RNA for preventing or treating tuberculosis

Abstract

The present disclosure provides agents and methods for preventing or treating tuberculosis using RNA. The RNA encoding variants of antigens of Mycobacterium tuberculosis ox of fragments thereof is formulated and administered in a way that the variants are produced by cells of a subject, in particular after intramuscular or intravenous administration of the RNA.

Description

[0001] RNA FOR PREVENTING OR TREATING TUBERCULOSIS

[0002] Related Applications

[0003] The present application claims priority to and the benefit of U.S. Provisional Application No. 63 / 708,040, filed on October 16, 2024, and U.S. Provisional Application No. 63 / 815,212, filed on May 30, 2025; the entirety of each of which are incorporated herein by reference.

[0004] Technical Field

[0005] The disclosure provides agents and methods for preventing or treating tuberculosis using RNA. The RNA encoding variants of antigens of Mycobacterium tuberculosis or of fragments thereof is formulated and administered in a way that the variants are produced by cells of a subject, in particular after intramuscular or intravenous administration of the RNA.

[0006] Background

[0007] The use of RNA to deliver foreign genetic information into target cells offers an attractive alternative to DNA. The advantages of RNA include transient expression and non-transforming character. RNA does not require nucleus infiltration for expression and moreover cannot integrate into the host genome, thereby eliminating the risk of oncogenesis.

[0008] The COVID-19 pandemic has showcased the utility and advantages of RNA technology for vaccination, as out of all COVID-19 vaccines under development, the first two to have received emergency use authorization by the FDA were RNA-based. The biotechnology response to the COVID-19 pandemic has highlighted the speed and flexibility of mRNA vaccines, and reveals mRNA therapeutics to be a powerful tool to address epidemic outbreaks caused by newly emerging viruses. The relative simplicity of the development process and flexibility of the manufacturing platform can markedly accelerate clinical development. As such, mRNA-based vaccine technology has attracted a lot of attention during the COVID-19 pandemic.

[0009] Tuberculosis (TB) is caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb) and, in rarer cases, by other pathogens from the Mycobacteriaceae family and is the leading cause of death from a single infectious agent. Mtb is a gram-positive, rod-shaped bacterium from the Mycobacteriaceae family. The more than 4,000 genes encoded within an approximately 4 million base pair genome render Mtb a complex pathogenic organism. This is further emphasized by the atypical composition of its cell wall, which has a high lipid content.

[0010] Despite the observed trend for reduction in TB cases and TB-related deaths for the last 20 years, 1.42 million people died from TB alone in 2019. In addition to the active form of TB, difficulties arise from latent TB infection (LTBI), when the infected patient doesn't present clinical symptoms. The estimated 2 billion latently infected individuals worldwide pose a huge and unpredictable reservoir of Mtb (WORLD HEALTH ORGANIZATION. Global tuberculosis report 2019. Geneva, WORLD HEALTH ORGANIZATION; 2019. ISBN: 978-92-4-156571-4). The high prevalence of HIV-1 infections further increases the risk for TB disease acquisition, activation of a latent TB infection, and death from HIV-TB co-infection. In 2009, 0.2 million deaths were related to HIV-TB comorbidity. The complexity of the Mtb cell wall makes the bacterium resistant to environmental impact and to therapy with certain antibiotics. The latter further complicates anti-TB treatment especially in low- and middle-income countries (WORLD HEALTH ORGANIZATION. Global tuberculosis report 2020. Geneva, WORLD HEALTH ORGANIZATION; 2020. ISBN: 978-92-4-001313-1).

[0011] An attenuated strain of Mycobacterium bovis, bacillus Calmette-Guerin (BCG), is the only licensed TB vaccine, introduced in 1921. The use of the live vaccine BCG is not recommended for immunocompromised individuals and the protective efficacy against pulmonary TB conferred by immunization with BCG is highly variable, ranging from 50-80%. Moreover, passaging of BCG over the decades further attenuated the currently used BCG strains, reducing its protective efficacy (Brosch R, et al. Proc. Natl. Acad. Sci. U.S.A., 2007; 104(13):5596-5601). Thus, there is an unmet medical need for a safer and more effective vaccine to prevent TB, especially for a vaccine that can be administered to immunocompromised individuals.

[0012] The pipeline of clinical trials for TB vaccine candidates comprises use of live, live-attenuated, and inactivated mycobacteria, and of Mtb antigens as recombinant protein (subunit vaccine) (TuBerculosis Vaccine Initiative (TBVI). Available from: https: / / www.tbvi.eu / what-we-do / pipeline-of-vaccines / ). The drawbacks from these vaccine platforms are i. their low safety, due to replication-competent live vaccines still being infectious, ii. low immunogenicity of inactivated vaccines, and iii. the need for addition of adjuvants to subunit vaccines to enhance immunogenicity. To date, most vaccine candidates have failed to demonstrate better protection from TB or from the development of TB compared to placebo in clinical trials.

[0013] For all these reasons, novel agents for preventing or treating tuberculosis are required.

[0014] Summary

[0015] The present disclosure provides compositions which are useful as tuberculosis (TB) vaccines. The compositions provided herein comprise RNA for delivering Mycobacterium tuberculosis (Mtb) antigens to a subject. The findings described herein demonstrate that RNA described herein, e.g., non-modified uridine containing mRNA (uRNA) or nucleoside modified mRNA (modRNA) encoding variants of the LpqH and / or PstSl antigens of Mycobacterium tuberculosis or of fragments thereof, is useful for preventing or treating tuberculosis. The RNA encoding variants of the LpqH and / or PstSl is formulated and administered in a way that the variants can be produced and either displayed on the membrane of the patient's cells or secreted by patient's cells to trigger an immune response, which prevents or combats tuberculosis.

[0016] Unlike the attenuated vaccine BCG, this TB vaccine candidate does not carry the risks associated with infection and may therefore be given to people who cannot be administered live organism (such as pregnant women and immunocompromised persons).

[0017] In addition, the variants according to the present disclosure have been designed to ensure more efficient expression and intracellular transport, as well as presentation or secretion of the LpqH and PstSl antigens of Mycobacterium tuberculosis or of fragments thereof.

[0018] In particular, the present inventors have recognized that the N-termini of LpqH and PstSl may be subject to 0- glycosylation in eukaryotic cells, which might lead to incorrect folding of the protein and may affect its immune properties. Accordingly, removal of amino acid residues that may be subject to O-glycosylation in eukaryotic cells leads to an improved immune response against LpqH and PstSl. Removal of the N-terminal cysteine can prevent N-terminal palmitoylation to occur intracellularly before the protein is exported and the signal peptide is cleaved. Such intracellular palmitoylation could result in reduced secretion or surface expression of the antigen, which would be detrimental for the induction of antibody and CD4 T cell responses. Furthermore, cysteines are well-known to form covalent bonds with other cysteines within the same, or other proximal proteins. For instance, the structure of folded domain of LpqH reveals a cysteine disulfide bond between residues at positions 67 and 158, which is likely essential to stabilize correct folding. Leaving an unmodified free cysteine in a mRNA encoded antigen version of such a protein could result in faulty formation of alternative sulfur bridges, or unwanted homodimerization with other copies of LpqH or heterodimerization with unknown proteins in the proximity. Lastly, efficient presentation of LpqH and PstSl epitopes to the immune system can be achieved by attachment of a transmembrane domain or multimerization domain to the variants.

[0019] These effects are achieved by the aspects and embodiments of the disclosure provided herein below.

[0020] In one aspect, the disclosure provides an RNA molecule comprising a nucleic acid sequence encoding an amino acid sequence comprising at least one variant of a Mycobacterium tuberculosis antigen or a fragment thereof, wherein the amino acid sequence of the at least one variant is characterized by one or more of the following modifications compared to the corresponding wildtype Mycobacterium tuberculosis antigen or fragment thereof: a) deletion or substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells, b) deletion or substitution of an N-terminal cysteine residue, and / or c) addition of a non-native transmembrane domain or multimerization domain.

[0021] In some embodiments, the Mycobacterium tuberculosis antigen is LpqH or PstSl.

[0022] In some embodiments of an RNA molecule, a variant is modified compared to the Mycobacterium tuberculosis antigen or fragment thereof in that the endogenous signal peptide has been replaced with a non-native signal peptide.

[0023] In some embodiments, LpqH comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0024] In some embodiments, at least one variant of LpqH comprises a deletion at one or more amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0025] In some embodiments, at least one variant of LpqH comprises a deletion of an amino acid sequence corresponding to positions 23 to 48 of SEQ ID NO 1 or SEQ ID NO: 2.

[0026] In some embodiments, at least one variant of LpqH comprises a substitution at one or more amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0027] In some embodiments, the N-terminal cysteine of LpqH is at an amino acid position corresponding to position 22 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0028] In some embodiments, an endogenous signal peptide of LpqH is at the N-terminal end of the antigen, optionally wherein the endogenous signal peptide is at amino acid positions corresponding to positions 1 to 21 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0029] In some embodiments, PstSl comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0030] In some embodiments, at least one variant of PstSl comprises a deletion at one or more amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0031] In some embodiments, at least one variant of PstSl comprises a deletion of an amino acid sequence corresponding to positions 25 to 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0032] In some embodiments, at least one variant of PstSl comprises a substitution at one or more amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0033] In some embodiments, the N-terminal cysteine of PstSl is at an amino acid position corresponding to position 24 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0034] In some embodiments, an endogenous signal peptide is at the N-terminal end of the antigen, optionally wherein the endogenous signal peptide is at amino acid positions corresponding to positions 1 to 23 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0035] In some embodiments, a non-native signal peptide is a viral signal peptide.

[0036] In some embodiments, a non-native signal peptide is an HSV-1 glycoprotein D signal peptide, a HSV-2 glycoprotein D signal peptide, a Japanese encephalitis PRM signal sequence or a VSVg protein signal sequence.

[0037] In some embodiments, the transmembrane domain is a transmembrane domain from Nipah virus (NiV) or a transmembrane domain from HSV, optionally a HSV-gDl-TM.

