments, 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 nitrogen form a five-, six-, or seven-membered heterocycloalkyl, R3 is CH3, and Y is C. In some embodiments, R1 is CH3, R2 and R3 are absent, and Y is O.

[0235] In an embodiment, X is wherein R4 and R5 are independently C with one or two cis-olefinic groups at one or both of the ω6 and ω9 positions 10-20 Hydrocarbon chain.

[0236] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0237] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0238] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0239] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2)p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C(-C 6-16 )-C 6-16 Saturated or unsaturated hydrocarbon chain.

[0240] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0241] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0242] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0243] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p'–O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0244] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0245] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )-C 6-16 Saturated or unsaturated hydrocarbon chain.

[0246] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0247] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p'–O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0248] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is -C 6-16 Saturated hydrocarbon chain; and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0249] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p' –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is -C 6-16 a saturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0250] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0251] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0252] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2)p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0253] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0254] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0255] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0256] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C(–C 6-16 )–C 6-16a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0257] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0258] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0259] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0260] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0261] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0262] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0263] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0264] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0265] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0266] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ -O-C(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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0267] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ -O-C(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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0268] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ -O-C(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; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0269] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ -O-C(O)-R8', p and p' are independently 0, 1, 2, 3 or 4; and R8 is -C 6-16Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0270] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ -O-C(O)-R8', p and p' are independently 0, 1, 2, 3 or 4; and R8 is -C 6-16 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0271] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –(CH2) p’ -O-C(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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0272] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0273] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0274] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p–O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0275] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0276] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0277] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0278] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions;8-20 Hydrocarbon chain.

[0279] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7–C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0280] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0281] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0282] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0283] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0284] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0285] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0286] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0287] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0288] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0289] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0290] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0291] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0292] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C6-16 Saturated hydrocarbon chain.

[0293] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0294] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0295] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0296] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0297] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –Cp’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0298] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0299] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0300] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0301] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –C p’–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0302] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0303] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0304] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0305] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0306] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, R7 is –Cp’ -R8', p and p' are independently 0, 1, 2, 3 or 4; and R8 is -C 6-16 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0307] In an embodiment, X is -CH(-R6)-R7, R6 is -(CH2) p –O–C(O)–R8, 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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0308] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0309] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0310] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0311] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0312] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0313] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C(O)-containing olefinic group having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions. 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0314] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0315] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3–C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0316] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0317] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0318] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0319] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0320] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0321] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0322] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0323] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0324] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C4-12 Saturated or unsaturated hydrocarbon chain.

[0325] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0326] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0327] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0328] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0329] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C(–C 6-16 )–C6-16 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0330] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0331] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0332] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0333] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0334] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0335] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0336] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0337] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ –O–C(O)–R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0338] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2)p’ -O-C(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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0339] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ -O-C(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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0340] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ -O-C(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; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0341] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ -O-C(O)-R8', p and p' are independently 0, 1, 2, 3 or 4; and R8 is -C 6-16 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0342] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –(CH2) p’ -O-C(O)-R8', p and p' are independently 0, 1, 2, 3 or 4; and R8 is -C 6-16 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0343] In an embodiment, X is -CH(-R6)-R7, R6 is -C p–R8, R7 is –(CH2) p’ -O-C(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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0344] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0345] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0346] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0347] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0348] In an embodiment, X is -CH(-R6)-R7, R6 is -Cp –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0349] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is a –C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0350] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7–C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0351] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0352] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7–C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0353] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0354] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0355] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7–C p x-R8', p and p' are independently 0, 1, 2, 3 or 4; R8 is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0356] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is a -C having one or two cis-olefin groups at one or both of the ω6 and ω9 positions. 8-20 Hydrocarbon chain.

[0357] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’–R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0358] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0359] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0360] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 Saturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0361] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; R8 is –C 6-16 a saturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0362] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C(–C 6-16 )–C 6-16a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0363] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p x-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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0364] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0365] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0366] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0367] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0368] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0369] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7–C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0370] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated hydrocarbon chain.

[0371] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0372] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7 is –C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0373] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7–C p’ –R8′, p and p′ are independently 0, 1, 2, 3 or 4; and R8 is –C[–C–O–C(O)–C 4-12 ]–C–O–C(O)–C 4-12 a saturated or unsaturated hydrocarbon chain; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0374] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is a -C having one or two cis-olefinic groups at one or both of the ω6 and ω9 positions; 8-20 Hydrocarbon chain.

[0375] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C 1-3 –C(–O–C 6-12 )–O–C 6-12 Saturated or unsaturated hydrocarbon chain.

[0376] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, R7–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; and R8' is -C 6-16Saturated hydrocarbon chain.

[0377] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C(-C 6-16 )–C 6-16 Saturated or unsaturated hydrocarbon chain.

[0378] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C[-C-O-C(O)-C 4-12 ]–C–O–C(O)–C 4-12 Saturated or unsaturated hydrocarbon chain.

[0379] In an embodiment, X is -CH(-R6)-R7, R6 is -C p –R8, 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; and R8' is -C 6-16 Saturated or unsaturated hydrocarbon chain.

[0380] In some embodiments, the cationic ionizable lipid comprises a cationic lipid from 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 / 07 5531, WO2018 / 081480, WO2015 / 074085, WO2018 / 1703322, U.S. Patent Application Publication Nos. 20220081392, 20220072155, 20220040285, 20210395188, 20210251898, 20210128488, 20210122703, 20210122702, 20210107861, 20200283372, 20200172472, 20200163878, 202001 21809, 20200046838, 20190359556, 20190314524, 20190274968, 20190270697, 20190022247, 20180185516, 20170283367, 20170157268, 20170119904, 20160376224, 20160317676, or 20150376115, U.S. Patent Nos. 61 / 905,724 or 15 / 614,499, or U.S. Patent 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, 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 therein as ionizable lipids).

[0381] In some embodiments, the cationic ionizable lipid comprises a first group and two biodegradable hydrophobic tails. In some embodiments, the first group comprises a central portion and a head group, wherein the first group can be positively charged. In some embodiments, the central portion is directly bonded to each of the two biodegradable groups. In some embodiments, the central portion is directly bonded to the head group. In some embodiments, the central portion 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: -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 wherein R a is hydrogen or C1-C 12In some embodiments, one of the two biodegradable tails or each of the two biodegradable tails: 1) has a terminal hydrophobic chain that is a branched alkyl group and the terminal end, 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 terminal end from the biodegradable group.

[0382] In some embodiments, the cationic ionizable lipid comprises bis(2-methacryloyl)oxyethyl disulfide (DSDMA, CAS No. 36837-97-5), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylamino propane (DLinDMA), 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenoyloxy-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-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), ICE (imidazolyl-based), HGT50 00, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) HGT4003, 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedioic acid (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-diol Methylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its 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)-heptatriacontane-6,9,28,31-tetraen-19-yl-4-(dimethyl)- amino)butyrate (MC3), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopentyl[d][1,3]dioxol-5-amine)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1 -yl)ethyl azanediyl) 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,N 16-double undecane-4,7,10,13-tetraazahexadecane-1,16-diamide), (6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), (Commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY), (commercially available cationic liposomes containing N-(1-(2,3-dioleoyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-trifluoroacetate (DOSPA), (DOPE), from GIBCO / BRL), or (commercially available cationic lipids comprising dioctadecylamido glycylcarboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the above. Other suitable cations include those described in International Patent Publication WO 2010 / 053572 (particularly CI 2-200 described in paragraph

[00225] ) and WO 2012 / 170930 (both of which are incorporated herein by reference), HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see U.S. Patent Application Publication No. 20150140070A1).

[0383] Representative cationic ionizable lipids include, but are not limited to, 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-linoleoyloxy-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-methylpiperazinyl)propane (DLin-MPZ), 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), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), MC3 (U.S. Patent Publication No. 20100324120).

[0384] Various amphiphilic lipids can form a bilayer in an aqueous environment to encapsulate the RNA-containing aqueous core as LNPs. These lipids can have anionic, cationic, or zwitterionic hydrophilic head groups. Some phospholipids are anionic, while others are zwitterionic and others are cationic. Suitable phospholipid classes include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol, with some useful phospholipids 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-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 1,2-dilinoleyloxy-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 are 1-2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and dodecylphosphocholine. The lipid can be saturated or unsaturated. Preferably, at least one unsaturated lipid is used to prepare liposomes. If the unsaturated lipid has two tails, both tails can be unsaturated, or it can have a saturated tail and an unsaturated tail.

[0385] Other useful LNPs are described in the following references: 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 the following references: WO2012 / 006376 and Geall et al. (2012) PNAS USA. Sep 4; 109(36): 14604-9.

[0386] polyethylene glycol-conjugated lipids

[0387] In some embodiments, the LNP comprises polyethylene glycol-conjugated (PEG-conjugated) lipids. In some embodiments, the PEG-conjugated lipids comprise polyethylene glycol (PEG) of various lengths and molecular weights.

[0388] In some embodiments, the PEG in the PEG-conjugated lipid has a median molecular weight of 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 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.

[0389] 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-glycero-3-methoxypolyethylene glycol.

[0390] Second lipid

[0391] In some embodiments, LNP also includes a second lipid, which includes an anionic lipid, a neutral lipid, or a zwitterionic lipid. In some embodiments, the neutral lipid includes a neutral zwitterionic lipid. In some embodiments, the anionic lipid, the neutral lipid, or the zwitterionic lipid includes a phosphate group (i.e., a phospholipid), choline, or a sphingolipid.

[0392] In some embodiments, the second lipid comprises 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (17:0PE), 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (06:0PE), 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (08:0PE), 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (10:0PE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0PE), 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (15:0PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (18:0PE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (14:0PE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (16:1PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (18:1(Δ9-Trans)PE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (18:2PE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (18:3 ...18:1(Δ9-Trans)PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:2PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:3PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1(Δ9-Trans)PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:2PE), 1,2 Docosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6PE), 1,2-arachidonic acid-sn-glycero-3-phosphoethanolamine (20:4PE), 1-pentadecanoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (15:0-18:1PE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1PE), 1-palmitoyl-2-arachidonic acid enoyl-sn-glycero-3-phosphoethanolamine (16:0-20:4PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2PE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6PE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1Lyso PE), 1-hydroxy-2-oleoyl-sn-glycero-3-phosphoethanolamine (2-18:1Lyso PE),1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (16:0Lyso PE), 1-tridecanoyl-sn-glycero-3-phosphoethanolamine (13:0Lyso PE), 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphoethanolamine (17:1Lyso PE), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:0Lyso PE), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (14:0Lyso PE), L-α-phosphatidylethanolamine, 1,2-dibutyryl-sn-glycero-3-phosphocholine (04:0PC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (7:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0PC), 1,2-diananoyl-sn-glycero-3-phosphocholine (9:0PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (11:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (12:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (13:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (14:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (15:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (16:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (17:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (18:0PC), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (19:0PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (1 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (11:0PC), 1,3-dipalmitoyl-rac-glycero-2-phosphocholine (16:02-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 (15:0PC), 1,2-dipalmitoyl-sn-glycero 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17:0PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (19:0PC), 1,2-diacaproyl-sn-glycero-3-phosphocholine (20:0PC), 1,2-dihexenoyl-sn-glycero-3-phosphocholine (21:0PC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (22: 0PC), 1,2-ditricosyl-sn-glycero-3-phosphocholine (23:0PC), 1,2-diicosanoyl-sn-glycero-3-phosphocholine (24:0PC), 1,2-diicosenoyl-sn-glycero-3-phosphocholine (18:1(11-cis)PC), 1,2-bis[(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-Disoleuroyl-sn-glycero-3-phosphocholine (18:1(Δ6-cis)PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-ditransoleoyl-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 1'-rac-glycerol), 1,2-dimyristoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dipentadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-distearoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-di-antioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-( 1'-rac-glycerol), 1,2-dilinolenoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-diamidolenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], 1,2-diicosahexenoyl-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-diicosenoyl-sn-glycero-3-phosphocholine (20:1(cis)PC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (2 0:4(cis)PC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (22:1(cis)PC), 1,2-dineuroyl-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 (15:0-18:1)PC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0PC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0PC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0PC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0PC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2PC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (16:0-22:6PC), 1-stearoyl-2-myristoyl sn-glycero-3-phosphocholine (18:0-14:0PC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0PC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1PC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6PC), 1-oleoyl-2-myristoyl-sn- Glycerol-3-phosphocholine (18:1-14:0PC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0PC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0PC), 1-(8Z-octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine (18:1(n10)-16:0PC), 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)P C), 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-tetracosanoyl-2-hydroxy-sn-glycero-3-phosphocholine, 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-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestyhemisuccinoyl-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 16.0PE), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), N-tetracosanoyl-D-erythro-sphingosine phosphoethanolamine, or sphingomyelin.