[0038] In some embodiments, the multimerization domain is a ferritin domain, optionally a ferritin domain from Helicobacter pylori (HP-ferritin).

[0039] In some embodiments, the multimerization domain is derived from a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase, the multimerization domain is a Mycobacterium tuberculosis 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, Mycobacterium bovis 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, Mycobacterium bovis BCG 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, or Mycobacterium leprae 6,7-dimethyl-8- ribityllumazine synthase or fragment thereof. In some embodiments, the / ^yco / ?acter / 'J776,7-dimethyl-8-ribityllumazine synthase is encoded by a Mycobacterium RibH gene.

[0040] In some embodiments, the multimerization domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or 100% identity to the amino acid sequence of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, or 132.

[0041] In some embodiments, a transmembrane domain and / or multimerization domain is attached to the N-terminus of the at least one variant and / or the C-terminus of the at least one variant, optionally with a linker, further optionally wherein the linker is a glycine-serine linker (GS linker).

[0042] In some embodiments, at least one variant of LpqH comprises the amino acid sequence of any one of SEQ ID NOs: 50, 56, 62, 66, 68, 70 or 72.

[0043] In some embodiments, at least one variant of PstSl comprises the amino acid sequence of any one of SEQ ID NOs: 76, 82, 88, 92, 94, 96 or 98.

[0044] In some embodiments, an RNA molecule comprises: a) a 5' cap, optionally having a capl structure or comprising m27'3'0Gppp( i2' °)ApG, b) a 5'-UTR, optionally comprising a modified human alpha-globin 5'-UTR or having the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 98%, 96%, 94%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104, c) a 3'-UTR, optionally comprising a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA or having the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105, d) a polyA sequence, wherein the polyA sequence optionally is an interrupted sequence of A nucleotides, comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a nucleotide linker sequence of 10 nucleotides or comprises the nucleotide sequence of SEQ ID NO: 106, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 106, and / or e) modified nucleotides, nucleosides or nucleobases, optionally comprising modified uridines in place of at least one or in place of all uridines, wherein the modified uridines further optionally are Nl-methyl- pseudouridine.

[0045] In some embodiments, the coding sequence of the RNA molecule is codon-optimized and / or is characterized in that its G / C content is decreased compared to the parental sequence.

[0046] In one aspect, the disclosure provides a protein encoded by any one of the RNA molecules disclosed herein.

[0047] In one aspect, the disclosure provides a DNA molecule encoding any one of the RNA molecules disclosed herein.

[0048] In one aspect, the disclosure provides a pharmaceutical composition comprising one or more RNA molecules disclosed herein.

[0049] In some embodiments of the pharmaceutical composition, the one or more RNA molecule is formulated in a lipid formulation, optionally wherein the one or more RNA molecule is formulated in lipid nanoparticles or liposomes.

[0050] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants, and wherein the adjuvants optionally comprise a) an RNA encoding one or more immunomodulating molecules, optionally wherein the one or more immunomodulating molecules comprise cytokines, and / or b) one or more immunity inducing or immunomodulating moieties, optionally comprising a peptidoglycan moiety.

[0051] In some embodiments, the one or more RNA molecules are in a liquid formulation.

[0052] In some embodiments, the one or more RNA molecules are in a frozen formulation.

[0053] In some embodiments, the one or more RNA molecules are in a lyophilized formulation.

[0054] In some embodiments, the one or more RNA molecules are formulated for injection. In some embodiments, the one or more RNA molecules are formulated for intramuscular administration.

[0055] In one aspect, the disclosure provides a kit comprising one or more pharmaceutical compositions disclosed herein.

[0056] In some embodiments, the kit comprises two or more pharmaceutical compositions which comprise the same or different RNA molecules disclosed herein in separate vials and optionally comprising instructions for use of the one or more pharmaceutical composition for treating or preventing tuberculosis.

[0057] In one aspect, the disclosure provides an RNA, protein, DNA, pharmaceutical composition or kit disclosed herein for use as a medicament.

[0058] In some embodiments of the RNA molecule, protein, DNA molecule, pharmaceutical composition or kit for use disclosed herein, the use comprises treating or preventing tuberculosis in a subject.

[0059] In one aspect, the disclosure provides a method for treating or preventing tuberculosis in a subject, wherein the method comprises administering a RNA, protein, DNA or pharmaceutical composition disclosed herein to the subject.

[0060] In some embodiments of the method, administration is intramuscular administration.

[0061] In some embodiments, the method comprises administering to the subject at least one dose of the RNA molecule, protein, DNA molecule, pharmaceutical composition or kit.

[0062] In some embodiments, the method comprises administering to the subject at least two doses of the RNA molecule, protein, DNA molecule, pharmaceutical composition or kit.

[0063] In some embodiments, the subject is a human.

[0064] In one aspect, the disclosure provides a use of the RNA, protein, DNA or pharmaceutical composition disclosed herein for the manufacture of a medicament for treating or preventing tuberculosis in a subject.

[0065] In some embodiments of the RNA molecule, protein, DNA molecule, pharmaceutical composition or kit for use disclosed herein, the method disclosed herein or the use disclosed herein, the tuberculosis is caused by an infection with a Mycobacterium, optionally selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium orygis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canetti and Mycobacterium pinnipedii.

[0066] Brief Description of the Figures

[0067] Fig. 1: Schematic representation of N-terminal segments of LpqH and PstSl

[0068] Provided are amino acid positions 1-53 of SEQ ID NO: 1 (LpqH) and amino acid positions 1-52 of SEQ ID NO: 3 (PstSl). Underlined amino acid residues are residues of LpqH and PstSl that may be subject to O-linked glycosylation in the mycobacterial form of these antigens. The amino acid residues shown in italics represent the secretory signal peptide and the Bold N in LpqH is a predicted N-linked glycosylation site.

[0069] Figs. 2A-2E: Deletion of the N-terminal cysteine has no negative effect on PstSl expression and localization.

[0070] Provided are graphs showing the expression and localization of variants of PstSl having a deletion of the N-terminal cysteine (corresponding to amino acid position 24 of SEQ ID NO: 3 or 4) following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total PstSl expression was quantified by flow cytometry using anti-PstSl (Fig. 2A) and anti-HiBiT (Fig. 2B) monoclonal antibodies (mAbs) after permeabilization. The surface expression was quantified using anti-PstSl (Fig. 2C) and anti-HiBiT mAbs (Fig. 2D). PstSl secretion levels in the supernatant of the HEK293T cultures were quantified using a HiBIT-Luciferase Assay (Fig. 2E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Nontransfected (blocked bars).

[0071] Figs. 3A-3E: Deletion of the N-terminal cysteine has no negative effect on LpqH expression and localization.

[0072] Provided are graphs showing the expression and localization of variants of LpqH having a deletion of the N-terminal cysteine (corresponding to amino acid position 22 of SEQ ID NO: 1 or 2) following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total LpqH expression was quantified by flow cytometry using anti-LpqH (Fig. 3A) and anti-HiBiT (Fig. 3B) monoclonal antibodies (mAbs) after permeabilization. Surface expression was quantified using anti-LpqH (Fig. 3C) and anti-HiBiT mAbs (Fig. 3D). LpqH secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 3E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Nontransfected (blocked bars).

[0073] Figs. 4A-4E: Deletion of sequence comprising amino acids subject to O-linked glycosylation has no negative effect on the expression level and localization of PstSl.

[0074] Provided are graphs showing the expression and localization of variants of PstSl having a deletion of an N-terminal sequence comprising amino acids subject to O-linked glycosylation (deletion of an amino acid sequence corresponding to positions 25 to 45 of SEQ ID NO: 3 or 4) and replacement of endogenous signal peptide with an HSV signal peptide following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total PstSl expression was quantified by flow cytometry using anti-PstSl (Fig. 4A) and anti-HiBiT (Fig. 4B) monoclonal antibodies (mAbs) after permeabilization. Furthermore, the surface expression was quantified using anti-PstSl (Fig. 4C) and anti-HiBiT (Fig. 4D) mAbs. PstSl secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 4E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Non-transfected (blocked bars).

[0075] Figs. 5A-5E: Deletion of sequence comprising amino acids subject to O-linked glycosylation has no negative effect on the expression level and localization of LpqH.

[0076] Provided are graphs showing the expression and localization of variants of LpqH having a deletion of an N-terminal sequence comprising amino acids subject to O-linked glycosylation (deletion of an amino acid sequence corresponding to positions 23 to 48 of SEQ ID NO: 1 or 2) and replacement of endogenous signal peptide with an HSV signal peptide following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total LpqH expression was quantified by flow cytometry using anti-LpqH (Fig. 5A) and anti-HiBiT (Fig. 5B) monoclonal antibodies (mAbs) after permeabilization. Surface expression was quantified using anti-LpqH (Fig. 5C) and anti-HiBiT mAbs (Fig. 5D). LpqH secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 5E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Non-transfected (blocked bars). Figs. 6A-6E: Addition of a non-native transmembrane domain leads to high surface expression of PstSl. Provided are graphs showing the expression and localization of variants of PstSl having an addition of a non-native transmembrane domain (from Nipah virus (NiV) or HSV-gDl) following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total PstSl expression was quantified by flow cytometry using anti-PstSl (Fig. 6A) and anti-HiBiT (Fig. 6B) monoclonal antibodies (mAbs) after permeabilization. Furthermore, the surface expression was quantified using anti-PstSl (Fig. 6C) and anti-HiBiT (Fig. 6D) mAbs. PstSl secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 6E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Nontransfected (blocked bars).

[0077] Figs. 7A-7E: Addition of a non-native transmembrane domain leads to high surface expression of LpqH. Provided are graphs showing the expression and localization of variants of LpqH having an addition of a non-native transmembrane domain (from Nipah virus (NiV) or HSV-gDl) following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total LpqH expression was quantified by flow cytometry using anti-LpqH (Fig. 7A) and anti-HiBiT (Fig. 7B) monoclonal antibodies (mAbs) after permeabilization. Surface expression was quantified using anti-LpqH (Fig. 7C) and anti-HiBiT (Fig. 7D) mAbs LpqH secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 7E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Non-transfected (blocked bars).