[0393] sterols

[0394] In some embodiments, the lipid nanoparticles further comprise a sterol. In some embodiments, the sterol comprises cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, symmetosterol, lathostheritol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydrolanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol 95, 4,4-dimethyl (d6)-cholester-8(9), 14-diene-3β-ol (dihydro-FF-MAS-d6), 4,4-dimethyl (d6)-cholester-8(9)- ene-3β-ol (dihydro T-MAS-d6), yeast enol, sitostanol, campesterol, campesterol (camperstanol), 7-dehydrodeaminostearate, pregnenolone, 4,4-dimethyl-cholest-8(9)-ene-3β-ol (dihydro T-MAS), Δ5-avenasterol, brassicasterol, dihydro FF-MAS, 24-methylene cholesterol, oxysterols, deuterated sterols, fluorinated sterols, sulfonated sterols, phosphorylated sterols, A-ring substituted sterols, cholester-5-ene-3β,4β-diol, 5α-cholest-3β-ol, 4-cholestene-3-one, cholester-8(9),24-dien-3-one, cholester-8(9),24-dien-3-one, 2,2, 3,4,4-pentadeuterium-5a-cholest-3β-ol, cholesterylphosphocholine, cholesteryl-d7 pentadecanoate, cholesteryl-d7 palmitate, B-ring substituted sterols, cholestanol, 5β,6β-epoxy-d7,3β-hydroxy-5-cholestene-7-one, 6α-hydroxy-5α-cholestane, cholestanol, 5α,6α-epoxy, cholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, cholestanol, 5α,6α-epoxy-d7,Δ5,7-cholesterol, cholest-5,8(9)-dien-3β-ol, cholest-5,8(14)-dien-3β-ol, 7α-hydroxy-4-cholestene-3-one, yeast enol-d7, yeast enol Alcohol, 7-dehydrodeaminosterol, 3b,5a-dihydroxycholest-6-one, D-ring substituted sterol, 3β-hydroxy-5α-cholest-8(14)-ene-15-one, 3β-hydroxy-5α-cholestane-15-one, 5α-cholest-8(14)-ene-3β,15α-diol, 5α-cholest-8(14)-ene-3β,15β,-diol, lanosterol 95, 5α-7,24-cholestadiene, 14-dehydroyeastenol, ergosta-5,7,9(11),22-tetraene-3β-ol, cholesta-5-ene-3β,25-diol, cholesta-(25R)-5-ene-3β,27-diol, 24(R / S), 25-epoxycholesterol, 24(S),25-Epoxycholesterol, 24(R / S), 25-Epoxycholesterol-d6, 5-cholestane-3β,22(S)-diol, 5-cholestane-3β,22(R)-diol, 5-cholestane-3β,24(S)-diol, 5-cholestane-3β,24(R)-diol, 27-hydroxy-4-cholestene-3-one, campestanol, N,N-dimethyl-3β-hydroxycholeamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3β-hydroxycholeamide, 25,27-dihydroxycholesterol, 5-cholestane-3β,20α-diol, 24S,25-epoxy-5α-cholestane-8(9)-ene-3β-ol, 24(S / R), 25-epoxylanost-8(9)-en-3β-ol, 7-keto-27-hydroxycholesterol, 7α,27-dihydroxy-4-cholesten-3-one, 7α,27-dihydroxycholesterol, 7β,27-dihydroxycholesterol, 5α,6β-dihydroxycholesterol, 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, estrogen, estradiol, corticosterone, cortisol, or 24S,27-dihydroxycholesterol.

[0395] Preparation of mRNA-encapsulated LNPs

[0396] As mentioned above, in some embodiments, the mRNA molecule is encapsulated in the LNP. In some embodiments, the lipid of mRNA and LNP can be mixed and / or purified, thereby providing described comprising or being encapsulated therein. In some embodiments, the lipid of mRNA and LNP can be mixed and / or purified, thereby providing the mRNA of the above-mentioned ratio that comprises or is encapsulated in the LNP.

[0397] In some aspects, a method for obtaining a composition is provided, the composition comprising mRNA and LNP, wherein the mRNA is encapsulated in LNP with the above ratio, and wherein the LNP comprises the above lipid; the method includes mixing a first solution comprising the recombinant RNA molecule and a second solution comprising the above lipid. In some embodiments, mixing is carried out at least by a T-type mixer, microfluidics or impact jet mixer. In some embodiments, the first solution also comprises a citrate buffer (e.g., sodium citrate) or an acetate buffer (e.g., sodium acetate).

[0398] 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 aliphatic alcohol (e.g., C1 to C8 alcohol). In some embodiments, the aliphatic alcohol comprises ethanol, propanol, isopropanol, butanol, tert-butyl alcohol, isobutanol, amyl alcohol, benzyl alcohol and hexanol. In some embodiments, the organic solvent comprises an alcoholic solution. In some embodiments, the organic alcohol solution comprises 70% to 100% by volume of ethanol.

[0399] In some embodiments, the method includes mixing a first solution of the above-mentioned lipid comprising a recombinant RNA molecule and LNP with a second solution being an aqueous solution. In some embodiments, the RNA of LNP and lipid 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 alcohol (e.g., C1 to C8 alcohol). In some embodiments, aliphatic alcohol includes ethanol, propanol, isopropanol, butanol, tert-butyl alcohol, isobutanol, amyl alcohol, benzyl alcohol and hexanol. In some embodiments, the organic solvent includes an alcoholic solution. In some embodiments, the organic alcohol solution includes 70% to 100% by volume of ethanol. In some embodiments, the organic alcohol solution includes 70% to 100% by volume of ethanol and 30% to 0% by volume of benzyl alcohol. 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 solution and the second solution 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.

[0400] In some embodiments, the mixing of the first and second solutions (i.e., any of the above two methods) is carried out at a pH of 4.5 to the pKa of the first lipid (e.g., a cation ionizable lipid), thereby obtaining a first mixture. In some embodiments, the mixing of the first and second solutions is carried out at a pH of 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 cation ionizable lipid), thereby obtaining a first mixture. In some embodiments, the method further comprises a first increase, which is to increase the pH of the first mixture to a pKa equal to or higher than the first lipid, thereby obtaining a pH-regulated first mixture. In some embodiments, the first increase results in a pH adjusted first mixture whose pH is from the pKa of the first lipid (e.g., a cationically ionizable 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.

[0401] In some embodiments, the first increase or purification includes cross-flow filtration or tangential flow filtration. In some embodiments, the first increase or purification also includes transferring the composition comprising LNP and recombinant RNA molecules to a third solution different from the first solution. In some embodiments, the third solution comprises phosphate buffered saline. In some embodiments, transfer includes dialysis. In some embodiments, tangential flow filtration includes using a hollow fiber filter. In some embodiments, the hollow fiber includes a polyethersulfone hollow fiber filter or a polysulfone hollow fiber filter.

[0402] In some embodiments, the first enrichment or purification comprises passing the LNP / RNA mixture through an ion exchange solid support prior to the filtration described above. In some embodiments, the ion exchange solid support comprises an anion exchange column or a cation exchange column.

[0403] In some embodiments, before mRNA is mixed with the lipid of LNP, the lipid of LNP is mixed with an organic solvent to obtain a concentrated stock solution (for example, a stock solution lipid / organic solvent mixture). In some embodiments, mixing (for example, a stock solution lipid / organic solvent mixture is stirred, shaken, vortexed, ultrasonically treated or agitated at 25 DEG C to 37 DEG C) for at least 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 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 or 40 minutes to form a homogeneous stock solution lipid / organic solvent mixture. In some embodiments, mixing (e.g., stirring, shaking, vortexing, sonicating, or agitating the stock lipid / organic solvent mixture at 25° C. to 37° C.) is performed for no more than 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 to form a homogenous stock lipid / organic solvent mixture. In view of the above embodiments, it is contemplated that any of the above "at least" amounts of time and "no more than" amounts of time 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. to 37° C. for 5 minutes to 19 minutes).

[0404] plan

[0405] The present invention relates to a method for treating chronic hepatitis B infection (CHB) by administering to a human 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 first and second mRNAs are co-administered. In a first embodiment of co-administration, the first and second mRNAs are in separate LNP formulations that are mixed into a single composition before administration. This can be done at the bedside immediately before administration. In a second embodiment of co-administration, the first and second mRNAs are co-formulated into a single LNP. In a third embodiment of co-administration, the first and second mRNAs are co-filled into a single vial.

[0406] 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 includes the resulting composition formed by mixing separate LNP formulations.

[0407] The present invention also 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 such compositions, the first and second mRNAs can be encapsulated by separate LNPs, or the first and second mRNAs can be formulated in the same LNP.

[0408] The present invention encompasses a method for treating chronic hepatitis B infection (CHB) by administering to a human a combination of an mRNA encoding at least one hepatitis B virus antigen and at least one recombinant hepatitis B polypeptide. Components (e.g., mRNA and recombinant hepatitis B polypeptide) can be administered sequentially in a heterologous primary immunization-boosting regimen. If a heterologous primary immunization-boosting regimen is used, mRNA is preferably administered as a primary immunization dose, and at least one recombinant hepatitis B polypeptide is administered as a booster dose. In this regimen, the method includes first administering mRNA, and then administering the recombinant hepatitis B polypeptide. In another aspect, at least one recombinant hepatitis B polypeptide is administered as a primary immunization dose, and mRNA is administered as a booster dose. In this regimen, the method includes administering the recombinant hepatitis B polypeptide, and then administering the mRNA. In a further aspect, at least one recombinant hepatitis B polypeptide is administered as a primary immunization dose, and mRNA is administered as a booster dose together with an adjuvanted recombinant protein. In this regimen, the method includes administering the recombinant hepatitis B polypeptide, and then administering the mRNA and recombinant protein.

[0409] In some embodiments, the at least one hepatitis B virus polypeptide is at least one of a recombinant hepatitis B surface antigen (HBs), a 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 surface antigen (HBs) and hepatitis B virus core (HBc) antigens. In this embodiment, the adjuvant is preferably AS01.

[0410] In such a scheme, there may be multiple priming and / or boosting doses. In one embodiment, there is a single priming of mRNA and multiple subsequent doses of at least one recombinant hepatitis B polypeptide. For example, two doses of recombinant hepatitis B polypeptide. In another embodiment, there are multiple priming doses of mRNA and multiple subsequent doses of recombinant hepatitis B polypeptide. For example, two doses of mRNA followed by two doses of recombinant hepatitis B polypeptide. In another embodiment, there is a single priming of at least one recombinant hepatitis B polypeptide and multiple subsequent doses of the mRNA. In yet another embodiment, there are multiple priming doses of recombinant hepatitis B polypeptide and multiple subsequent doses of mRNA.

[0411] In other embodiments, mRNA is administered simultaneously with at least one recombinant hepatitis B polypeptide. Further doses of these components may be subsequently administered at a later time. In some embodiments, mRNA is administered simultaneously with at least one recombinant hepatitis B polypeptide. At least one recombinant hepatitis B polypeptide is at least one of recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc), or a combination thereof. Recombinant HBc can be full-length or truncated, preferably truncated. At least one recombinant hepatitis B polypeptide may be administered with or without an adjuvant. In one embodiment, mRNA is administered simultaneously with an adjuvanted recombinant hepatitis B polypeptide, wherein the recombinant hepatitis B polypeptide includes both hepatitis B surface antigen (HBs) and hepatitis B virus core (HBc) antigens. In this case, the adjuvant is preferably AS01.

[0412] 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 virus core antigen (HBc) may have the amino acid sequence of SEQ ID NO: 2 or 11. Preferably, HBc has the amino acid sequence of SEQ ID NO: 2.

[0413] In all of these regimens, at least one recombinant hepatitis B polypeptide can be administered with a suitable adjuvant. Suitable adjuvants are those that can enhance the immune response in subjects with chronic diseases and compromised immune capacity. CHB patients are characterized by their inability to mount effective innate and adaptive immune responses to the virus, making effective vaccine development challenging. In these patients, a key function of adjuvanted vaccine formulations should be to direct the cell-mediated immune response toward the T helper 1 (Th1) spectrum, which is believed to be critical for eliminating intracellular pathogens.

[0414] Examples of suitable adjuvants include, but are not limited to, 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., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immunostimulating complexes (ISCOMS), liposomes, or biodegradable microspheres), viral particles, bacterial adjuvants (e.g., monophosphoryl lipid A (MPL) ( For example, 3-O-deacylated monophosphoryl lipid A (3D-MPL) or muramyl peptide), synthetic adjuvants (e.g., non-ionic block copolymers, muramyl peptide analogs or synthetic lipid A), synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine) 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 or squalene) and / or a bacterial adjuvant (e.g., monophosphoryl lipid A (MPL) (e.g., 3-O-deacylated monophosphoryl lipid A (3D-MPL))).