[0078] Figs. 8A-8E: Variants of PstSl having a multimerization domain are expressed and can be found in cellular supernatant.

[0079] Provided are graphs showing the expression and localization of variants of PstSl having an addition of a multimerization domain (ferritin from H. pylori) following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total PstSl expression was quantified by flow cytometry using anti-PstSl (Fig. 8A) and anti-HiBiT (Fig. 8B) monoclonal antibodies (mAbs) after permeabilization. Surface expression was quantified using anti-PstSl (Fig. 8C) and anti-HiBiT (Fig. 8D) mAbs. PstSl secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 8E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Non-transfected (blocked bars).

[0080] Figs. 9A-9E: Variants of LpqH having a multimerization domain are expressed and can be found in the cellular supernatant.

[0081] Provided are graphs showing the expression and localization of variants of LpqH having an addition of a multimerization domain (ferritin from H. pylori) following transfection of corresponding mRNA constructs in HEK293T cells (0.5 pg of the mRNA construct) using MessengerMax. Total LpqH expression was quantified by flow cytometry using anti-LpqH (Fig. 9A) and anti-HiBiT (Fig. 9B) monoclonal antibodies (mAbs) after permeabilization. Furthermore, the surface expression was quantified using anti-LpqH (Fig. 9C) and anti-HiBiT (Fig. 9D) mAbs. LpqH secretion levels in the supernatant of the HEK293T cultures were quantified using the HiBiT-Luciferase Assay (Fig. 9E). Bars depict the average Median fluorescent intensity (MFI) values of three independent transfections. Empty bars depict untagged constructs and black bars HiBit-tagged constructs. Error bars depict the standard deviation. NT = Non-transfected (blocked bars).

[0082] Figs. 10A-10B: Variants of LpqH having a multimerization domain form secreted multimers.

[0083] Supernatants of HEK293T cells transfected with the indicated constructs. Protein samples were analyzed either by SDS- PAGE and western blot in normal reducing conditions (Fig. 10A) or were analyzed by Blue-Native PAGE (Fig. 10B). For the BN-PAGE, samples were carefully thawed on ice, mixed with native sample buffer and Wpl of the sample were loaded onto a 3-12% native PAGE gel. Next, 10 pl of the native was added, and the gel was run at 4°C for 90-120 minutes at 150V. Blotting was performed using a PVDF membrane that was incubated in methanol, rinsed with ddH20, and incubated in transfer buffer before being transferred onto the membrane using a Keutz-Kammer for 180 minutes at 84A. The PVDF membrane was then washed with PBS-Tween and blocked with 5% milk in PBST-T for 1 hour at RT before the primary anti-HiBiT and secondary peroxidase conjugated anti-mouse.

[0084] Fig. 11: Mutation of N-glycosylation sites within PstSl improves formation of conformational epitopes.

[0085] HEK293T cells were transfected with the mRNA constructs encoding variants of PstSl having mutated N-glycosyylation sites. Expression was determined by flow cytometry after staining permeabilized cells with the anti-PstSl antibody P4- 163. Transfection was depicted either as the percentage of positive cells (left y-axis, X-shaped icons), or as the median fluorescence intensity of the population (Right y-axis, Bars). The constructs Tbl9 and Tb21 with mutated N- glycosylation sites exhibited better recognition by the P4-163 antibody. These data suggest that removal of the N- linked glycosylation sites within PstSl may improve correct folding of the antigen.

[0086] Figs. 12A-12C: Induction of PstSl-specific IgG following immunization with mRNAs encoding PstSl antigens.

[0087] An immunization and serum collection scheme is provided (Fig. 12A). Serum samples were collected seven days after booster immunization, and IgG concentrations were measured using ELISA (Fig. 12B). Meta bolically active H37Ra (BSL- II lab strain) were incubated with serum from mice immunized with mRNA constructs and the surface binding capacity of IgG antibodies was quantified through flow cytometry (Fig. 12C).

[0088] Figs. 13A-13B: Induction of LpqH-specific IgG following immunization with mRNAs encoding LpqH antigens.

[0089] Serum samples were collected seven days after booster immunization, and IgG concentrations were measured using ELISA (Fig. 13A). Metabolically active H37Ra (BSL-II lab strain) were incubated with serum from mice immunized with mRNA constructs and the surface binding capacity of IgG antibodies were quantified through flow cytometry (Fig. 13B).

[0090] Figs. 14A-14B: Induction of germinal center B cells (GCB) following immunization with mRNAs encoding PstSl antigens.

[0091] The overall GCB frequencies in the lymph nodes of mice immunized with mRNAs encoding PstSl antigens were similar. However, frequencies of PstSl-reactive GCB were notably higher in mice immunized with the mRNA encoding monomeric secreted PstSl. Lymph node samples were harvested seven days after the booster immunization, and the GCB response was quantified by flow cytometry. Fig. 14A: Total frequencies of GCB. Fig. 14B: Frequencies of antigenreactive GCB. Figs. 14A and 14B: Statistical differences were assessed by Kruskal-Wallis test followed by multiple comparisons against a single control where that control is the benchmark PstSl. * = p<0.05; ** = p<0.01, *** = p<0.001. Figs. 15A-15B: Induction of germinal center B cells (GCB) following immunization with mRNAs encoding LpqH antigens.

[0092] The overall and antigen-reactive GCB frequencies in the lymph nodes of mice immunized with mRNAs encoding LpqH antigens were similar. Lymph node samples were harvested seven days after the booster immunization, and the GCB response was quantified by flow cytometry. Fig. 15A: Total frequencies of GCB. Statistical differences between the immunized groups were assessed using Kruskal-Wallis test no significant difference was detected by ANOVA. Fig. 15B: Frequencies of antigen-reactive GCB. Significant differences detected by Kruskal-Wallis test are depicted. * = p<0.05; ** = p<0.01.

[0093] Fig. 16: Induction of PstSl-reactive memory B cells following immunization with mRNAs encoding PstSl antigens.

[0094] The highest frequencies of PstSl-reactive memory B cells were induced when immunizing with mRNAs encoding a construct comprising PstSl and LpqH connected to a RibH multimerization domain. Spleen samples were harvested seven days after the booster immunization and the memory B cell response was analyzed by flow cytometry.

[0095] Figs. 17A-17E: Splenocyte responses following immunization with mRNAs encoding LpqH antigens or mRNAs encoding PstSl antigens.

[0096] C57BL / 6 mice were immunized with the indicated constructs in two separate experiments (Figs. 17A-17C and 17D-17F) as described in Fig. 12A. At Day 35 of the experiment splenocytes were harvested and seeded at the indicated cell densities (Figs 17A-17C: 5xl05, Figs. 17D-17F: 3xl05) and re-stimulated with overlapping peptide pools of PstSl (Figs. 17A-17C) or LpqH (Figs. 17D-17F). Each circle represents the value for one animal averaged over three (Figs. 17A- 17C) or two (Figs. 17D-17F) technical replicates. Interferon gamma (IFNy) responses against PstSl (Fig. 17A) reached the upper limit of detection (ULOD) for many samples and are therefore depicted as 2000 SFU. Statistics were performed with Kruskal-Wallis test of multiple comparisons against a single control, excluding the buffer control group and setting the benchmark as the monomeric PstSl (Figs. 17A-17C) or LpqH (Figs. 17D-17F) respectively. For groups displayed in Figs. 17A-17C, ANOVA was not significant and therefore no specific comparisons are depicted. Ns = not significant (p=0.05-1.0), ** = p<0.01, *** = pcO.OOl.

[0097] Fig. 18: Functional binding of antibodies.

[0098] To assess functional binding of antibodies, first, serum binding to lysate of Mycobacterium tuberculosis H37Rv was assessed in a lysate ELISA. Equivalent, although moderately increased IgG binding was seen for all optimized PstSl constructs (Tb29, Tb39, Tb57, and Tb59) compared to the flag-tagged benchmark. In the LpqH-based constructs, the benchmark exhibited varied responses, which seemed slightly lower in the optimized monomeric secreted (Tb30) construct, equivalent in the LpqH-RibH-LpqH (Tb60) multimeric construct and slightly higher in the membrane-bound construct (Tb36). However, the best responses among all groups were found for the chimeric, multimeric construct PstSl-RibH-LpqH (Tb61).

[0099] Detailed Description

[0100] Mycobacterium tuberculosis (Mtb) is a non-motile, slowly growing and rod shaped (2-4 pm in length and 0.2-0.5 pm in width) bacterium. Mtb is gram-positive, obligate aerobe, requires a host for growth and reproduction, and does not form spores.

[0101] The term "tuberculosis" or "TB" is used to describe the infection caused by the infective agent "Mycobacterium tuberculosis or "Mtb'. Tuberculosis is a potentially fatal contagious disease that can affect almost any part of the body but is most frequently an infection of the lungs. While the majority of tuberculosis infections are caused by Mycobacterium tuberculosis, there are other Mycobacterium species that can cause tuberculosis as well. These species include Mycobacterium bovis, Mycobacterium caprae, Mycobacterium orygis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canetti and Mycobacterium pinnipedii. Mycobacterium tuberculosis and some other mycobacteria that are transmitted by airborne droplet nuclei produced when an individual with active disease coughs, speaks, or sneezes. When inhaled, the droplet nuclei reach the alveoli of the lung. In susceptible individuals, the organisms may then multiply and spread through lymphatics to the lymph nodes, and through the bloodstream to other sites such as the lung apices, bone marrow, kidneys, and meninges. Infections with other Mycobacterium species, such as Mycobacterium bovis or Mycobacterium caprae, are also associated with the consumption of un-pasteurized milk from infected animals. The development of acquired immunity in 2 to 10 weeks results in a halt to bacterial multiplication. Lesions heal and the individual remains asymptomatic. Mycobacteria can remain dormant (latent TB) in the body after infection for years, concealed in the phagocytosed cells, and never develop into the disease. Such an individual is said to have tuberculous infection without disease, and will show a positive tuberculin test. The clinical status of latent TB is traditionally associated with the transition of Mtb to a dormant state in response to non-optimal growth conditions in vivo due to activation of the host immune response. Dormancy is a specific physiological state characterized by significant cessation of metabolic activity and growth, whereas resuscitation from dormancy is a process of restoring cell activity followed by bacterial multiplication, which in case of Mtb can lead to disease progression. The risk of developing active disease with clinical symptoms diminishes with time and may never occur, but is a lifelong risk. Approximately 5% of individuals with tuberculous infection progress to active disease.