[0415] A suitable adjuvant is monophosphoryl lipid A (MPL), in particular 3-O-deacylated monophosphoryl lipid A (3D-MPL). Chemically, it is typically provided as a mixture of 3-O-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2122204B, which also discloses the preparation of diphosphoryl lipid A and its 3-O-deacylated variants. Other purified and synthetic lipopolysaccharides have been described [U.S. Patent No. 6,005,099 and EP0729473B1; Hilgers, 1986; Hilgers, 1987; and EP0549074B1].

[0416] Saponins are also suitable adjuvants [Lacaille-Dubois, 1996]. For example, the saponin Quil A (derived from the South American soapberry tree) and its fractions are described in U.S. Pat. No. 5,057,540 and Kensil, 1996; and EP 0 362 279 B1. Purified fractions of Quil A are also known as immunostimulants, such as QS21 and QS17; their preparation methods are disclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1. The use of QS21 is further described in Kensil, 1991. Combinations of QS21 and polysorbates or cyclodextrins are also known (WO 99 / 10008). Particulate adjuvant systems containing Quil A fractions such as QS21 and QS7 are described in WO 96 / 33739 and WO 96 / 11711.

[0417] Adjuvants such as those described above can 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 can be formulated with aluminum hydroxide (EP 0 689 454) or oil-in-water emulsions (WO 95 / 17210); QS21 can be formulated with cholesterol-containing liposomes (WO 96 / 33739), oil-in-water emulsions (WO 95 / 17210) or alum (WO 98 / 15287).

[0418] Combinations of adjuvants can be used in the disclosed compositions, particularly combinations of monophosphoryl lipid A and saponin derivatives (see, for example, WO 94 / 00153; WO 95 / 17210; WO 96 / 33739; WO 98 / 56414; WO 99 / 12565; WO 99 / 11241), more particularly combinations of QS21 and 3D-MPL as disclosed in WO 94 / 00153, or compositions in which QS21 is quenched in cholesterol-containing liposomes (DQ) as disclosed in WO 96 / 33739. A potent adjuvant formulation involving QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion is described in WO 95 / 17210 and is another formulation that can 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). Another exemplary adjuvant comprises QS21 and / or MPL and / or CpG. As disclosed in WO 96 / 33739, QS21 can be quenched in cholesterol-containing liposomes.

[0419] Therefore, a suitable adjuvant for use with at least one recombinant hepatitis B polypeptide is AS01 (sometimes referred to as "AS-01"), which is a liposome-based adjuvant containing MPL and QS-21. The liposomes, which serve as a vehicle for the MPL and QS-21 immunopotentiators, are composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in a phosphate-buffered saline solution. AS01 B-4 It is a particularly preferred variant of the AS01 adjuvant, consisting of the immunopotentiators QS-21 (a triterpene glycoside purified from the bark of the Quillaja sapodilla tree) and MPL (3-D monophosphoryl lipid A), as well as DOPC / cholesterol liposomes (as a vehicle for these immunopotentiators) and sorbitol in a PBS solution. In particular, a single human dose of AS01 B-4 (0.5mL) contains 50μg QS-21 and 50μg MPL. AS01 E-4 Equivalent to AS01 B-4 A two-fold dilution of , i.e. it contains 25 μg QS-21 and 25 μg MPL per human dose.

[0420] In all of these schemes, in a preferred embodiment, the mRNA encodes a hepatitis B core (HBc) polypeptide fused with or without HLI. 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 with HLI. In another preferred embodiment, the mRNA encodes a full-length hepatitis B core (HBc) antigen fused with or without HLI, and a hepatitis B surface protein (HBsAg) fused with or without HLI. Preferably, HBsAg is a hepatitis B small surface protein (HBs) fused with or without HLI. In another preferred embodiment, the hepatitis B small surface protein (HBs) is fused with HLI.

[0421] HBc encoded by mRNA and / or HBs encoded by mRNA are preferably fused to hIi.

[0422] The present invention also includes treating chronic hepatitis B infection (CHB) by administering an adenoviral vector to a human, wherein the adenoviral vector comprises a combination of a polynucleotide encoding a hepatitis B polypeptide and an mRNA encoding at least one hepatitis B virus antigen. Components can be administered in a heterologous primary immunization-boosting regimen. If a primary immunization-boosting regimen is used, the adenoviral vector is preferably administered as a primary immunization dose, and mRNA is administered as a first booster dose. In such a regimen, multiple primary immunizations and / or booster doses may be present. In one embodiment, there is a single primary immunization of an adenoviral vector (such as a replication-defective chimpanzee adenovirus (ChAd) vector), and multiple subsequent booster doses comprising mRNA and / or recombinant HBV polypeptides.

[0423] In an alternative embodiment of a heterologous prime-boost regimen, mRNA is used as the first priming dose and an adenoviral vector (such as a replication-defective chimpanzee adenovirus (ChAd) vector) is used as the first boosting dose.

[0424] The present invention also includes treating chronic hepatitis B infection (CHB) by administering to a human (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 virus antigen.

[0425] The present invention may also include administering multiple subsequent doses of mRNA. In such embodiments, the mRNA used for the priming and boosting doses is preferably the same.

[0426] Subjects

[0427] The present invention is intended for use in human subjects.The subject to be treated using the methods of the present invention can be of any age.

[0428] The method of the present invention is suitable for treating HBV, ie, for administering to a subject infected with hepatitis B virus. The subject may be infected with hepatitis B virus alone, or concurrently with hepatitis B and hepatitis D viruses.

[0429] Formulation and administration

[0430] The mRNA can be administered by various suitable routes, including parenteral administration, such as intramuscular or subcutaneous administration. Suitably, the mRNA is administered intramuscularly.

[0431] The mRNA may be provided in liquid or dried (e.g. lyophilized) form. The preferred form will depend on factors such as the precise nature of the mRNA, such as whether the mRNA is easy to dry, or other components that may be present.

[0432] Preferably, the mRNA is provided in liquid form.

[0433] Compositions comprising mRNA for combination with other compositions prior to administration need not themselves have a physiologically acceptable pH or physiologically acceptable tonicity; formulations for administration should have a physiologically acceptable pH and physiologically acceptable osmotic pressure.

[0434] The pH of the liquid formulation is adjusted according to the components of the composition and the necessary suitability for administration to human subjects.

[0435] For parenteral administration, the solution should have a physiologically acceptable osmotic pressure to avoid excessive cell deformation or lysis. Physiologically acceptable osmotic pressure generally refers to a solution whose osmotic pressure is close to isotonic or slightly hypertonic. Osmolality can be measured according to techniques known in the art, for example, by using a commercially available osmometer, such as those available from Advanced Model 2020 acquired from Instruments Inc. (USA).

[0436] The liquid used for reconstitution is essentially aqueous, such as water for injection, phosphate buffered saline, etc. As described above, the need for a buffer and / or tonicity adjusting agent will depend on the contents of the container being reconstituted and the subsequent use of the reconstituted contents. The buffer can be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate, and TRIS. The buffer can be a phosphate buffer, such as Na / Na2PO4, Na / K2PO4, or K / K2PO4.

[0437] mRNA can be provided in a variety of physical containers, such as vials or prefilled syringes.

[0438] In some embodiments, the mRNA is provided in a single dose. In other embodiments, the mRNA is provided in a multiple dose form, e.g., comprising 2, 5, or 10 doses.

[0439] Typically, liquids are transferred between containers, such as from a vial to a syringe, to provide an "overage" to ensure that the full required volume can be conveniently transferred. The level of overage required will vary depending on the circumstances, but overage should be avoided to reduce waste, and underage overage may cause practical difficulties. Overages can range from 20 to 100 μl per dose, for example, 30 μl or 50 μl.

[0440] Stabilizers may be present. Stabilizers may be particularly important when multi-dose containers are provided, since doses of the final formulation may be administered to a subject over a period of time.

[0441] The formulation is preferably sterile.

[0442] The method for setting up strong and lasting immunity generally comprises repeated immunization, i.e., enhancing immune response by administering one or more further dosages. This further administration can be carried out with the same immunogenic composition (homologous boosting) or different immunogenic compositions (heterologous boosting). The present invention can be used as a part of a homologous or heterologous initial / boosting scheme, as a primary immunization or booster immunization application.

[0443] Therefore, the administration of mRNA can be part of a multiple-dose administration regimen. For example, mRNA can be provided as a primary immunization dose in a multiple-dose regimen, such as a regimen of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more doses, particularly a six-dose regimen administered within six months. mRNA can be provided as a booster dose in a multiple-dose regimen, particularly two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more doses, such as a six-dose regimen administered within six months. In a specific embodiment, mRNA is administered as a four-dose regimen.

[0444] Initial immunization and booster dose can be homologous or heterologous.Therefore, mRNA can be provided as initial immunization dose and booster dose in homologous multiple dose scheme, particularly two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, eleven doses, twelve doses or more of the scheme, particularly six dose schemes used in six months.In one embodiment, mRNA is used as four dose schemes.Alternately, mRNA can be provided as initial immunization dose or booster dose in heterologous multiple dose scheme, particularly two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, eleven doses, twelve doses or more of the scheme, particularly six dose schemes used in six months, and one or more booster doses can be different (such as mRNA; Or alternative antigen presentation such as protein or viral vector antigen-with or without adjuvant, such as AS01 or squalene emulsion adjuvant).In one embodiment, mRNA is used as four dose schemes.

[0445] The time between doses can be from two weeks to six months, for example, from three weeks to three months. Preferably, two doses are administered simultaneously each month - a priming dose and a booster dose - for six months. Regular long-term booster doses can also be provided, for example, every 2 to 10 years.

[0446] 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 in separate LNP formulations.

[0447] In another embodiment, 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 by separate LNPs, or the first and second mRNAs can be co-formulated in the same LNP.

[0448] In some examples, the combination or composition comprises equal amounts of the first and second mRNAs by weight. However, in other examples, the combination or composition may comprise unequal amounts of the first and second mRNAs 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 contains more of the first mRNA than the second mRNA by weight.

[0449] In embodiments of formulations containing both HBc and HBs mRNA, the composition contains equal amounts (by weight) of HBc and HBs mRNA. However, in one embodiment, the composition contains more mRNA encoding HBc ("HBc-mRNA") by weight than mRNA encoding HBs (HBs-mRNA). In one embodiment, the composition contains 1.25 to 2 times the amount of HBc-mRNA, such as 1.5 to 2 times the amount of mRNA, when compared to HBs-mRNA. In a specific embodiment, the composition contains 1.5 times the weight of HBc-mRNA as HBs-mRNA.

[0450] In another embodiment, an immunogenic combination is provided comprising:

[0451] 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

[0452] A second composition comprising recombinant hepatitis B core protein (HBc), recombinant hepatitis B small surface protein (HBs), and AS01.

[0453] The combination can be used in a method for treating chronic hepatitis B (CHB) by sequential or concomitant administration of the first and second compositions. The first composition can comprise mRNA encoding HBc and HBs co-formulated in a single LNP. Alternatively, the mRNA encoding HBc and HBs can be formulated into separate LNPs, and these LNPs can be co-filled into a single vial.

[0454] definition

[0455] Unless defined otherwise, all technical and scientific terms used herein generally have the same meanings as commonly understood by one of ordinary skill in the art.

[0456] Throughout the specification, including the claims, where the context permits, the term "comprising" and variations such as "comprises" should be interpreted as including the stated element or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers). Thus, a composition "comprising" X may consist solely of X, or may include something else, such as X+Y.

[0457] The expression "substantially" does not exclude "completely", for example, a composition "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0458] The term "about" or "approximately" with respect to a value x is optional and means, for example, x ± 10% of the given value, such as x ± 5% of the given value.

[0459] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0460] Unless otherwise specified, a method comprising the step of mixing two or more components does not require any particular order of mixing. Thus, the components can be mixed in any order. If there are three components, two components can be combined with each other, and then that combination can be combined with the third component, and so on.

[0461] The terms "protein," "polypeptide," "antigen," and "peptide" are used interchangeably herein and refer to any 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 comprising two or more peptide-linked proteins. A fusion protein is produced by linking two or more genes that originally encoded separate proteins. Translation of the fusion gene produces a single fusion protein.

[0462] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric macromolecules prepared from nucleotide monomers. Suitably, the polynucleotides of the present invention are recombinant. Recombinant means that the polynucleotide is the product of at least one of a cloning, restriction digestion or ligation step or other procedure that produces a polynucleotide different from that found in nature.

[0463] A heterologous nucleic acid sequence refers to any nucleic acid sequence that is not isolated, derived from, or based on a naturally occurring nucleic acid sequence found in a host organism. "Naturally occurring" means a sequence that is found in nature and is not synthetically prepared or modified. A sequence is "derived from" a source when it is isolated from the source but modified (e.g., by deletion, substitution (mutation), insertion, or other modification), appropriately without disrupting the normal function of the source gene.

[0464] Suitably, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is a polynucleotide that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the coexisting substances in the natural system. A polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of its natural environment or if it is contained in a cDNA.