[0102] Embodiments of variants of Mycobacterium tuberculosis (Mtb) antigens

[0103] The present disclosure describes variants of Mtb antigens or of fragments of the Mtb antigens (referred to as "Mtb antigens" herein) and RNA encoding these antigens. Mtb antigens described herein include LpqH and PstSl.

[0104] Both PstSl (a 38kDa lipoprotein) and LpqH (a 19kDa lipoprotein) are known mycobacterial lipoproteins. Both antigens contain bacterial secretory signal peptides on their N-terminal ends. The PstSl signal peptide spans residues 1-23 of SEQ ID NO: 1 (MKIRLHTLLAVLTAAPLLLAAAG; SEQ ID NO: 107); and the LpqH signal peptide spans residues 1-22 of SEQ ID NO: 2 (MKRGLTVAVAGAAILVAGLSG; SEQ ID NO: 108). In some databases, the starting amino acids of both the proteins are annotated as valines, since they are encoded by the DNA nucleotides GTG. However, in Mycobacteria a GTG sequence can encode an N-terminal Methionine, which is therefore considered as the wild-type sequence here. These secretory signal peptides target the proteins towards the Sec-translocon complexes SecYEG in the Mycobacterial host. The energy for this translocation is thought to come from the secAl essential ATP-ase in Mycobacteria (Miller BK.Zulauf KE.Braunstein M. 2017. The Sec Pathways and Exportomes of Mycobacterium tuberculosis. Microbiol Spectr 5:10.1128 / microbiolspec.tbtb2-0013-2016.). The first N-terminal residue after the cleavage site of the signal peptide is a cysteine, that together with the lipobox motif spanning that residue, targets the protein for lipidation (D.B. Young, T.R. Garbe, Lipoprotein antigens of Mycobacterium tuberculosis, Research in Microbiology, Volume 142, Issue 1, 1991, Pages 55-65.) after translocation over the inner membrane. Generally, this is a multistep process resulting in a triacylated lipoprotein that is located either in the periplasm of the Mycobacteria or transported to the surface and maintained in the outer membrane layer by unknown mechanisms (Nakayama, H., Kurokawa, K. and Lee, B.L. (2012), Lipoproteins in bacteria: structures and biosynthetic pathways. FEES J, 279: 4247-4268.). Indeed experimental data confirm C16 and C19 fatty acid modifications of the N-terminal cysteine of LpqH and other lipoproteins in slow growing Mycobacteria. Specifically, such lipidations included tuberculostearic acid or palmitic acid (Briille, J.K., Tschumi, A. & Sander, P. Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are / ^acylated by Apolipoprotein N- Acyltransferase BCG_2070c. BMC Microbiol 13, 223 (2013).).

[0105] There are some similarities in the process of protein modification between Mycobacteria and eukaryotes. These characteristics can be relevant when a mRNA encoding these proteins is expressed in human or other eukaryotic expression systems. Specifically, Eukaryotic cells can translocate proteins using the sec-translocon, but it is unclear if the bacterial secretory signal peptides would be functional in a Eukaryotic system; and palmitic acid modifications are known to be frequent on human proteins and they can have profound impacts on protein function localization or immunogenicity. Indeed, palmitic acid modification are well described in Eukaryotes. In the cases that this modification occurs on the on N-terminal cysteine residue, the palmitate moves from the side chain to the free amino-group making this modification irreversible (Linder, M., Deschenes, R. Palmitoylation: policing protein stability and traffic. Nat Rev Mo! Cel! Biol 8, 74-84 (2007). https: / / doi.org / 10.1038 / nrm2084).

[0106] The inventors of the present disclosure realized that it is essential to understand whether the bacterial signal peptide targets the proteins for secretion in a eukaryotic cell when expressed as mRNA. Secretion of proteins over the cell membrane, resulting in soluble protein that can be taken up by immune cells or by surface localized antigen, is essential for efficient induction of CD4 T cell responses and B cell responses (including germinal center and memory B cell responses) leading to antibodies. Expressing a wild-type protein where the signal peptide is not functional would be highly detrimental. Therefore, the inventors of the present disclosure investigated whether the endogenous signal peptide can be replaced with a viral SP and whether this optimizes or maintains protein secretion.

[0107] The inventors of the present disclosure further identified that pal ytoilation of the N-terminal cysteine could occur intracellularly before the protein is exported and the signal peptide is cleaved. This could result in reduced secretion or surface expression of the antigen, which would be detrimental for the induction of antibody and CD4 T cell responses. Even if lipid modification of the N-terminal cysteine were found not to occur in human cells, removing or replacing this N-terminal cysteine this could be preferred to avoid unpredictable complications on protein folding in vivo. Cysteines are well-known to form covalent bonds with other cysteines within the same, or other proximal proteins. For instance, the structure of folded domain of LpqH reveals a cysteine disulfide bond between residues 67 and 158, which is likely essential to stabilize correct folding. Leaving an unmodified free cysteine in a mRNA encoded antigen version of such a protein could result in faulty formation of alternative sulfur bridges, or unwanted homodimerization with other copies of LpqH or Heterodimerization with unknown proteins in the proximity.

[0108] In addition to the foregoing, it had been previously described that removal of N-linked glycosylation sites is beneficial for the expression of correctly folded PstSl and LpqH antigen (PCT / US2024 / 024498). Indeed, for PstSl, the inventors found that binding of the antibody P4-163, which recognizes a conformational epitope of PstSl, was improved in HEK293T cells transfected with constructs with mutated N-linked glycosylation sites. This was both reflected in a higher percentage of the cells expressing correctly folded antigen, as well as higher median fluorescence intensity reflecting antibody binding (Fig. 11).

[0109] Importantly, both bioinformatic predictions and published literature suggest that LpqH and PstSl both contain a heavily O-linked glycosylated domain in their N-terminus. Based on analyses performed by the inventors, it was determined that the residues underlined in the sequences shown in Fig. 1 would be O-glycosylated in the mycobacterial form of these antigens. The residues shown in italics represent the secretory signal peptide and the Bold N in LpqH is a predicted N-linked glycosylation site.

[0110] Since this O-linked glycosylation is not expected to occur when these antigens are expressed in eukaryotic expression systems, it forms an inherent risk of producing a protein with a different quaternary structure compared to its counterpart in the bacterium. Since for both these antigens the O-linked glycosylation sites are exclusively present in the N-terminal unstructured part of the protein, the inventors realized that they could get equivalent or better expression with a correct confirmation when using an antigen form lacking this domain.

[0111] The inventors have further demonstrated that these exemplary PstSl and LpqH constructs of the disclosure e.g., having deletion or substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells, deletion or substitution of an N-terminal cysteine residue, and / or addition of a non-native transmembrane domain or multimerization domain) provide improved immune responses (e.g., B cell responses) in vivo. Specifically, the exemplary PstSl and LpqH constructs of the disclosure result in increased responses from germinal center (GC) B cells and memory B cells, including increased relative abundance of antigen-specific GC B cells and memory B cells.

[0112] LBQH

[0113] In some embodiments, the Mtb antigen LpqH comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2. A full-length antigen representing the antigen LpqH is characterized in that it comprises the full-length amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2 or an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2. An antigen fragment representing the antigen LpqH is characterized in that it comprises an amino acid sequence which is only a part of SEQ ID NO: 1 or 2 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2, but which still is able to induce an immune reaction to LpqH, when delivered to a subject.

[0114] Accordingly, a fragment of LpqH comprises at least one B-cell epitope and optionally one or more T-cell epitopes which can give rise to an immune reaction to LpqH.

[0115] In some embodiments, a fragment of LpqH comprises a C-terminal deletion, an N-terminal deletion and / or an internal deletion of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the deletion covers 1 or more, 2 or more, 5 or more 10 or more, 20 or more, 50 or more, 70 or more, 80 or more, 90 or more, 100 or more or up to 120 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1 or 2 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2. Conversely, in some embodiments, a fragment of LpqH may comprise 20 or more, 30 or more, 40 or more, 50 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more or up to 158 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1 or 2 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0116] A variant of the Mtb antigen LpqH or of a fragment thereof is characterized by one or more of the following modifications compared to LpqH or the fragment thereof: a) deletion of one or more amino acids subject to O-linked glycosylation in eukaryotic cells or deletion of an amino acid sequence comprising one or more amino acids subject to O-linked glycosylation in eukaryotic cells, b) substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells, c) deletion or substitution of an N-terminal cysteine residue, and d) addition of a non-native transmembrane domain or multimerization domain.

[0117] In some embodiments a variant is modified compared to LpqH or the fragment thereof by deletion of one or more amino acids subject to O-linked glycosylation in eukaryotic cells. In some embodiments, the one or more amino acids subject to O-linked glycosylation in eukaryotic cells are serine or threonine residues. In some embodiments, the one or more amino acids subject to O-linked glycosylation in eukaryotic cells are at amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments a variant of LpqH comprises a single deletion at an amino acid position corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments a variant of LpqH comprises deletions at 2, 3, 4, 5, 6, 7, or all of the amino acid position corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0118] In some embodiments a variant is modified compared to LpqH or the fragment thereof by deletion of one or more amino acid sequences comprising one or more amino acids subject to O-linked glycosylation in eukaryotic cells, wherein the deletion encompasses 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more or up to 50 consecutive amino acids from the amino acid sequence of SEQ ID NO: 1 or 2 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2 and covering 1, 2, 3, 4, 5, 6, 7 or all of the amino acid sequence positions corresponding to position 23, 24, 27, 28, 29, 31, 3, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2. Some embodiments comprise a combination of one or more deletions affecting single amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2 and one or more deletions of amino acid sequences covering one or more additional of these amino acid positions.

[0119] In some embodiments a variant is modified compared to LpqH or the fragment thereof by substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells. In some embodiments 1, 2, 3, 4, 5, 6, 7, or all of the amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2 are substituted with an amino acid, which is neither serine nor threonine.

[0120] In some embodiments a variant is modified compared to LpqH or the fragment thereof by deletion or substitution of an N-terminal cysteine residue. In some embodiments, the N-terminal cysteine residue of LpqH or of a fragment thereof is located at an amino acid position corresponding to position 22 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0121] In some embodiments a variant is modified compared to LpqH or the fragment thereof by the addition of a non-native transmembrane domain. In some embodiments, the non-native transmembrane domain is selected from the group comprising the Nipah or HSV transmembrane domain, the CorA or KdpF transmembrane domain from Mycobacterium tuberculosis or the M2 transmembrane domain from the influenza A virus. In certain embodiments, the transmembrane domain is a Nipah-transmembrane domain having the sequence according to SEQ ID NO: 100. In some embodiments, the transmembrane domain is a HSV-transmembrane domain having the sequence according to SEQ ID NO: 101. In certain embodiments, the transmembrane domain is located at the C-terminus or at the N-terminus of the variant.

[0122] In some embodiments a variant is modified compared to LpqH or the fragment thereof by the addition of a non-native multimerization domain. In some embodiments, the non-native multimerization domain is selected from Hp-Ferritin or any of the protein domains disclosed in Bale, Jacob B et al. Science (New York, N.Y.) vol. 353,6297 (2016): 389-94. doi : 10.1126 / science.aaf8818.

[0123] In some embodiments, the multimerization domain is Hp-ferritin having the sequence according to SEQ ID NO: 102.

[0124] In some embodiments, the multimerization domain is derived from a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase. In some embodiments the multimerization domain is a Mycobacterium tuberculosis 6,7-dimethyl-8- ribityll umazine synthase or fragment thereof, Mycobacterium / ?ows6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, Mycobacterium bovis BCG 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, or Mycobacterium leprae 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof. In some embodiments, the Mycobacterium 6,7- dimethyl-8-ribityllumazine synthase is encoded by a Mycobacterium RibH gene.

[0125] In some embodiments, the multimerization domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or 100% identity to the amino acid sequence of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, or 132.

[0126] In some embodiments, the multimerization domain is located at the C-terminus or at the N-terminus of the variant.

[0127] In some embodiments, the transmembrane domain or the multimerization domain is connected to the variant by a protein linker. In some embodiments, the linker is a GS-linker. In some embodiments the GS-linker has the sequence according to SEQ ID NO: 103 (GGSGGGGSGGGGSGG).

[0128] In some embodiments, the variant has an amino acid sequence selected from SEQ ID NO: 50, 56, 62, 66, 68, 70 or 72. In some embodiments, the variant has an amino acid sequence that is as described in Table 1.

[0129] In some embodiments, the variant is encoded by a nucleic acid sequence selected from SEQ ID NO: 51, 57, 63, 67, 69, 71 or 73. In some embodiments, the variant is encoded by a nucleic acid sequence selected from Table 2. PstSl

[0130] In some embodiments, the Mtb antigen PstSl comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4. A full-length antigen representing the antigen PstSl is characterized in that it comprises the full-length amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 4 or an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4. An antigen fragment representing the antigen PstSl is characterized in that it comprises an amino acid sequence which is only a part of SEQ ID NO: 3 or 4 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4, but which still is able to induce an immune reaction to PstSl, when delivered to a subject.

[0131] Accordingly, a fragment of PstSl comprises at least one B-cell epitope and optionally one or more T-cell epitopes which can give rise to an immune reaction to PstSl.

[0132] In some embodiments, a fragment of PstSl comprises a C-terminal deletion, an N-terminal deletion and / or an internal deletion of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the deletion covers 1 or more, 2 or more, 5 or more 10 or more, 20 or more, 50 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more or up to 340 consecutive amino acids from the amino acid sequence of SEQ ID NO: 3 or 4 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4. Conversely, in some embodiments, a fragment of PstSl may comprise 20 or more, 30 or more, 40 or more, 50 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more or up to 373 consecutive amino acids from the amino acid sequence of SEQ ID NO: 3 or 4 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0133] A variant of the Mtb antigen PstSl or of a fragment thereof is characterized by one or more of the following modifications compared to PstSl or the fragment thereof: a) deletion of one or more amino acids subject to O-linked glycosylation in eukaryotic cells or deletion of an amino acid sequence comprising one or more amino acids subject to O-linked glycosylation in eukaryotic cells, b) substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells, c) deletion or substitution of an N-terminal cysteine residue, and d) addition of a non-native transmembrane domain or multimerization domain.

[0134] In some embodiments a variant is modified compared to PstSl or the fragment thereof by deletion of one or more amino acids subject to O-linked glycosylation in eukaryotic cells. In some embodiments, the one or more amino acids subject to O-linked glycosylation in eukaryotic cells are serine or threonine residues. In some embodiments, the one or more amino acids subject to O-linked glycosylation in eukaryotic cells are at amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments a variant of PstSl comprises a single deletion at an amino acid position corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments a variant of PstSl comprises deletions at 2, 3, 4, 5, 6 or all of the amino acid position corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0135] In some embodiments a variant is modified compared to PstSl or the fragment thereof by deletion of one or more amino acid sequences comprising one or more amino acids subject to O-linked glycosylation in eukaryotic cells, wherein the deletion encompasses 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more or up to 50 consecutive amino acids from the amino acid sequence of SEQ ID NO: 3 or 4 or of an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4 and covering 1, 2, 3, 4, 5, 6 or all of the amino acid sequence positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4. Some embodiments comprise a combination of one or more deletions affecting single amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4 and one or more deletions of amino acid sequences covering one or more additional of these amino acid positions.

[0136] In some embodiments a variant is modified compared to PstSl or the fragment thereof by substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells. In some embodiments 1, 2, 3, 4, 5, 6 or all of the amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4 are substituted with an amino acid, which is neither serine nor threonine.

[0137] In some embodiments a variant is modified compared to PstSl or the fragment thereof by deletion or substitution of an N-terminal cysteine residue. In some embodiments, the N-terminal cysteine residue of PstSl or of a fragment thereof is located at an amino acid position corresponding to position 24 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0138] In some embodiments a variant is modified compared to PstSl or the fragment thereof by the addition of a non-native transmembrane domain. In some embodiments, the non-native transmembrane domain is selected from the group comprising the Nipah virus or HSV transmembrane domain, the CorA or KdpF transmembrane domain from Mycobacterium tuberculosis or the M2 transmembrane domain from the influenza A virus. In certain embodiments, the transmembrane domain is a Nipah-transmembrane domain having the sequence according to SEQ ID NO: 100. In some embodiments, the transmembrane domain is a HSV-transmembrane domain having the sequence according to SEQ ID NO: 101. In certain embodiments, the transmembrane domain is located at the C-terminus or at the N-terminus of the variant.

[0139] In some embodiments a variant is modified compared to PstSl or the fragment thereof by the addition of a non-native multimerization domain. In some embodiments, the non-native multimerization domain is selected from Hp-Ferritin or any of the protein domains disclosed in Bale, Jacob B et al. Science (New York, N.Y.) vol. 353,6297 (2016): 389-94. doi:10.1126 / science.aaf8818.

[0140] In some embodiments, the multimerization domain is Hp-ferritin having the sequence according to SEQ ID NO: 102.

[0141] In some embodiments, the multimerization domain is derived from a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase. In some embodiments, the multimerization domain is a Mycobacterium tuberculosis 6,7-dimethyl-8- ribityllumazine synthase or fragment thereof, Mycobacterium do / s6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, Mycobacterium bovis BCG 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, or Mycobacterium leprae 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof. In some embodiments, the Mycobacterium 6,7- dimethyl-8-ribityllumazine synthase is encoded by a Mycobacterium RibH gene.

[0142] In some embodiments, the multimerization domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or 100% identity to the amino acid sequence of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, or 132.

[0143] In some embodiments, the multimerization domain is located at the C-terminus or at the N-terminus of the variant.

[0144] In some embodiments, the transmembrane domain or the multimerization domain is connected to the variant by a protein linker. In some embodiments, the linker is a GS-linker. In some embodiments the GS-linker has the sequence according to SEQ ID NO: 103 (GGSGGGGSGGGGSGG).

[0145] As used herein, the term "multimerization domain" refers to a specific amino acid sequence or structural motif within a polypeptide that mediates multimerization, i.e., a process by which two or more of the chimeric proteins described herein associate with one another to form a protein complex having higher molecular order. Multimerization may occur through covalent interactions (e.g., disulfide bonds) or non-covalent interactions (e.g., hydrophobic, electrostatic, hydrogen-bonding, or van der Waals forces). Multimerization can result in the formation of dimers, trimers, tetramers, pentamers, decamers, or higher-order oligomeric structures. In certain embodiments, the multimerization domain disclosed herein leads to formation of pentamers of the chimeric proteins. In certain embodiments, two pentamers of the chimeric proteins are assembled into a decamer. In certain embodiments, a multimerization domain disclosed herein is a "self-assembling multimerization domain", i.e., it is capable of spontaneously associating with identical domains under physiological or near-physiological conditions, without requiring external scaffolding or enzymatic facilitation.

[0146] In some embodiments, the multimerization domain described herein is derived from a Mycobacterium 6,7-dimethyl-8- ribityllumazine synthase. A multimerization domain that is derived from a AfycoAacter / i / m 6,7-dimethyl-8-ribityllumazine synthase, may be a full-length Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase, a variant of a full-length Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase, a fragment of a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase or fragment of a variant of a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase.

[0147] A 6,7-dimethyl-8-ribityllumazine synthase described herein originates from an organism of the genus Mycobacterium.