[0465] As used herein, "concomitant" administration refers to administration during the same ongoing immune response. Preferably, the two components are administered simultaneously (e.g., concomitant administration of a composition comprising the vector and a composition comprising the protein), however, one component can be administered within a few minutes (e.g., at the same medical appointment or doctor's visit) or within a few hours. Such administration is also referred to as co-administration. In some embodiments, concomitant administration can 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, e.g., a poxvirus such as MVA. In other embodiments, co-administration refers to administration of an adenoviral vector and a protein component, wherein the protein component is adjuvanted.

[0466] "Sequential" administration refers to the administration of a first composition followed by administration of a second composition after a significant period of time, e.g., not during the ongoing immune response generated by the first administration. Thus, sequential administration encompasses the first and subsequent administrations in a prime-boost scenario. The time period between two consecutive administrations is, for example, 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks. More specifically, it is 4 weeks or 8 weeks.

[0467] The term "adjuvant" refers to an agent that enhances, stimulates, activates, strengthens or modulates the immune response to the antigen of the composition at the cellular or humoral level, for example, an immune adjuvant stimulates the immune system's response to the antigen, but does not itself have an immunological effect. The immunogenic compositions disclosed herein may include an adjuvant as a separate ingredient in the formulation, regardless of whether the vector (or another component thereof) contained in the composition also encodes a "genetic adjuvant", such as hIi.

[0468] Example

[0469] Preclinical data comparing the immunogenicity of SAM-HBV with or without human invariant chains have been generated in mice (Example 1 below). In addition, preclinical data 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 have been generated (Example 2 below). Preclinical data observing the immunogenicity of co-administered LNP-mRNA in HLA-A2 / DRB1 naive mice have also been generated (Example 3 below).

[0470] These experiments showed that LNP-formulated SAM-HBV containing a human constant chain (hIi) fused to the hepatitis B core antigen (HBc) induced 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 without human constant chains (Example 1 below). In addition, it has been shown that the use of SAM-hIi-HBV can induce a higher frequency of HBc- and HBs-specific CD8+ T cell responses in a mouse model of chronic HBV infection (Example 2 below). In addition, experiments have shown a preferred ratio of hli-HBc and hli-Hbs mRNA for co-administration of LNP-formulated hli-HBc (Example 3 below).

[0471] In all of these experiments, HLA.A2 / DRB1 mice (transgenic for human HLA-A2 and HLA-DRB1 molecules) were used to assess the ability of HBV mRNA vaccines to induce HBc-specific CD8+ T cell responses. HBV-specific CD4+ T cells and antibodies were assessed in the same HLA.A2 / DRB1 mice.

[0472] Production of ChAd155-hIi-HBV Drug Substance:

[0473] The production of the ChAd155-hIi-HBV drug substance involves culturing Procell-92.S cells to a defined cell density. The cells are then infected with a ChAd155-hIi-HBV master virus seed (MVS) at a defined multiplicity of infection. The ChAd155-hIi-HBV viral harvest is purified by a multi-step process based on anion exchange chromatography.

[0474] Vaccine preparation and filling of ChAd155-hIi-HBV:

[0475] The purified ChAd155-hIi-HBV bulk drug substance was then processed as follows:

[0476] • Dilute purified ChAd155-hIi-HBV drug substance in formulation buffer.

[0477] Sterile filtration.

[0478] Filling into final container.

[0479] The ChAd155-hIi-HBV vaccine is a liquid formulation contained in vials.

[0480] Production of MVA-HBV Drug Substance:

[0481] MVA-HBV drug substance was produced in primary cell cultures of chicken embryo fibroblasts (CEFs) to a defined cell density and then infected with MVA-HBV master virus seeds (MVS) at a defined multiplicity of infection. The MVA-HBV viral harvest was purified by a multi-step process based on step gradient centrifugation.

[0482] MVA-HBV vaccine preparation and filling:

[0483] The purified MVA-HBV bulk drug substance is then processed as follows:

[0484] • Dilute purified MVA-HBV DS in formulation buffer.

[0485] Filling into final container.

[0486] MVA-HBV vaccine is a liquid formulation supplied in vials with a 0.5 mL withdrawable volume.

[0487] Production of HBc drug substance:

[0488] The HBc DS production process consists of inoculating pre-culture flasks 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.

[0489] Production of HBs drug substance:

[0490] The HBs DS production process consists of inoculating pre-culture flasks with recombinant Saccharomyces cerevisiae working seeds, followed by a fermentation process and a multi-step purification process including harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0491] HBc and HBs vaccine preparation and filling:

[0492] Purified HBs and HBc DS were diluted in a formulation buffer including sucrose as a cryoprotectant and poloxamer as a surfactant, filled into 4 mL clear glass vials and lyophilized.

[0493] Dosage of AS01 adjuvant system:

[0494] The AS01B-4 adjuvant system consists of the immunopotentiators QS-21 (a triterpene glycoside purified from the bark of the Quillaja sapodilla tree) and MPL (3-D monophosphoryl lipid A), using liposomes as a vehicle for these immunopotentiators, and sorbitol. Specifically, a single clinical dose container (0.5 mL) of AS01B-4 contains 50 μg of QS-21 and 50 μg of MPL. This is 1 / 10 the human dose, meaning 50 μl is the volume injected in mice (corresponding to 5 μg of QS-21 and MPL).

[0495] Generation of mRNA constructs:

[0496] The plasmid was linearized with the BspQI restriction endonuclease to generate 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 treatment, phosphatase treatment, and silica gel column purification. Newly synthesized mRNA was verified by capillary gel electrophoresis and denaturing agarose gel analysis.

[0497] Generation of SAM-HBV and SAM-hIi-HBV constructs:

[0498] HBV and hli-HBV sequences were codon-optimized for human protein expression, synthesized, and cloned into the SAM plasmid using GENEWIZ. RNA was synthesized by in vitro transcription. Briefly, the DNA plasmid encoding the SAM replicon was linearized by restriction digestion with BspQI at the 3' end of the poly(A) tail and purified by phenol-chloroform extraction. The linearized DNA was used as a template for an in vitro transcription reaction using T7 RNA polymerase. Following in vitro transcription, RNA was capped using a vaccinia capping kit and purified by LiCl precipitation and resuspended in nuclease-free water.

[0499] The preparation of LNP and SAM follows the established method of preparing LNP by microfluid mixing, wherein lipid (cationic lipid, zwitterionic lipid, cholesterol and PEG-lipid conjugate) is dissolved in ethanolic solution, and SAM is dissolved in aqueous buffer solution.Microfluidic mixing chamber is used that ethanolic solution and aqueous solution are mixed together quickly.The lipid nanoparticles of encapsulating SAM are spontaneously formed by the nucleation of supersaturated lipid in the mixture.Condensation and precipitation of lipid capture SAM and form lipid nanoparticles.After the LNP of short maturity, then the buffer exchange of SAM-LNP is in storage buffer.Size, lipid content, RNA retention and the in vitro efficacy of SAM-LNP solution are characterized.

[0500] The SAM vector VEE TC-83 was used as a background construct for cloning in the examples. The background empty construct had the nucleic acid sequence SEQ ID NO: 16.

[0501] Figure 14 The design of the HBV-SAM construct includes a sequence encoding an HBV antigen cloned under a subgenomic promoter in the SAM vector. Modifications to the SAM HBV construct include codon optimization of the antigen encoding sequence.

[0502] The SAM constructs were evaluated for robust antigen production and antigenicity, and further tested for immunogenicity and efficacy using in vivo models.

[0503] 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.

[0504] Characterization of SAM-HBV and SAM-hIi-HBV constructs:

[0505] RNA pattern homogeneity assessment

[0506] To investigate the homogeneity of RNA patterns, RNA samples were analyzed on 1% agarose gels. RNA samples were prepared as follows: 100-250 ng 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. The samples were denatured at 50° C. for 20 minutes. The agarose gel was run at 130 V for 45 minutes in NorthernMax-Gly gel running buffer (Invitrogen™). No major RNA degradation was observed, and similar patterns were obtained between the two constructs.

[0507] Assessment of protein expression by western blotting

[0508] The ability of cells to express a given antigen from different HBV SAM constructs was assessed according to the following method.

[0509] On day 0, baby hamster kidney (BHK) cells were seeded at 1×10 7 in T225 flasks in growth medium (DMEM high glucose (Gibco™), 1% L-glutamine, 1% Pen-Strep 5% FBS (Gibco™). For trypsinization, remove the culture medium and wash the cells with 5 mL of PBS. Remove the PBS wash solution, add 5 mL of preheated trypsin, and spread it thoroughly on the plate. Remove the trypsin and incubate the plate at 37°C for 1-2 minutes. Then resuspend the cells in 10 mL of growth medium. Count the cells and seed them into a new flask at the desired concentration. Then incubate the cells at 37°C, 5% CO2 for approximately 20 hours.

[0510] On the first day, prepare the plate by adding 2mL growth medium (DMEM high glucose, 1% L-glutamine, 1% Pen-Strep, 1% FBS) to each well of a 6-well plate (one well / electroporation). The plate is kept warm in a 37°C incubator. Prepare an electroporator to deliver 120V, 25ms pulses, 0.0 pulse interval, 1 pulse (for a 2mm cuvette). The cuvette is labeled and stored on ice. Cells in the growth phase are harvested in BHK growth medium and counted using a cell counter. According to the same trypsinization protocol as described above, cells are trypsinized. The cells are then centrifuged at 462x g for 3 minutes. Aspirate the culture medium and wash the cells once with 20mL cold Opti-MEM culture medium (Gibco™). The cells are again centrifuged at 462x g for 5 minutes. Aspirate the culture medium and resuspend the cells in Opti-MEM culture medium to 0.25mL / 1x106 cells / electroporation. Standards and negative controls were also prepared.

[0511] For each sample, 2ug RNA was mixed with 250μL cells and the mixture was gently pipetted 4-5 times. The cells and RNA mixture were transferred to a 2mm cuvette and an electroporation pulse was performed using the above parameters. The cells were allowed to stand at room temperature for 10 minutes. Cells from a cuvette were added to a well of a preheated 6-well plate, and the plate was tilted back and forth, then tilted at a 45° angle to evenly distribute the cells. On the second day (17h after electroporation), cell culture supernatants were collected and analyzed by Western blotting at different concentrations. The cell monolayer was separated and resuspended in 1mL of 20mM HEPES, 150mM NaCl, 5% glycerol pH 7.6 buffer supplemented with cOmplete™ protease inhibitor cocktail (Roche, catalog number 11697498001), and then cell lysis was performed by ultrasonic treatment. After cell lysis, the intracellular fraction was analyzed by Western blotting. The primary antibodies used were mouse anti-HBc monoclonal antibody and rabbit anti-HBs polyclonal serum (produced in-house).

[0512] In vitro efficacy of SAM after LNP formulation

[0513] In vitro potency assays were also performed after LNP formulation.

[0514] Cellular Immune Response - Intracellular Cytokine Staining (ICS):

[0515] Fresh pools of peripheral blood leukocytes (PBLs), splenocytes, or liver-infiltrating lymphocytes collected at different time points were stimulated ex vivo for 6 hours with a pool of 15-mers overlapping 11aa (covering the HBc or HBs sequence). HBc- and HBs-specific cellular responses were assessed by ICS, measuring the number of CD4+ or CD8+ T cells expressing IFN-γ and / or IL-2 and / or tumor necrosis factor (TNF)-α. Technical acceptance criteria for ICS results included a minimum number of CD8+ or CD4+ T cells >3000 events.

[0516] Humoral immune response - Enzyme-linked immunosorbent assay (ELISA):

[0517] HBc- and HBs-specific antibody responses were measured by ELISA on serum from immunized mice at different time points. Briefly, 96-well ELISA plates were coated with purified hepatitis B core antigen (HBc) or purified hepatitis B surface antigen (HBs). Serum from vaccinated mice was serially diluted and incubated. Serial dilutions of standard and control materials were used to calculate the anti-HBc or anti-HBs antibody standard titers of the tested serum and to ensure the validity of the test. After each incubation step, the plates were washed with PBS 0.1% tween20 buffer. Horseradish peroxidase goat anti-mouse IgG (H+L) antibody was then added, and the antibody complex was revealed by incubation with tetramethylbenzidine liquid substrate (TMB). Optical density (OD) was recorded at 450-620 nm. The anti-HBc or HBs antibody titer of each individual mouse serum was determined by the standard curve of ELISA using a regression model. The geometric mean titer (GMT) of each group of mice was then calculated. For each time point and each antigen (HBc, HBs), an analysis of variance (ANOVA) model was fitted on the log10 titer, including group, study, and interaction as fixed effects, and using a heterogeneous variance model (not assuming equal variance between groups). This model was used to estimate the geometric mean (and its 95% CI) and the ratio of geometric means and its 95% CI. Since no predefined criteria were set, the analysis was descriptive, and the 95% CI of the ratio between groups was calculated without adjusting for multiplicity.