[0148] In certain embodiments, the 6,7-dimethyl-8-ribityllumazine synthase originates from the species Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, or Mycobacterium leprae. In certain embodiments, the Mycobacterium 6,7-dimethyl-8-ribltyllumazine synthase is encoded by the Mycobacterium RibH gene. In certain embodiments, the Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase corresponds to the amino acid sequence encoded in the coding sequence of the Mycobacterium RibH gene. In some embodiments, the RibH gene is from the species Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, or Mycobacterium leprae. In some embodiments, the full-length Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase has an amino acid sequence of any one of SEQ ID NOs.: 125, 126, 127, 128, 129, 130, 131, or 132.

[0149] In some embodiments, the multimerization domain is a variant of a full-length 6,7-dimethyl-8-ribityllumazine synthase, a fragment of a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase or fragment of a variant of a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase.

[0150] The inventors have found that fusion constructs involving a 6,7-dimethyl-8-ribityllumazine synthase domain can selfassemble into homopentamers of homodecamers comprising two rings that present payload polypeptide sequences attached to the N-terminus and / or the C-terminus of the 6,7-dimethyl-8-ribityllumazine synthase domain in outward directed equatorial positions relative to the homodecamer structure.

[0151] This orientation generates two attachment sites per protein-complex subunit, makes the payload sequences particularly accessible to the environment and, at the same time, reduces or entirely avoids steric interference inside the protein complex, which might interfere with the structure of the payload proteins or multimerization. A further advantage follows from the fact that the number of subunits inside the protein complex is very limited compared to, e.g., ferritin multimerization domains, which form protein complexes with 24 subunits. Relative to Ferritin, the homopentamers assembled based on the 6,7-dimethyl-8-ribityllumazine synthase domain, which then may further assemble into homodecamers, result in the formation of a far greater number of particles, increase the speed with which particles are generated or, depending on the specific setting, may be configured to reduce the strain imposed on the expression system during protein translation relative to the number of protein complexes produced.

[0152] In some embodiments, the multimerization domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 125, 126, 127, 128, 129, 130, 131, or 132.

[0153] In some embodiments, the multimerization domain is encoded by the nucleotide sequence of SEQ ID NO: 133 or SEQ ID NO: 134.

[0154] In some embodiments, the variant has an amino acid sequence selected from SEQ ID NO: 76, 82, 88, 92, 94, 96 or 98. In some embodiments, the variant has an amino acid sequence that is as described in Table 1. In some embodiments, the variant is encoded by a nucleic acid sequence selected from SEQ ID NO: 77, 83, 89, 93, 95, 97 or 99. In some embodiments, the variant is encoded by a nucleic acid sequence selected from Table 2.

[0155] able 1: Amino acid sequences of Mycobacterium tuberculosis antigens used

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] able 2: Nucleotide sequences of Mycobacterium tuberculosis antigens used

[0163] - 25 -

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] able 3: Exemplary transmembrane and multimerization domains

[0177]

[0178] Nucleic Acids

[0179] The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a nucleic acid is DNA. In some embodiments, a nucleic acid is RNA. In some embodiments, a nucleic acid is a mixture of DNA and RNA. A nucleic acid may be present as a singlestranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0180] The term "nucleoside" (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0181] The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.

[0182] A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups, e.g., one or more Cm alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl- uracil, and N(l)-alkyl-uracil, such as N7-CI-4alkyl-guanine, N6-CI-4alkyl-adenine, 5-Cwalkyl-cytosine, 5-CI-4alkyluracil, and N(1)-CI-4alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl- uracil, and N(l)-methyl-uracil.

[0183] DNA

[0184] Herein, the term "DNA" relates to a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'- position of a p-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nudeotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be nonstandard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (Ze., naturally occurring) nucleotide residues or analogs thereof).

[0185] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0186] RNA

[0187] The term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (Ze., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (Ze., naturally occurring) nucleotide residues or analogs thereof).

[0188] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0189] The term "in vitro transcription" or "IVT" as used herein means that the transcription i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).

[0190] According to the present disclosure, the term "’RNA" includes "mRNA". According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide. mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0191] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0192] In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide or polypeptide.

[0193] In some embodiments, the mRNA which preferably encodes a peptide or polypeptide has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0194] As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 4thEdition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified mRNA, correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0195] In some embodiments, RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in i / Ztrotranscription. The cDNA may be obtained by reverse transcription of RNA.

[0196] In some embodiments of the present disclosure, the RNA is "replicon RNA" or simply a "replicon", in particular "selfreplicating RNA" or "self-amplifying RNA". In certain embodiments, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose etai., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail. The genome of alphaviruses encodes non- structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould eta!., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

[0197] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0198] In some embodiments of the present disclosure, the RNA (in particular, mRNA) described herein (e.g., contained in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in order to increase expression of the RNA (in particular, mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and / or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization e.g., to alter, preferably increase, the GC content of the RNA). A combination of the above described modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 51- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (MJ) or N(l)-methylpseudouridine (mlMJ) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (MJ) or N(l)- methyl pseudouridine (mlMJ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[0199] 5'-Cao

[0200] In some embodiments, the RNA (in particular, mRNA) described herein comprises a 5'-cap structure. In some embodiments, the RNA does not have uncapped 5'-triphosphates. In some embodiments, the RNA (in particular, mRNA) may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5'-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the RNA). The guanosine may be methylated at position N7(resulting in the cap structure m7Gppp). The term "5'-cap analog" includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3'-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphoroth ioate modified 5'-cap analogs at the p-phosphate (such as m27'2’°G(5')ppSp(5')G (referred to as beta-S-ARCA or p-S-ARCA)), as described in PC17EP2019 / 056502. Providing an RNA (in particular, mRNA) with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated RNA (in particular, mRNA) strand, or the RNA (in particular, mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0201] In some embodiments, the RNA (in particular, mRNA) comprises a 5'-cap structure selected from the group consisting of m27'2'°G(5')ppSp(5')G (in particular its DI diastereomer), m27'3'°G(5')ppp(5')G, and m27'3‘0Gppp(mi2’’ °)ApG. In some embodiments, RNA comprises m27'2'°G(5')ppSp(5')G (in particular its DI diastereomer) as 5'-cap structure. In some embodiments, RNA comprises m27'3''0Gppp(mi2'0)ApG as 5'-cap structure.

[0202] In some embodiments, the RNA (in particular, mRNA) comprises a capO, capl, or cap2, preferably capl or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0203] The 5'-cap analog beta-S-ARCA (P-S-ARCA) has the following structure:

[0204] The "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml / min can be applied. In some embodiments, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0205] The 5'-cap analog m27'3''0Gppp(mi2' °)ApG (also referred to as m27'3'0G(5')ppp(5')m2' °ApG) which is a building block of a capl has the following structure:

[0206]

[0207] An exemplary capO mRNA comprising p-S-ARCA and mRNA has the following structure:

[0208] An exemplary capO mRNA comprising m27'3 OG(5')ppp(5')G and mRNA has the following structure:

[0209] An exemplary capl mRNA comprising m27-3'0Gppp(mi2L°)ApG and mRNA has the following structure:

[0210]

[0211] Poly- A tai!

[0212] As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA (in particular, mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs (in particular, mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (in particular, mRNAs) disclosed herein can have a poly-A tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0213] It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (S') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0214] The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0215] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

[0216] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. co / / and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0217] In some embodiments, the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of 10 nucleotides.

[0218] In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.

[0219] In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail shown in SEQ ID NO: 106. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.

[0220] Untranslated regions (UTR)

[0221] In some embodiments, RNA (in particular, mRNA) described in present disclosure comprises a 5'-UTR and / or a 3'- UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5 -UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 5 -UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5 -UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3'-end, downstream of the termination codon of a proteinencoding region, but the term "3'-UTR" does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'- UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp eta!., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3 -UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alphal- globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin. For example, the RNA (e.g., mRNA) may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'- UTR derived from a globin gene, such as alpha2-globin, alpha 1-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0222] In some embodiments, a 5'-UTR is or comprises a modified human alpha-globin 5'-UTR. A particularly preferred 5'- UTR comprises the nucleotide sequence of SEQ ID NO: 104. In some embodiments, a 3'-UTR comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 105. In some embodiments, RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104.

[0223] In some embodiments, RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105.

[0224] Table 4: Exemplary untranslated RNA sequences

[0225] Chemical modification

[0226] The RNA (in particular, mRNA) described herein may have modified ribonucleotides in order to increase its stability and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in some embodiments, uridine in the RNA (in particular, mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0227] In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine ( j), Nl-methyl-pseudouridine (mlip), 5-methyl-uridine (m5U), and combinations thereof.

[0228] In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza- uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5- oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl- pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1- taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls4qi), 4- thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3i ), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza- pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl- pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 i ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a- thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (i m), 2-thio- 2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O- methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5llm), 1-thio-uridine, deoxythymidine, 2'-F-ara- uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

[0229] An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) Is referred to herein as "MJ-modified", whereas the term "mli -modified" means that the RNA (preferably mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (replacing partially or completely, preferably completely, uridine). Such MJ- or mlUJ- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(l)-methylpseudouridine replacing completely uridine.

[0230] Codon optimization and GC content

[0231] The codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to decrease the GC content of the RNA and / or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is decreased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or decreased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, the codonoptimization and / or the decrease in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0232] The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon-optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codonoptimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".

[0233] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the RNA (in particular, mRNA) described herein is decreased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine) / C (cytosine) content are more stable than sequences having an increased A (adenosinej / U (uracil) content.

[0234] In the context of the claimed Mycobacterium tuberculosis antigens LpqH and PstSl, however, the wildtype coding sequence has a relatively high GC-content. Accordingly, stability of the RNA sequences is not a concern. Instead, the inventors have found that decreasing the GC content to a target value between 50 and 65%, more preferably between 55 and 60% may be particularly beneficial for expression in human cells.

[0235] With respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleotides.