[0518] ALT / AST measurement:

[0519] Quantify the levels of ALT and AST in mouse serum using the following commercially available kits:

[0520] Alanine aminotransferase activity assay kit Sigma Aldrich catalog number MAK052

[0521] Aspartate aminotransferase activity assay kit Sigma Aldrich catalog number MAK055

[0522] Serum HBs antigen quantification

[0523] Circulating HBs antigen in mouse sera was quantified using the Monolisa Anti-HBs PLUS commercial kit from BIO-RAD (Cat. No. 72566) and an international standard (Abbott Diagnostics).

[0524] Example 1: SAM-HBV with or without human invariant chain

[0525] In this experiment, male and female HLA.A2 / DR1 naive mice received intramuscular injections on day 0 and day 28. The compositions administered to the different groups on day 0 and day 28 are detailed in Table 1 below.

[0526] Table 1:

[0527]

[0528]

[0529] (Note: SAM-hIi-HBV is the construct of SEQ ID NO: 17, and SAM-HBV is the construct of SEQ ID NO: 19)

[0530] Except control group 7, all groups all use 14 mice (N=14).After the first injection 14 days (14dpI), put to death 2 mice from each group, so that spleen sample and serum sample can be collected and T cell response is measured at this time point, and serum sample is collected from all mice.After the second injection (12 / 13dpII) 12 and 13 days, the remaining animals are all put to death, and spleen, liver and serum sample are collected.

[0531] For groups 1 to 6, all mice were primed with ChAd155-hIi-HBV and boosted with SAM-HBV (±hIi). The same dose of ChAd155-hIi-HBV was administered: 10 8 vp / mouse. However, three different doses of SAM-HBV (± hIi) were used: 2.5 μg, 1 μg, and 0.1 μg. The specific doses used in each group are specified in Table 1 above.

[0532] The HBc- and HBs-specific CD4+ and CD8+ T cell responses and HBc- and HBs-specific antibody responses generated 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.

[0533] like Figure 2A and 2B As shown, the SAM construct containing the invariant chain (SAM-hIi-HBV) induced a significantly higher frequency of HBc-specific CD8+ T cell responses against HBc (geometric mean ratio, GMR = 4.1, 95% CI [1.96-8.40]) compared to the construct without the invariant chain (SAM-HBV). The geometric mean of the HBc-specific CD8+ T cell responses of the group of mice immunized with SAM-hIi-HBV was first calculated, and then the geometric mean of the group of mice immunized with SAM-HBV was calculated. The ratio of these two geometric means was then calculated. In this case, we observed that SAM-hIi-HBV induced a 4-fold higher HBc-specific CD8+ T cell response.

[0534] Effective 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 major role in the control and regression of HBV infection.

[0535] Several published studies have compared HBV antigen-specific T cells in different segments of patients affected by HBV (after acute infection, patients recovering from chronic infection, active chronic infection, and inactive carriers), emphasizing the necessity of inducing strong multispecific T cell responses against HBV antigens, particularly HBc antigen, to promote clearance of HBV infection. Consistent with this, comparison of T cells from patients with resolved chronic HBV infection compared to patients with non-resolved chronic HBV infection showed higher CD4+ T cells and CD8+ T cells specific for HBc antigen in patients with resolved infection [Boni, 2012; Li, 2011; Liang, 2011].

[0536] Furthermore, the role of functional CD8+ T cells appears to be critical. During acute HBV infection, depletion of CD8+ T cells in chimpanzees leads to persistent viremia [Thimme, 2003]. In humans, clearance of HBV during acute hepatitis B is associated with strong, polyclonal, polyspecific CD8+ T cell responses to viral nucleocapsid, envelope, and polymerase proteins that persist for decades after clinical recovery. In contrast, patients with chronic hepatitis B (CHB) typically fail to mount a robust CD8+ T cell response to the virus. CHB patients who experience spontaneous or interferon-induced remission mount CD8+ T cell responses to HBV that are similar in intensity and specificity to those in patients who recover from acute hepatitis [Rehermann, 1996].

[0537] Since SAM constructs containing invariant chains (SAM-hli-HBV) were shown to induce greater CD8+ T cell responses against HBc and HBs antigens, these constructs were selected for use in Example 2.

[0538] Example 2: Substitution of MVA-HBV or ChAd155-HBV with SAM-hIi-HBV in HLA.A2 / DR1-transduced mice Assessment of both hIi-HBV and MVA-HBV

[0539] Due to the maximum capacity of each experimental group, two independent experiments were planned. Both experiments contained the animals detailed in Table 2 in each group.

[0540] In this study, male and female HLA.A2 / DR1-transduced mice were used. The AAV2 / 8-HBV-transduced HLA.A2 / DR1 murine model recapitulates the virological and immunological features of chronic HBV infection. It was selected to evaluate the immunogenicity of different vaccine regimens, the impact of liver-infiltrating HBc-specific CD8+ T cells (which potentially target HBcAg-expressing hepatocytes), and potential vaccine-associated liver inflammation by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities.

[0541] Therefore, in these experiments, male and female HLA.A2 / DR1 mice (Groups 1-6 and 8) were injected intravenously on day 0 with 10 μg of adeno-associated virus serotype 2 / 8 (AAV2 / 8HBV) vector carrying a replication-competent HBV DNA genome. 10 A viral genome (vg).

[0542] Based on the level of circulating HBs antigen detected in the serum on day 21 / 22, age and sex ratio, HLA.A2 / DR1 mice were randomly divided into 7 different groups (Groups 1-6 and Group 8) before immunization.

[0543] Mice from Group 7 were not transduced with AAV2 / 8-HBV viral vectors but were immunized intramuscularly (IM) using the co-administered vaccine regimen. This group served as a positive control for immunological readouts.

[0544] HLA.A2 / DR1 transduced mice received intramuscular (gastrocnemius) injections of various formulations containing HBc and HBs antigens (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 the presentation of the figures and statistical analysis of the results.

[0545] In all cases, use the same dose of each combination:

[0546] ChAd155-hIi-HBV was added at 108 vp / mouse dose was administered to mice.

[0547] Mice vaccinated with MVA-HBV received a dose of 10 7 pfu / mouse,

[0548] Administering SAM-hIi-HBV to mice at a dose of 1 μg / mouse, and

[0549] The group receiving adjuvanted protein received a dose of 4 μg HBc, 1 μg HBs and AS01 containing 5 μg MPL and 5 μg QS21 per mouse.

[0550] The aim of this experiment was to evaluate whether SAM-hIi-HBV could replace MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV in sequential or co-administered vaccine regimens by inducing at least the same level of HBc-specific CD8+ T cell responses as the MVA-HBV vaccine regimen.

[0551] Table 2:

[0552]

[0553] (Note: SAM-hIi-HBV is the construct of SEQ ID NO: 17, and SAM-HBV is the construct of SEQ ID NO: 19)

[0554] Mice from Groups 1 to 6 and 8 were transduced with AAV2 / 8-HBV 31 or 33 days before the first injection. As mentioned above, for practical reasons related to running the experiments, each group was divided into two and two separate experiments were performed. All results shown are the combined output from the two experiments.

[0555] 13 and 14 days after the second injection (13 / 14 dpII), 15 mice from each group of groups 1 to 6 (from each experiment), 6 and 4 mice from groups 7 and 8 of experiment 20200719, and 4 and 5 mice from groups 7 and 8 of experiment 20200720 were sacrificed so that spleen samples could be collected from all mice and serum samples could be collected. 22 days after the fourth injection (22 dPIV), all the remaining animals were sacrificed and spleen, liver and serum samples were collected.

[0556] The CD8+ T cell and antibody responses generated by each group in Example 2 were as follows: Figure 4A and 4BAs shown in Figure 4, the results show that 14 days after the second immunization, priming with ChAd155-hIi-HBV and boosting with SAM-hIi-HBV produced a 7.36-fold higher HBc-specific CD8+ T cell response than priming with ChAd155-hIi-HBV and boosting with MVA-HBV (GMR = 7.36, 90% CI [3.96-13.70]). Similarly, the results also showed that 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]) than priming with ChAd155-hIi-HBV and boosting with MVA-HBV.

[0557] Similar results were shown for CD8+ T cells. Figure 5A and 5B Here, 14 days after the second immunization, priming with ChAd155-hIi-HBV and boosting with SAM-hIi-HBV induced a 3.6-fold higher HBs-specific CD8+ T cell response than priming with ChAd155-hIi-HBV and boosting with MVA-HBV (GMR = 3.64, 90% CI [2.34-5.67]). The results also showed that priming with SAM-hIi-HBV and boosting with SAM-hIi-HBV induced a 7.78-fold higher HBs-specific CD8+ T cell response than priming with ChAd155-hIi-HBV and boosting with MVA-HBV (GMR = 7.78, 90% CI [5-12.12]).

[0558] Interestingly, replacing MVA-HBV with SAM-hIi-HBV vaccine or both ChAd155-hIi-HBV and MVA-HBV induced more polyfunctional HBV-specific CD8+ T cells, as demonstrated by cytokine co-expression profiles ( Figure 13A 、 13B The majority of HBV-specific CD8+ T cells expressed primarily a combination of IFN-g and TNFα, and this population was further increased when homologous priming with SAM-hIi-HBV was used.

[0559] Figures 6 and 7 show CD4+ responses measured in the spleen. 14 days after the second immunization, all vaccine regimens elicited very low to undetectable HBc-specific CD4+ T cells (≤0.1%) in AAV2 / 8-HBV-transduced HLA-A2 / DR1 mice, and slightly higher HBc-specific CD4+ T cells were induced only after administration of HBc-HBs / AS01 alone or in combination with both vectors.

[0560] 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 a 7.12-fold higher HBs-specific CD4+ T cell response compared to priming with ChAd155-hIi-HBV and boosting with MVA-HBV (geometric mean ratio (GMR) = 7.12, 90% CI [4.61-11]). Of note, replacing both ChAd and MVA with SAM-hIi-HBV did not induce a higher response. However, the results showed that priming with SAM-hIi-HBV and boosting with SAM-hIi-HBV induced a 2.56-fold higher HBs-specific CD4+ T cell response compared to priming with ChAd155-hIi-HBV and boosting with MVA-HBV (GMR = 2.56, 90% CI [1.66-3.95]). As previously observed for HBc-specific CD4+ T cell responses, strong HBs-specific CD4+ T cells were induced only after administration of HBc-HBs / AS01 alone or in combination with both vectors.

[0561] Regarding humoral immune responses, replacing 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.

[0562] Interestingly, immunization with Th HBV vaccine showed a ±1.5-fold decrease in serum HBs antigen in all groups, regardless of the regimen used (sequential or co-administration), with no between-group differences ( Figure 10 and 11 Finally, as liver-related inflammatory parameters, serum activity of AST and ALT was measured in mouse sera after the second and fourth immunizations. For each group, ALT levels were stable throughout the study period and were not significantly affected by replacing MVA-HBV with SAM-hIi-HBV or by replacing both ChAd155-hIi-HBV and MVA-HBV. AST levels were slightly higher in all groups, with no intergroup differences ( Figure 12 ).

[0563] Overall conclusion:

[0564] Substituting SAM-hIi-HBV for MVA-HBV or both ChAd155-hIi-HBV and MVA-HBV induced significantly higher HBc- and HBs-specific CD8+ T cell responses: SAM / SAM > ChAd / SAM > ChAd / MVA, with a slight positive effect on HBs-specific CD4+ T cell responses. No significant effect was observed on the levels of anti-HBc and HBs IgG antibody responses.

[0565] Regarding circulating HBs antigen, there was a trend toward a ±1.5-fold decrease in circulating HBs antigen in all groups, with no between-group differences.

[0566] Furthermore, when potential vaccine-associated liver inflammation was assessed by measuring serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), no increase in liver enzymes was detected in the vaccine group compared with the unvaccinated group.

[0567] Example 3: Evaluation of the immunogenicity of co-administered LNP-mRNA in HLA-A2 / DRB1 naive mice

[0568] The details of the experiment are given in Table 3. The LNP-mRNA construct contained the UTR4 backbone and RV39 LNP. The formulation also included 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), polyethylene glycol-conjugated (PEG-conjugated) lipids, and cholesterol.

[0569] Naive HLA-A2 / DRB1 mice (54 males, 47 females) aged 8-12 weeks were used in this experiment. The schedule included injections via intramuscular immunization on days 0, 21, 42, and 63. The doses used were:

[0570] mRNA: As shown in the table,

[0571] ChAd155-hIi-HBV: 10 8 vp / mouse,

[0572] MVA-HBV: 10 7 pfu / mouse,

[0573] HBc-HBs: 4-1 μg / AS01, ie, 4 μg HBc and 1 μg HBs (for adjuvanted protein administered simultaneously with mRNA in Group 7 of Table 3).

[0574] 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.