[0236] In various embodiments, the G / C content of the coding region of the RNA (in particular, mRNA) described herein is decreased by at least 2 percentage points, at least 5 percentage points, at least 10 percentage points, or more compared to the G / C content of the coding region of the wild type RNA.

[0237] Non-immunoaenic RNA

[0238] The term "non-immunogenic RNA" (such as "non-immunogenic mRNA") as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and / or limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA), e.g., during in vitro transcription, and / or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription. In certain embodiments, non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and / or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.

[0239] For rendering the non-immunogenic RNA (especially mRNA) non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nudeobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl- uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U),

[0240] 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5- halo-uridine e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5- methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2- thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2- thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U),

[0241] 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1- methyl-4-thio-pseudouridine (m^i ), 4-thio-l-methyi-pseudouridine, 3-methyl-pseudouridine (m3i ), 2-thio-l- methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3qj), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2- thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl- pseudouridine (ipm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mcmsUm), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio- uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5- [3-(l-E-propenylamino)uridine. In certain embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (qj), Nl-methyl-pseudouridine (mlqj) or 5-methyl-uridine (m5U), in particular Nl-methyl- pseudouridine.

[0242] In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0243] During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. Formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP may be added once or several times during synthesis of mRNA. Also, dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using £ coii RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524.

[0244] As the term is used herein, "remove" or "removal" refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances.

[0245] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, e.g., dsRNA (especially dsmRNA) is removed from non-immunogenic RNA , such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non- immunogenic RNA (especially mRNA) is free or essentially free of dsRNA. In some embodiments, the non- immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises singlestranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, , at least 99.993%,, at least 99.994%, , at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.). Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with dsRNA-specific antibody and the amount of antibody binding to RNA may be taken as a measure for the amount of dsRNA in the sample. A sample containing a known amount of dsRNA may be used as a reference.

[0246] For example, RNA may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCI, 0.1% (v / v) TWEEN-20) containing 5% (w / v) skim milk powder. For detection of dsRNA, the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.

[0247] In some embodiments, the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5- fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10- fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000- fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0248] In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by an 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100- fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.

[0249] The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non- immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the decrease is such that the non- immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0250] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.

[0251] Antiaen-coding RNA and use thereof for inducing an immune response

[0252] Generally, RNA (in particular, mRNA) described in the present disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide comprising one or more variants of Mycobacterium tuberculosis antigens or of fragments thereof, for inducing an immune response against Mycobacterium tuberculosis in a subject. The peptide or polypeptide for inducing an immune response is also designated herein as "vaccine antigen" or simply "antigen". In some embodiments, the RNA (in particular, mRNA) is translated into the respective protein upon entering cells of a subject being administered the RNA, e.g., muscle cells or antigen-presenting cells (APCs).

[0253] In some embodiments, the RNA encoding the vaccine antigen is expressed in cells of the subject to provide the vaccine antigen. In some embodiments, the RNA encoding the vaccine antigen is transiently expressed in cells of the subject. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, the vaccine antigen is secreted by cells of the subject.

[0254] In some embodiments, the RNA encoding the vaccine antigen is administered intramuscularly.

[0255] In some embodiments, the RNA encoding the vaccine antigen is administered systemically, e.g., intravenously. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in spleen occurs. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in antigen presenting cells, preferably professional antigen presenting cells occurs. In some embodiments, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, no or essentially no expression of the RNA encoding the vaccine antigen in lung and / or liver occurs. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in spleen is at least 5-fold the amount of expression in lung.

[0256] A vaccine antigen comprises an epitope for inducing an immune response against a disease-associated antigen, e.g., a protein of an infectious agent (e.g., Mtb antigen), in a subject. Accordingly, the vaccine antigen comprises an antigenic sequence for inducing an immune response against a disease-associated antigen in a subject. Such antigenic sequence may correspond to a target antigen or disease-associated antigen, an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or the immunogenic variant thereof. Thus, the antigenic sequence may comprise at least an epitope of a target antigen or disease- associated antigen or an immunogenic variant thereof.

[0257] The antigenic sequences, e.g., epitopes, suitable for use according to the disclosure typically may be derived from a target antigen, i.e. the antigen against which an immune response is to be elicited. For example, the antigenic sequences contained within the vaccine antigen may be a target antigen or a fragment or variant of a target antigen. The antigenic sequence or a procession product thereof, e.g., a fragment thereof, may bind to an antigen receptor such as TCR carried by immune effector cells. In some embodiments, the antigenic sequence is selected from the group consisting of the antigen expressed by a target cell to which the immune effector cells are targeted or a fragment thereof, or a variant of the antigenic sequence or the fragment.

[0258] A vaccine antigen which may be provided to a subject according to the present disclosure by administering RNA encoding the vaccine antigen, preferably results in the induction of an immune response, e.g., in the stimulation, priming and / or expansion of immune effector cells, in the subject being provided the vaccine antigen. Said immune response, e.g., stimulated, primed and / or expanded immune effector cells, is preferably directed against a target antigen, in particular a target antigen expressed in diseased cells, tissues and / or organs, i.e., a disease-associated antigen. Thus, a vaccine antigen may comprise the disease-associated antigen, or a fragment or variant thereof. In some embodiments, such fragment or variant is immunologically equivalent to the disease-associated antigen.

[0259] The term "immunologically equivalent" means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence. Thus, in some embodiments, a molecule which is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effects regarding the stimulation, priming and / or expansion of T cells as the antigen to which the T cells are targeted.

[0260] In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent which results in the induction of an immune response, e.g., in the stimulation, priming and / or expansion of immune effector cells, which immune response, e.g., stimulated, primed and / or expanded immune effector cells, targets the antigen, i.e. a disease-associated antigen, in particular when presented by diseased cells, tissues and / or organs. Thus, the vaccine antigen may correspond to or may comprise the disease-associated antigen, may correspond to or may comprise a fragment of the disease-associated antigen or may correspond to or may comprise an antigen which is homologous to the disease-associated antigen or a fragment thereof. If the vaccine antigen comprises a fragment of the disease-associated antigen or an amino acid sequence which is homologous to a fragment of the disease-associated antigen said fragment or amino acid sequence may comprise an epitope of the disease-associated antigen to which the antigen receptor of the immune effector cells is targeted or a sequence which is homologous to an epitope of the disease-associated antigen. Thus, according to the disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence being homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against, e.g., stimulating, priming and / or expanding immune effector cells carrying an antigen receptor binding to, the antigen or cells expressing the antigen. It is preferred that the vaccine antigen (similar to the disease-associated antigen) provides the relevant epitope for binding by the antigen receptor present on the immune effector cells. In some embodiments, the vaccine antigen or a fragment thereof (similar to the disease- associated antigen) is expressed on the surface of a cell such as an antigen-presenting cell (optionally in the context of MHC) so as to provide the relevant epitope for binding by immune effector cells. The vaccine antigen may be a recombinant antigen. In some embodiments of all aspects described herein, the RNA encoding the vaccine antigen is expressed in cells of a subject to provide the antigen or a procession product thereof for binding by the antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and / or expansion of the immune effector cells.

[0261] An "antigen" according to the present disclosure covers any substance that will elicit an immune response and / or any substance against which an immune response or an immune mechanism such as a cellular response and / or humoral response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an "antigen" relates to any substance, such as a peptide or polypeptide, that reacts specifically with antibodies or T-lymphocytes (T-cells). The term "antigen" may comprise a molecule that comprises at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule which, optionally after processing, induces an immune reaction, which may be specific for the antigen (including cells expressing the antigen). In some embodiments, an antigen is a disease-associated antigen, such as an Mtb antigen.

[0262] In some embodiments, an antigen is presented or present on the surface of cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. An antigen or a procession product thereof such as a T cell epitope is in some embodiments bound by an antigen receptor. Accordingly, an antigen or a procession product thereof may react specifically with immune effector cells such as T-lymphocytes (T cells).

[0263] According to the present disclosure, an antigen or a combination of antigens described herein may induce an immune response, wherein the immune response may comprise a humoral or cellular immune response, or both. In the context of some embodiments of the present disclosure, the antigen is presented by a cell, such as by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen. An antigen may be a product which corresponds to or is derived from a naturally occurring antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product.

[0264] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. In some embodiments, a disease-associated antigen is a molecule which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens, e.g., Mtb antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0265] The term "bacterial antigen" refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasm membrane of the bacterium. The term "bacterial antigen" includes Mtb antigens, e.g., Mtb antigens as described herein.

[0266] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies, T cells or B cells, in particular when presented in the context of MHC molecules. An epitope of a protein may comprises a continuous or discontinuous portion of said protein and, e.g., may be between about 5 and about 100, between about 5 and about 50, between about 8 and about 30, or about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, the epitope in the context of the present disclosure is a T cell epitope.

[0267] Terms such as "epitope", "fragment of an antigen", "immunogenic peptide" and "antigen peptide" are used interchangeably herein and, e.g., may relate to an incomplete representation of an antigen which is, e.g., capable of eliciting an immune response against the antigen or a cell expressing or comprising and presenting the antigen. In some embodiments, the terms relate to an immunogenic portion of an antigen. In some embodiments, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. The term "epitope" refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, between about 8 and about 30, or between about 8 and about 25 amino acids in length, for example, the epitope may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0268] The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules, including epitopes predicted by bioinformatic means. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.

[0269] The peptide and polypeptide antigen can be 2 to 100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0270] The peptide or polypeptide antigen can be any peptide or polypeptide that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or polypeptide.

[0271] In some embodiments, vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response, is recognized by an immune effector cell. In some embodiments, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen may be, e.g., presented or present on the surface of a cell, such as an antigen presenting cell.

[0272] In some embodiments, an antigen is expressed in a diseased cell (such as an infected cell).

[0273] In some embodiments, an antigen is presented by a diseased cell (such as an infected cell). In some embodiments, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In some embodiments, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In some embodiments, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells release cytotoxic factors, e.g., perforins and granzymes.