[0575] The primary objective of this study was to investigate immune interference between co-administered hli-HBc and hLI-HBs mRNAs (i.e., "hIi-HBc + hIi-HBs"). Three co-administered mRNAs (including hIi-HBc and hIi-HBs) were compared to a formulation containing only a single mRNA type. In this early study, it was observed that co-administration negatively impacted HBc- and HBs-specific CD8+ T cell responses (see Figure 17 In particular, when these mRNAs were co-administered, the HBc-specific response was 6.7-fold lower and the HBs-specific response was 2-fold lower.

[0576] The first change made was to co-administer only HBc and HBs mRNA (i.e., without the use of a third mRNA). Furthermore, since the effect on HBc-specific CD8+ responses was greatest, the amount of HBs mRNA was reduced relative to HBc mRNA. This resulted in the different ratios of HBc mRNA to HBs mRNA observed in Groups 2 to 5 of Table 3:

[0577] A composition containing 7 μg HBc mRNA and 7 μg HBs mRNA was administered to mice in Group 2.

[0578] • For Group 3, HBs mRNA was diluted 1.5-fold, resulting in 4.6 μg (rounded to 1 decimal place) of HBs mRNA being administered to mice.

[0579] o Therefore, the ratio of mRNA used in this composition is 1.5HBc mRNA:1HBs mRNA.

[0580] For Group 4, 4.6 μg of the HBs mRNA composition was diluted 1.5-fold, resulting in 3.1 μg (rounded to 1 decimal place) of the composition being administered to the mice.

[0581] o Therefore, the ratio of mRNA used in this composition is 2.3HBc mRNA:1HBs mRNA.

[0582] For Group 5, 3.1 μg of HBs mRNA composition was further diluted 1.5 times, resulting in 2.0 μg (rounded to 1 decimal place) of composition administered to mice.

[0583] o Therefore, the ratio of mRNA used in this composition is 3.5HBc mRNA:1HBs mRNA.

[0584] To select the co-administrations (from Groups 2, 3, 4 and 5) containing the ratio of LNP-mRNA associated with the lowest level of immune interference compared to LNP-mRNA alone (i.e. Groups 8, 9, 10, 11 and 12), we therefore:

[0585] Potential negative effects on HBc-specific CD8+ T cell responses were assessed.

[0586] Potential negative effects on HBs-specific CD8+ T cell responses were assessed.

[0587] To assess immune interference, HBc- and HBs-specific CD8+ T cell responses in the spleen were measured on days 75 and 77 (see Figure 18 、 19 and 20).

[0588] The success criteria were defined as: "An assessment of the non-inferiority of co-administration of different LNP-mRNA ratios compared to a single LNP-mRNA formulation. Non-inferiority was demonstrated if the lower limit of the 90% confidence interval for the ratio of geometric means was above 0.33. If non-inferiority was demonstrated statistically, scientists would assess for biological relevance."

[0589] At least 80% of the patients were able to demonstrate 3-fold non-inferiority at a 5% α level with a sample size of 8 mice and a SD of less than 0.36. Eight mice were assigned to each group, and 5 mice were assigned to the NaCl group, for a total of 101 mice.

[0590] The results showed that reducing the amount of hIi-HBs mRNA relative to hIi-HBc mRNA improved HBc-specific CD8+ responses compared to the control group (mice immunized with only 7 μg hIi-HBc mRNA) (see Figure 19 The highest HBc-specific CD8+ response was observed when 4.6 μg hIi-HBs mRNA was used.

[0591] Interestingly, it was also found that the composition with 7 μg hIi-HBc and 4.6 μg hIi-HBs produced a similar level of HBs-specific CD8+ T cell response as the composition with 7 μg hIi-HBc and 7 μg hIi-HBs, and this response was similar to the response detected in the group of mice immunized with only 4.6 μg HBs hIi-HBs mRNA. Although the 7 μg-3.1 μg and 7 μg-2 μg compositions resulted in higher HBc-specific T cell responses than the 7 μg-7 μg, significantly lower HBs-specific CD8+ T cell responses were observed for these two compositions.

[0592] Therefore, it was found that co-administration containing 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 a preferred composition because the immune response induced by the co-administered mRNAs was similar to the immune response induced by each mRNA alone.

[0593] The results of this experiment also showed that:

[0594] Co-administration has no negative impact on HBc-specific CD4+ T cell responses, and

[0595] • Co-administration (hIi-HBc + hIi-HBs) did not negatively interfere with HBc-specific IgG responses.

[0596] The experiment was also designed to:

[0597] (i) Direct comparison of prime-boost immunization using co-administered mRNA (hli-HBc + hIi-HBs) versus ChAd / MVA (comparison between Group 1 and Groups 2, 3, 4, and 5 in Table 3).

[0598] (ii) Evaluation of 4 doses versus 2 doses of co-administered mRNA

[0599] Immunogenicity of (hIi-HBc+hIi-HBs) (comparison between Group 4 and Group 6 in Table 3).

[0600] (iii) To investigate any immune interference between the co-administered mRNA (hIi-HBc + hIi-HBs) and AS01 adjuvanted protein (comparison of Groups 6 and 7 in Table 3). In Group 7, the "hIi-HBc + hIi-HBs" and "HBc-HBs / AS01" compositions were administered simultaneously but as two separate injections into two different limbs of mice.

[0601] The secondary endpoints were HBc- and HBs-specific CD4+ and CD8+ T cell responses in the spleen measured by ICS on days 75 / 77, and HBc- and HBs-specific antibody responses measured by ELISA on days 75 and 77 (see Figure 18 、 20 and 21).

[0602] The results are as follows:

[0603] (i) For 2 doses of mRNA compared to ChAd / MVA prime boost:

[0604] Two doses of mRNA were found to induce higher CD8+ T cell responses than ChAd / MVA prime boost. In particular, CD8+ T cell responses to HBc were found to increase by 3-fold and CD8+ T cell responses to HBs by 2-fold (see Figure 18 ).

[0605] It was also found that both doses of mRNA induced an 8-fold higher HBc-specific CD4+ T cell response. Neither mRNA nor ChAd / MVA induced HBs-CD4+ T cell responses (see Figure 19 ).

[0606] It was also found that two doses of mRNA induced an 8-fold higher HBc-specific IgG response (see Figure 21 Neither mRNA nor ChAd / MVA induced HBs-specific IgG responses.

[0607] (ii) For 4 doses compared to 2 doses of mRNA:

[0608] Two additional immunizations with co-administered mRNA were found to induce CD8+ T cell responses. In particular, a 2-fold increase in CD8+ T cell responses against HBc and a 3-fold increase in CD8+ T cell responses against HBs were found (see Figure 18 ).

[0609] It was also found that four doses of mRNA induced a 3.5-fold higher HBc-specific CD4+ T cell response. No HBs-specific CD4+ T cell response was induced after 4 or 2 doses (see Figure 19 ).

[0610] It was also found that four doses of mRNA induced a 2-fold higher HBc-specific IgG response (see Figure 21 ). Neither 4 doses nor 2 doses induced HBs-specific IgG responses.

[0611] (iii) For co-administration of mRNA and adjuvanted protein:

[0612] Co-administration of adjuvanted protein and co-administration of mRNA (hIi-HBc + hIi-HBs) was found to have a negative impact on HBc-specific CD8+ T cell responses. However, this co-administration regimen was not found to have a negative impact on HBs-specific CD8+ T cell responses (see Figure 18 ).

[0613] • This co-administration regimen was found to negatively impact HBc-specific CD4+ T cell responses. However, inclusion of adjuvanted protein in the formulation also resulted in the induction of HBs-specific CD4+ T cell responses (see Figure 19 ).

[0614] • The co-administration regimen was also found to have a positive effect on HBc-specific IgG responses (see Figure 21 ). Furthermore, the inclusion of adjuvanted protein in the formulation resulted in the induction of HBs-specific IgG responses.

[0615] Table 3:

[0616]

[0617]

[0618] Embodiments of the present invention

[0619] The embodiments of the present invention are described below in three groups of embodiments. Where appropriate, features from the three groups can be combined to form separate embodiments.

[0620] Group 1 of embodiments describes:

[0621] Embodiment A. 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 lipid nanoparticles (LNPs).

[0622] Embodiment B. The composition of embodiment A, wherein the hepatitis B virus core antigen (HBc) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:11.

[0623] Embodiment C. The composition of any preceding embodiment, wherein the hepatitis B virus core antigen (HBc) is fused to a human constant chain (hli).

[0624] Embodiment D. The composition of any preceding embodiment, wherein the composition further comprises mRNA encoding the hepatitis B small surface protein (HBs).

[0625] Embodiment E. The composition of embodiment D, wherein the hepatitis B small surface protein (HBs) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:1.

[0626] Embodiment F. The composition of any preceding embodiment, wherein the hepatitis B small surface antigen (HBs) is fused to a human invariant chain (hli).

[0627] Embodiment G. A composition for treating chronic hepatitis B infection, comprising mRNA encoding at least hepatitis B virus surface protein (HBsAg), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

[0628] Embodiment H. The composition of embodiment G, wherein the HBsAg is hepatitis B small surface protein (HBs).

[0629] Embodiment I. The composition of embodiment H, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0630] Embodiment J. The composition of any one of Embodiments G to I, wherein the HBsAg is fused to a human constant chain (hli).

[0631] Embodiment K. The composition of any preceding embodiment, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.

[0632] Embodiment L. The composition of embodiment K, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:12.

[0633] Embodiment M. The composition of any preceding embodiment, wherein the composition is administered sequentially or simultaneously with one or more recombinant hepatitis B polypeptides.

[0634] 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).

[0635] Embodiment O. The composition of embodiment M or N, wherein the HBc comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:2.

[0636] Embodiment P. The composition of embodiment M or N, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:1.

[0637] Embodiment Q. The composition of any one of Embodiments M to P, wherein the one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

[0638] Embodiment R. The composition of embodiment Q, wherein the adjuvant is AS01.

[0639] 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 virus antigen is administered as a prime dose and one or more recombinant hepatitis B polypeptides are administered as a boost dose.

[0640] Embodiment T. The method of embodiment S, wherein the mRNA encodes at least one hepatitis B virus antigen selected from hepatitis B core antigen (HBc) and hepatitis B surface antigen (HBsAg).

[0641] Embodiment U. The method of embodiment T, wherein the hepatitis B surface antigen (HBsAg) is the hepatitis B small surface protein (HBs).

[0642] Embodiment V. The method of any one of Embodiments S to U, wherein the hepatitis B virus antigen is fused to hli.

[0643] Embodiment W. The method of any one of Embodiments S to U, wherein the recombinant hepatitis B polypeptide comprises recombinant hepatitis B core protein (HBc) and recombinant hepatitis B small surface protein (HBs).

[0644] Embodiment X. The method of any one of Embodiments S to W, wherein the one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

[0645] Embodiment Y. The method of embodiment X, wherein the adjuvant is AS01.

[0646] Group 2 of embodiments describes:

[0647] 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).

[0648] Embodiment ii. The composition of embodiment ii, wherein the hepatitis B virus core antigen (HBc) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:11.

[0649] Embodiment iii. The composition of any preceding embodiment, wherein the hepatitis B virus core antigen (HBc) is fused to a human constant chain (hli).

[0650] Embodiment iv. The composition of any preceding embodiment, wherein the composition further comprises a second mRNA encoding the hepatitis B small surface protein (HBs).

[0651] Embodiment v. The composition of embodiment iv, wherein the first mRNA encoding HBc ("HBcmRNA") and the second mRNA encoding HBs ("HBs mRNA") are encapsulated in different LNPs.

[0652] Embodiment vi. The composition of embodiment iv, wherein the first mRNA encoding HBc ("HBcmRNA") and the second mRNA encoding HBs ("HBs mRNA") are encapsulated in the same LNP.

[0653] Embodiment vii. The composition of any one of embodiments iv to vi, wherein the hepatitis B small surface protein (HBs) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0654] Embodiment viii. The composition of any one of embodiments iv to vii, wherein the hepatitis B small surface antigen (HBs) is fused to a human invariant chain (hIi).

[0655] Embodiment ix. The composition of any one of embodiments iv to viii, wherein the first mRNA in the composition is greater than the second mRNA by weight.

[0656] Embodiment x. The composition of any one of embodiments iv to ix, wherein the first mRNA and the second mRNA are each present in a ratio of 1.5:1 by weight.

[0657] Embodiment xi. A composition for treating chronic hepatitis B infection, comprising a first mRNA encoding at least hepatitis B small surface protein (HBs), wherein the first mRNA is encapsulated in a lipid nanoparticle (LNP).

[0658] Embodiment xii. The composition of embodiment xi, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0659] Embodiment xiii. The composition of embodiment xi or xxii, wherein the HBs is fused to a human constant chain (hli).

[0660] Embodiment xiv. The composition of any one of embodiments xi to xiii, wherein the composition further comprises a second mRNA encoding hepatitis B virus core antigen (HBc).

[0661] Embodiment xv. The composition of embodiment xii, wherein the hepatitis B virus core antigen (HBc) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:11.

[0662] Embodiment xvi. The composition of embodiment xiv or xv, wherein the hepatitis B virus core antigen (HBc) is fused to a human constant chain (hli).

[0663] Embodiment xvii. The composition of any preceding embodiment, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12.

[0664] Embodiment xviii. The composition of embodiment xvii, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 12.

[0665] Embodiment xix. The composition of any preceding embodiment, wherein the composition is administered sequentially or simultaneously with one or more recombinant hepatitis B polypeptides.

[0666] 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).

[0667] Embodiment xxi. The composition of embodiment xix or xx, wherein the HBc comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:2.

[0668] Embodiment xxii. The composition of embodiment xix or xx, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0669] Embodiment xxiii. The composition of any one of embodiments xix to xxii, wherein the one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

[0670] Embodiment xxiv. The composition of embodiment xxiii, wherein the adjuvant is AS01.

[0671] Group 3 of embodiments describes:

[0672] 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 lipid nanoparticles (LNPs).

[0673] Embodiment 2. The mRNA of any preceding embodiment, wherein the hepatitis B virus core antigen (HBc) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:11.

[0674] Embodiment 3. The mRNA of any preceding embodiment, wherein the hepatitis B virus core antigen (HBc) is fused to human constant chain (hli).

[0675] Embodiment 4. The mRNA of any preceding embodiment, wherein the mRNA further encodes the hepatitis B small surface protein (HBs).

[0676] Embodiment 5. The mRNA of any preceding embodiment, wherein the hepatitis B small surface protein (HBs) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0677] Embodiment 6. The mRNA of any one of embodiments 4 or 5, wherein the mRNA encoding HBc (HBcmRNA) is more than the mRNA encoding HBs (HBs mRNA) by weight.

[0678] Embodiment 7. The mRNA of any one of embodiments 4 to 6, wherein the HBc mRNA and the HBs mRNA are each present in a ratio of 1.5:1 by weight.

[0679] Embodiment 8. A composition for treating chronic hepatitis B infection, comprising mRNA encoding at least hepatitis B virus surface protein (HBsAg), wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

[0680] Embodiment 9. The mRNA of embodiment 8, wherein the HBsAg is hepatitis B small surface protein (HBs).

[0681] Embodiment 10. The mRNA of embodiment 9, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0682] Embodiment 11. The mRNA of embodiments 8 to 10, wherein the HBsAg is fused to human constant chain (hli).

[0683] Embodiment 12. The mRNA of any preceding embodiment, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12.

[0684] Embodiment 13. The mRNA of any preceding embodiment, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 12.

[0685] Embodiment 14. The mRNA of any preceding embodiment, wherein the composition is administered sequentially or simultaneously with one or more recombinant hepatitis B polypeptides.

[0686] 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).

[0687] Embodiment 16. The mRNA of any preceding embodiment, wherein the HBc comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:2.

[0688] Embodiment 17. The mRNA of any preceding embodiment, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0689] Embodiment 18. The mRNA of any preceding embodiment, wherein the one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

[0690] Embodiment 19. The mRNA of embodiment 18, wherein the adjuvant is AS01.

[0691] Embodiment 20. A method of treating chronic hepatitis B infection comprising administering a prime-boost regimen to a human, wherein mRNA encoding at least one hepatitis B virus antigen is administered as a prime dose and mRNA encoding at least one hepatitis B virus antigen is administered as a boost dose.

[0692] Embodiment 21. The method of embodiment 20, comprising administering four consecutive doses of mRNA to the human.

[0693] Embodiment 22. A method as described in embodiment 20 or 21, wherein a separate composition comprising one or more adjuvanted recombinant hepatitis B polypeptides is administered simultaneously with the mRNA, wherein the recombinant hepatitis B polypeptides comprise recombinant hepatitis B core protein (HBc) and recombinant hepatitis B small surface protein (HBs).

[0694] Embodiment 23. 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 virus antigen is administered as a prime dose and one or more recombinant hepatitis B polypeptides are administered as a boost dose.

[0695] Embodiment 24. The method of embodiment 23, wherein the hepatitis B virus antigen is selected from hepatitis B core antigen (HBc) or hepatitis B surface antigen (HBsAg).

[0696] Embodiment 25. The method of embodiment 23, wherein the hepatitis B surface antigen (HBsAg) is the hepatitis B small surface protein (HBs).

[0697] Embodiment 26. The method of embodiments 23-25, wherein the hepatitis B virus antigen is fused to hli.

[0698] Embodiment 27. The method 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).

[0699] Embodiment 28. The method of embodiments 23-27, wherein the one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

[0700] 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.

[0701] 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.

[0702] 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).

[0703] Embodiment 32. The mRNA of any preceding embodiment, wherein the sterol is cholesterol.

[0704] Embodiment 33. The mRNA of any preceding embodiment, wherein the LNP comprises about 0.5 to 15 mol% of a PEG-modified lipid, about 5 to 25 mol% of a non-cationic lipid, about 25 to 55 mol% of a sterol, and about 20 to 60 mol% of an ionizable cationic lipid.

[0705] Embodiment 34. The mRNA of any preceding embodiment, wherein the LNP has a diameter of 50 to 200 μm.

[0706] Embodiment 35. The mRNA of any preceding embodiment, wherein the LNP has a polydispersity of 0.4 or less, such as 0.3 or less.

[0707] 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.

[0708] Embodiment 37. The mRNA of any preceding embodiment, wherein at least half of the mRNA is encapsulated in the LNP, suitably at least 85%, especially at least 95%, such as 100%.

[0709] Embodiment 38. The mRNA of any preceding embodiment, wherein the mRNA is a non-replicating or self-replicating mRNA (SAM).

[0710] Embodiment 39. The mRNA of any preceding embodiment, wherein the self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and (ii) the hepatitis B polypeptide.

[0711] Embodiment 40. The mRNA of any preceding embodiment, wherein the mRNA has the configuration 5' cap-5' UTR-nonstructural protein (NSP) 1-4-subgenomic promoter-hepatitis B polypeptide-3' UTR-poly A.

[0712] Embodiment 41. The mRNA of any preceding embodiment, for administration to a human subject suffering from chronic hepatitis B infection.

[0713] Embodiment 42. The mRNA of any preceding embodiment, wherein the mRNA is a non-replicating mRNA.

[0714] Embodiment 43. The mRNA of any preceding embodiment, wherein the one or more recombinant hepatitis B polypeptides are administered with AS01 adjuvant.

[0715] 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.

[0716] Embodiment 45. The mRNA of any preceding embodiment, wherein the method comprises a prime-boost regimen, wherein the mRNA is administered as a prime dose and the one or more recombinant hepatitis B polypeptides are administered as a boost dose.

[0717] Embodiment 46. The mRNA of embodiment 45, wherein the method comprises a single priming dose of the mRNA and multiple subsequent booster doses of the recombinant hepatitis B polypeptide.

[0718] Embodiment 47. The mRNA of embodiment 46, wherein the method comprises two or three subsequent booster doses of the one or more recombinant hepatitis B polypeptides.

[0719] Embodiment 48. The mRNA of embodiment 45, wherein the method comprises multiple priming doses of the mRNA and multiple subsequent boosting doses of the recombinant hepatitis B polypeptide.

[0720] Embodiment 49. The mRNA of embodiment 48, wherein the method comprises two priming doses of the mRNA and two subsequent boosting doses of the recombinant hepatitis B polypeptide.

[0721] Embodiment 50. An immunogenic composition comprising the mRNA of any preceding embodiment.

[0722] Embodiment 51. The immunogenic composition of embodiment 50, further comprising the one or more recombinant hepatitis B polypeptides.

[0723] Embodiment 52. An immunogenic combination comprising:

[0724] (a) the mRNA of any one of embodiments 1 to 49; and

[0725] (b) one or more recombinant hepatitis B polypeptides administered together with the mRNA.

[0726] 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.

[0727] Embodiment 54. The immunogenic combination of embodiment 53, wherein the one or more recombinant hepatitis B polypeptides are combined with AS01 adjuvant.

[0728] Embodiment 55. The immunogenic combination of any one of embodiments 52 to 54, further comprising an adenoviral vector, which may be a replication-defective chimpanzee adenovirus (ChAd) vector encoding a hepatitis B polypeptide.

[0729] Embodiment 56. The immunogenic combination of embodiment 55, wherein the adenoviral vector encodes hepatitis B virus core antigen (HBc) fused to human invariant chain (hli).

[0730] Embodiment 57. The immunogenic combination of embodiment 56, wherein the adenoviral vector further encodes hepatitis B virus surface antigen (HBs).

[0731] 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 that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 15.

[0732] Embodiment 59. The immunogenic combination of any one of embodiments 55 to 58, wherein the adenoviral vector encodes a polypeptide consisting of an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 15.

[0733] 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 shown in SEQ ID NO: 15.

[0734] 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 shown in SEQ ID NO: 15.

[0735] Embodiment 62. An immunogenic combination comprising:

[0736] 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

[0737] • A second composition comprising recombinant hepatitis B core protein (HBc) and recombinant hepatitis B small surface protein (HBs) and an adjuvant.

[0738] Embodiment 63. The immunogenic combination of embodiment 62, wherein the second composition comprises AS01 adjuvant.

[0739] Embodiment 64. The combination of embodiment 62 or 63, for use in a method of treating chronic hepatitis B (CHB) by sequential or concomitant administration of said composition.

[0740] Embodiment 65. A method of treating chronic hepatitis B (CHB) infection in a human, wherein the method comprises administering to the human the mRNA of any one of embodiments 1 to 49 sequentially or simultaneously with the one or more recombinant hepatitis B polypeptides.

[0741] Embodiment 66. The method for treating chronic hepatitis B infection (CHB) in a human as described in Embodiment 62, wherein the one or more recombinant hepatitis B polypeptides is recombinant hepatitis B virus core antigen (HBc).

[0742] Embodiment 67. The method for treating chronic hepatitis B infection (CHB) in a human as described in Embodiment 63, wherein the composition further comprises recombinant hepatitis B surface antigen (HBs) and an adjuvant.

[0743] Embodiment 68. The method of treating chronic hepatitis B infection (CHB) in a human as described in embodiment 62 or 63, wherein the composition further comprises an adjuvant.

[0744] Embodiment 69. The method of treating chronic hepatitis B infection (CHB) in a human as described in embodiment 65, wherein the adjuvant comprises MPL and QS-21.

[0745] Embodiment 70. The method for treating chronic hepatitis B infection (CHB) in a human as described in Embodiment 63, wherein the recombinant hepatitis B surface antigen (HBs) is a C-terminally truncated recombinant hepatitis B virus core antigen (HBc).

[0746] Embodiment 71. A method of treating chronic hepatitis B (CHB) infection in a human as described in any one of Embodiments 62 to 67, wherein the method further comprises administering to the human an adenoviral vector comprising a polynucleotide encoding a hepatitis B polypeptide.

[0747] Embodiment 72. The method of treating chronic hepatitis B infection (CHB) in a human as described in embodiment 68, wherein the adenoviral vector is a replication-defective chimpanzee adenovirus (ChAd) vector.

[0748] Embodiment 73. A method for treating chronic hepatitis B infection (CHB) in a human as described in embodiment 68 or 69, wherein the adenoviral vector encodes a hepatitis B polypeptide fused to a human invariant chain (hIi).

[0749] Embodiment 74. The method of treating chronic hepatitis B infection (CHB) in a human according to any one of embodiments 68 to 70, wherein the adenoviral vector encodes hepatitis B core antigen (HBc).

[0750] Embodiment 75. The method for treating chronic hepatitis B infection (CHB) in a human as described in Embodiment 71, wherein the adenoviral vector further encodes hepatitis B surface antigen (HBs).

[0751] Embodiment 76. Use of the mRNA of any one of embodiments 1 to 49 or the immunogenic combination of any one of embodiments 50 to 61 in the treatment of HBV.

[0752] Embodiment 77. Use of the mRNA of any one of embodiments 1 to 49 or the immunogenic combination of any one of embodiments 50 to 61 for reducing the level of circulating hepatitis B surface antigen (HBs) in a patient infected with HBV.

[0753] Embodiment 78. Use of the mRNA of any one of embodiments 1 to 49 or the immunogenic combination of any one of embodiments 50 to 61 for the preparation of a medicament.

[0754] Embodiment 79. Use of the mRNA of any one of embodiments 1 to 5449 or the immunogenic combination of any one of embodiments 50 to 61 for the preparation of a medicament for the treatment of HBV.

[0755] Embodiment 80. A kit comprising the following components:

[0756] (a) the mRNA of any one of embodiments 1 to 49; and

[0757] (b) one or more recombinant hepatitis B polypeptides administered together with the mRNA.

[0758] References

[0759] World Health Organization(WHO).Global Hepatitis report,2017.http: / / apps.who.int / iris / bitstream / 10665 / 255016 / 1 / 9789241565455-eng.pdf? ua=1.Accessed November 2017.

[0760] Block TM, Locarnini S, McMahon BJ, Rehermann B, Peters MG. Use of Current and New Endpoints in the Evaluation of Experimental Hepatitis BTherapeutics. Clin Infect Dis. 2017;64(9):1283-1288.

[0761] Cornberg M, Wong VW, Locarnini S, Brunetto M, Janssen HL, Chan HL. The role of quantitative hepatitis B surface antigen revisited. J Hepatol. 2017;66(2):398-411.

[0762] EASL 2017.Clinical Practice Guidelines on the management of hepatitisB virus infection.J Hepatol 2017;67(2),370-398.

[0763] Borghese F,Clanchy FI.CD74:an emerging opportunity as a therapeutictarget in cancer and autoimmune disease.Expert Opin Ther Targets.2011;15(3):237-51.

[0764] Jiang S,et al.,Lipidoid-Coated Iron Oxide Nanoparticles for EfficientDNA and siRNA delivery Nano Lett.,13:1059-1064(2013).

[0765] Zhang,C et al.Advances in mRNA vaccines for infectious diseases,Front.Immunol.2019,10:594.

[0766] Boni C,Laccabue D,Lampertico P,et al.Restored function of HBV-specific T cells afterlong-term effective therapy with nucleos(t)ideanalogues.Gastroenterology.2012;143(4):963-73.e9.

[0767] Rehermann B,Ferrari C,Pasquinelli C,Chisari FV.The hepatitis B viruspersists for decades after patients'recovery from acute viral hepatitisdespite active maintenance of a cytotoxic T-lymphocyte response.Nat Med.1996;2(10):1104-8.

[0768] Thimme R, Wieland S, Steiger C, et al. CD8+ T Cells Mediate Viral Clearance and Disease Pathogenesis during Acute Hepatitis B Virus Infection. Journal of Virology 2003;77(1):68-76.

[0769] WO2005 / 113782

[0770] WO2011 / 076807

[0771] WO2012 / 006376

[0772] WO2012 / 006378

[0773] WO2012 / 006380

[0774] WO2012 / 030901

[0775] WO2012 / 031043

[0776] WO2012 / 031046

[0777] WO2013 / 006825

[0778] WO2013 / 006834

[0779] WO2013 / 006837

[0780] WO2013 / 033563

[0781] WO2014 / 136086

[0782] WO2015 / 095340

[0783] WO2015 / 095346

[0784] WO2016 / 037053

[0785] WO2017 / 070620

[0786] WO2017 / 162265

[0787] Sequence Listing

[0788] SEQ ID NO:1: Amino acid sequence of HBs

[0789] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS TTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

[0790] SEQ ID NO: 2: Amino acid sequence of HBc truncate

[0791] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV

[0792] SEQ ID NO: 3: Amino acid sequence of the spacer region of the foot-and-mouth disease virus 2A cleavage region

[0793] APVKQTLNFDLLKLAGDVESNPGP

[0794] SEQ ID NO: 4: Nucleotide sequence encoding a spacer region incorporated into the foot-and-mouth disease virus 2A cleavage region

[0795] GCCCCTGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAATCCCGGCCCT

[0796] SEQ ID NO: 5: Amino acid sequence of HBc-2A-HBs

[0797] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCAPVKQTLNFDLLKLAGDVESNPGPMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

[0798] SEQ ID NO:6: Nucleotide sequence encoding HBc-2A-HBs

[0799]

[0800] SEQ ID NO: 7: Amino acid sequence of hIi

[0801] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPKPVSKMRMATPLLM QALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGGVTKQDLGPVPM

[0802] SEQ ID NO: 8: Nucleotide sequence encoding hIi

[0803] ATGCACAGGAGGAGGAGCAGGAGCTGCAGGGAGGACCAGAAGCCCGTGATGGACGACCAGCGCGACCTGATCAGCAACAACGAGCAGCTGCCAATGCTGGGCAGGAGGCCCGGAGCACCCGAAAGCAAGTGCAGCAGGGGCGCCCTGTACACCGGCTTCAGCATCCTGGTGACCCTCCTGCTGGCCGGCCAGGCCACCACCGCCTATTTCCTGTACCAGCAGCAGGGCAGGCTCGATAAGCTGACCGTGACCTCCCAGAACCTGCAGCTGGAGAACCTGAGGATGAAGCTGCCCAAGCCCCCCAAGCCCGTGAGCAAGATGAGGATGGCCACCCCCCTGCTGATGCAGGCTCTGCCCATGGGGGCCCTGCCCCAGGGCCCCATGCAGAACGCCACCAAATACGGCAACATGACCGAGGACCACGTGATGCACCTGCTGCAGAACGCCGATCCTCTGAAGGTGTACCCACCCCTGAAAGGCAGCTTCCCCGAGAACCTCAGGCACCTGAAGAACACCATGGAGACCATCGACTGGAAGGTGTTCGAGAGCTGGATGCACCACTGGCTGCTGTTCGAGATGAGCCGGCACAGCCTGGAGCAGAAGCCCACCGACGCCCCTCCCAAGGAGAGCCTCGAGCTCGAGGACCCAAGCAGCGGCCTGGGCGTGACCAAGCAGGACCTGGGCCCCGTGCCCATG

[0804] SEQ ID NO:9: Amino acid sequence of hIi-HBc-2A-HBs

[0805] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGGVTKQDLGPVPMMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVAPVKQTLNFDLLKLAGDVESNPGPMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

[0806] SEQ ID NO:10: Nucleotide sequence encoding hIi-HBc-2A-HBs

[0807]

[0808] SEQ ID NO: 11: Amino acid sequence of HBc

[0809] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0810] SEQ ID NO: 12: Amino acid sequence of hIi substitution variant

[0811] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLM QALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVP

[0812] SEQ ID NO: 13: Nucleotide sequence encoding hIi substitution variant

[0813] ATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATGGATGACCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGGCCGGCTGGACAAACTGACAGTCACCTCCCAGAACCTGCAGCTGGAGAACCTGCGCATGAAGCTTCCCAAGCCTCCCAAGCCTGTGAGCAAGATGCGCATGGCCACCCCGCTGCTGATGCAGGCGCTGCCCATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACAGAGGACCATGTGATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCCGCCACTGAAGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACCATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGAAATGAGCAGGCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAGAGTCACTGGAACTGGAGGACCCGTCTTCTGGGCTGGGTGTGACCAAGCAGGATCTGGGCCCAGTCCCC

[0814] SEQ ID NO:14: Alternative nucleic acid sequence of hIi-HBc-2A-HBs

[0815]

[0816] SEQ ID NO:15: Alternative amino acid sequence of hIi-HBc-2A-HBs

[0817] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCAPVKQTLNFDLLKLAGDVESNPGPMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

[0818] SEQ ID NO:16: Nucleic acid sequence of empty SAM vector

[0819] 1

[0820] 1 The insertion starts after nucleotide 7561

[0821] SEQ ID NO: 17: Human codon-optimized (Genewiz) nucleic acid sequence encoding the hIi_HBc_2A_HBs SAM transgene

[0822]

[0823] SEQ ID NO: 18: hli_HBc_2A_HBs in AA098, SAM plasmid sequence

[0824]

[0825] SEQ ID NO: 19: Human codon-optimized (Genewiz) nucleic acid sequence encoding the HBc_2A_HBs SAM transgene

[0826]

[0827] SEQ ID NO: 20: HBc_2A_HBs in AA098, SAM plasmid sequence

[0828]

[0829] SEQ ID NO:21: Amino acid sequence of hli-HBc

[0830] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0831] SEQ ID NO:22: Nucleotide sequence encoding hli-HBc

[0832]

[0833] SEQ ID NO: 23: hli-HBc plasmid sequence (UTR4)

[0834]

[0835] SEQ ID NO:24: Nucleotide sequence encoding HBs

[0836] ATGGAGAATATCACCAGCGGCTTCCTCGGCCCCCTCTTAGTGCTGCAGGCCGGCTTCTTCCTCCTGACACGGATCCTGACCATCCCGCAGTCCCTGGACTCATGGTGGACCTCCCTGAACTTCCTGGGCGGCTCCCCCGTGTGCCTGGGCCAGAACTCCCAGAGCCCCACCAGCAACCACTCCCCCACCAGCTGCCCCCCCATCTGCCCCGGCTACAGGTGGATGTGCCTGCGGCGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCTCCACCACCACCAACACCGGCCCCTGCAAGACCTGCACCACCCCCGCCCAGGGGAACAGCATGTTCCCCTCCTGCTGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCCCTCCAGCTGGGCCTTCGCCAAGTACCTGTGGGAGTGGGCCTCCGTGCGGTTCAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGTCCCCCACCGTGTGGCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAGCATCGTGAGCCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATC

[0837] SEQ ID NO:25: HBs plasmid sequence (UTR4)

[0838]

[0839] SEQ ID NO:26: Amino acid sequence of hli-HBs

[0840] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

[0841] SEQ ID NO:27: Nucleotide sequence encoding hli-HBs

[0842]

[0843] SEQ ID NO: 28: hli-HBs plasmid sequence (UTR4)

[0844]

[0845] SEQ ID NO:29: IRES nucleotide sequence

[0846] TGATTAAAACAGCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGCACTCTGATTTTACGAAATCCTTGTGCGCCTGTTTTATATCCCTTCCCTAATTCGAAACGTAGAAGCAATGCGCACCACTGATCAATAGTAGGCGTAACGCGCCAGTTACGTCATGATCAAGCATATCTGTTCCCCCGGACTGAGTATCAATAGACTGCTTACGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAGCCCAGTAACACCATGGAAGCTGCAGGGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGCCACTGCAATCCCCACAGGTGACTGTGGCAGTGGCTGCGTTGGCGGCCTGCCTATGGGGAGACCCATAGGACGCTCTAATGTGGACATGGTGCGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATGCCTTCAACCCAGAGGGTAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTTATTCTTATATTGGCTGCTTATGGTGACAATTACAGAATTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTGTAATAGAGCTGTTATATACCTATTTGTTGGCTTTGTACCACTAACTTTAAAATCTATAACTACCCTCAACTTTATATTAACCCTCAATACAGTTGACC

[0847] SEQ ID NO:30: Nucleic acid sequence of human codon-optimized (CodeRNA2) encoding hIi_HBc mRNA transgenic

[0848]

[0849] SEQ ID NO:31: Nucleic acid sequence of human codon-optimized (CodeRNA2) encoding HBs mRNA transgenic

[0850] ATGGAGAATATCACCAGCGGCTTCCTCGGCCCCCTCTTAGTGCTGCAGGCCGGCTTCTTCCTCCTGACACGGATCCTGACCATCCCGCAGTCCCTGGACTCATGGTGGACCTCCCTGAACTTCCTGGGCGGCTCCCCCGTGTGCCTGGGCCAGAACTCCCAGAGCCCCACCAGCAACCACTCCCCCACCAGCTGCCCCCCCATCTGCCCCGGCTACAGGTGGATGTGCCTGCGGCGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCTCCACCACCACCAACACCGGCCCCTGCAAGACCTGCACCACCCCCGCCCAGGGGAACAGCATGTTCCCCTCCTGCTGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCCCTCCAGCTGGGCCTTCGCCAAGTACCTGTGGGAGTGGGCCTCCGTGCGGTTCAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGTCCCCCACCGTGTGGCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAGCATCGTGAGCCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATC

[0851] SEQ ID NO:32: Nucleic acid sequence of human codon-optimized (CodeRNA2) encoding hIi_HBs mRNA transgenic

[0852]

Claims

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 lipid nanoparticles (LNPs).

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

11.

3. The composition of any one of the preceding claims, wherein the hepatitis B virus core antigen (HBc) is fused to a human invariant chain (hIi).

4. The composition of any one of the preceding claims, wherein the composition further comprises mRNA encoding the hepatitis B small surface protein (HBs).

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

1.

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

7. The composition of any one of claims 4 to 6, wherein the composition comprises more mRNA encoding HBc (HBc-mRNA) than mRNA encoding HBs (HBs-mRNA) by weight.

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

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

10. The composition of claim 9, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

1.

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

12. The composition of claim 3, 6 or 11, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO:

12.

13. The composition of claim 12, wherein the human constant chain (hIi) comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

12.

14. The composition of any one of the preceding claims, wherein the composition is administered sequentially or simultaneously with one or more recombinant hepatitis B polypeptides.

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

16. The composition of claim 15, wherein the HBc comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

2.

17. The composition of claim 15 or 16, wherein the HBs comprises an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

1.

18. The composition of any one of claims 14 to 17, wherein the one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

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

20. 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 virus antigen is administered as a prime dose and one or more recombinant hepatitis B polypeptides are administered as a boost dose.

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

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

23. The method of any one of claims 20 to 22, wherein the recombinant hepatitis B polypeptide comprises 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 one or more recombinant hepatitis B polypeptides are administered with an adjuvant.

25. The method of claim 24, wherein the adjuvant is AS01.

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