[0274] In some embodiments, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In some embodiments, an antibody or B cell receptor binds to native epitopes of an antigen. The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells. The term "antigen-specific T cell" or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted, in particular when presented on the surface of antigen presenting cells or diseased cells in the context of MHC molecules and preferably exerts effector functions of T cells. T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be measured.

[0275] In some embodiments, the term "target" shall mean an agent such as a cell or tissue which is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In some embodiments, the target cell is a cell expressing an antigen and presenting said antigen with class I MHC.

[0276] "Antigen processing" refers to the degradation of an antigen into processing products which are fragments of said antigen (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments e.g., via binding) with MHC molecules for presentation by cells, such as antigen-presenting cells to specific T-cells. Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells.

[0277] The term "professional antigen presenting cells" relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.

[0278] The term "non-professional antigen presenting cells" relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.

[0279] The term "dendritic cell" (DC) refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen- presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In some embodiments, the dendritic cells are splenic dendritic cells.

[0280] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are splenic macrophages.

[0281] By "antigen-responsive CTL" is meant a CD8+T-cell that is responsive to an antigen or a peptide derived from said antigen, which is presented with class I MHC on the surface of antigen presenting cells.

[0282] According to the disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as Interferon gamma (IFN~y) and Tumor Necrosis Factor alpha (TNF-a), up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.

[0283] "Activation" or "stimulation", as used herein, refers to the state of a cell that has been sufficiently stimulated to induce detectable cellular proliferation, such as an immune effector cell such as T cell. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers to, among other things, immune effector cells that are undergoing cell division.

[0284] The term "priming” refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

[0285] The term "expansion" refers to a process wherein a specific entity is multiplied. In some embodiments, the term is used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. In some embodiments, expansion leads to differentiation of the immune effector cells.

[0286] The terms "immune response" and "immune reaction" are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the disclosure, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, such as CD4+and CD8+T-lymphocytes, e.g. CD8+T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.

[0287] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventive / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigen peptide, such as against a pathogen-associated antigen (e.g., an antigen of Mtb). "Inducing" in this context may mean that there was no immune response against a particular antigen or pathogen before induction, but it may also mean that there was a certain level of immune response against a particular antigen or pathogen before induction and after induction said immune response is enhanced. Thus, "inducing the immune response" in this context also includes "enhancing the immune response". In some embodiments, after inducing an immune response in an individual, said individual is protected from developing a disease such as an infectious disease or the disease condition is ameliorated by inducing an immune response.

[0288] The terms "cellular immune response", "cellular response", "cell-mediated immunity" or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen and / or presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes which act as either "helpers" or "killers". The helper T cells (also termed CD4+T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+T cells or CTLs) kill cells such as diseased cells.

[0289] The term "humoral immune response" refers to a process in living organisms wherein antibodies are produced in response to agents and organisms, which they ultimately neutralize and / or eliminate. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind antigen of a single specificity. Following binding of an appropriate antigen and receipt of various other activating signals, B lymphocytes divide, which produces memory B cells as well as antibody secreting plasma cell clones, each producing antibodies that recognize the identical antigenic epitope as was recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the resulting escalation in antibody response when re-exposed to a specific antigen. The term "antibody" as used herein, refers to an immunoglobulin molecule, which is able to specifically bind to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3, the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy-terminus in the following order: CHI, CH2, CH3). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

[0290] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, such as to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the Vi (variable light chain) domain, CL (constant light chain) domain, VH(variable heavy chain) domain, and the CH(constant heavy chain) domains CH1, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, i.e., a, 5, c, y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.

[0291] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s).

[0292] By "cell characterized by presentation of an antigen" or "cell presenting an antigen" or "MHC molecules which present an antigen on the surface of an antigen presenting cell" or similar expressions is meant a cell such as a diseased cell, in particular an infected cell, or an antigen presenting cell presenting the antigen or an antigen peptide, either directly or following processing, in the context of MHC molecules, such as MHC class I and / or MHC class II molecules. In some embodiments, the MHC molecules are MHC class I molecules.

[0293] Embodiments of antigen-coding RNA

[0294] Generally, at least four formats useful for RNA pharmaceutical compositions may be used herein, namely nonmodified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA), self-amplifying RNA (saRNA), and trans-amplifying RNAs.

[0295] Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus augmented polypeptide {e.g., protein) expression.

[0296] Features of self-amplifying platform may include, for example, long duration of polypeptide {e.g., protein) expression, good tolerability and safety, higher likelihood for efficacy with very low RNA dose.

[0297] In some embodiments, a self-amplifying platform {e.g., RNA) comprises two nucleic acid molecules, wherein one nucleic acid molecule encodes a replicase {e.g., a viral replicase) and the other nucleic acid molecule is capable of being replicated {e.g., a replicon) by said replicase in trans (frans-replication system). In some embodiments, a self-amplifying platform {e.g., RNA) comprises a plurality of nucleic acid molecules, wherein said nucleic acids encode a plurality of replicases and / or replicons.

[0298] In some embodiments, a fra / rs-replication system comprises the presence of both nucleic acid molecules in a single host cell.

[0299] In some such embodiments, a nucleic acid encoding a replicase {e.g., a viral replicase) is not capable of selfreplication in a target cell and / or target organism. In some such embodiments, a nucleic acid encoding a replicase {e.g., a viral replicase) lacks at least one conserved sequence element important for (-) strand synthesis based on a (+) strand template and / or for (+) strand synthesis based on a (-) strand template.

[0300] In some embodiments, a self-amplifying RNA comprises a 3' untranslated region (UTR), a 5' UTR, a cap structure, a poly adenine (polyA) tail, and any combinations thereof.

[0301] In some embodiments, a self-amplifying platform does not require propagation of virus particles {e.g., is not associated with undesired virus-particle formation). In some embodiments, a self-amplifying platform is not capable of forming virus particles.

[0302] In some embodiments, RNA {e.g., a single stranded RNA) described herein has a length of at least 500 ribonucleotides (such as, e.g., at least 600 ribonucleotides, at least 700 ribonucleotides, at least 800 ribonucleotides, at least 900 ribonucleotides, at least 1000 ribonucleotides, at least 1250 ribonucleotides, at least 1500 ribonucleotides, at least 1750 ribonucleotides, at least 2000 ribonucleotides, at least 2500 ribonucleotides, at least 3000 ribonucleotides, at least 3500 ribonucleotides, at least 4000 ribonucleotides, at least 4500 ribonucleotides, at least 5000 ribonucleotides, or longer). In some embodiments, RNA described herein is single-stranded RNA having a length of about 800 ribonucleotides to 5000 ribonucleotides.

[0303] In some embodiments, a relevant RNA includes a polypeptide-encoding portion or a plurality of polypeptide- encoding portions. In some particular embodiments, such a portion or portions encode one or more polypeptides which are not endogenous (i.e., it is foreign) to the subject treated.

[0304] In some embodiments, the RNA described herein (e.g., contained in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) is single-stranded RNA (in particular, mRNA) that may be translated into the respective protein upon entering cells, e.g., cells of a recipient, e.g., muscle cells or antigen-presenting cells (APCs). In addition to wild-type or codon-optimized sequences encoding an antigen sequence, the RNA may contain one or more structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A)-tail). In some embodiments, the RNA contains all of these elements. In some embodiments, beta-S-ARCA(Dl) (m27,2' °GppSpG) or m27'3’0Gppp(mi2'°)ApG may be utilized as specific capping structure at the 5'-end of the RNA. As 5 -UTR sequence, the 5'-UTR sequence of the human alpha-globin mRNA, optionally with an optimized 'Kozak sequence' to increase translational efficiency may be used. As 3'-UTR sequence, a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) placed between the coding sequence and the poly(A)-tail to assure higher maximum protein levels and prolonged persistence of the mRNA may be used (see WO 2017 / 060314, herein incorporated by reference). Furthermore, a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues may be used. In some embodiments, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104. In some embodiments, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 106, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 106.

[0305] In some embodiments, the RNA described herein is not chemically modified, i.e. it solely contains naturally occurring nucleosides, and preferably has the composition of naturally occurring RNA.

[0306] In some embodiments, the RNA described herein is modified for optimized efficacy of the RNA (e.g., increased translation efficacy, decreased immunogenicity, and / or decreased cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (in particular uridine) with synthetic nucleosides (e.g., modified nucleosides, e.g., selected from the group consisting of pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine); and / or codon-optimization). In some embodiments, the RNA comprises a modified nucleoside in place of uridine. In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine is selected from the group consisting of pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine. In some embodiments, the RNA encoding the vaccine antigen has a coding sequence (a) which is codon-optimized, (b) the G / C content of which is increased compared to the wild type coding sequence, or (c) both (a) and (b).

[0307] In some embodiments, the RNA described herein comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly(A) sequence (e.g., as described above); is modified by replacing (partially or completely, preferably completely) uridine with modified nucleosides, e.g., selected from the group consisting of pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine; and has a coding sequence which is codon-optimized, and the G / C content of which is increased compared to the wild type coding sequence. In some embodiments, if the present disclosure provides for a mixture of different RNA molecules, a composition comprising different RNA molecules or an administration of different RNA molecules, these different RNA molecules are present in approximately the same amount. Such different RNA molecules may be formulated in individual particulate formulations, mixed particulate formulations, or combined particulate formulations as described herein. The present disclosure provides RNA (in particular, mRNA) comprising a nucleic acid sequence encoding a variant of an Mtb antigen or of a fragment of the Mtb antigen.

[0308] In some embodiments, RNA (in particular, mRNA) described in the present disclosure comprises a nucleic acid sequence encoding a variant of an Mtb antigen or of a fragment of the Mtb antigen, and is capable of expressing said variant of an Mtb antigen or of a fragment of the Mtb antigen, in particular if transferred into a cell or subject, preferably a human cell or subject. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure contains a coding region (open reading frame (ORF)) encoding a variant of an Mtb antigen or of a fragment of the Mtb antigen.

[0309] In some embodiments, RNA comprises a nucleic acid sequence encoding more than one variant of an Mtb antigen or of a fragment of the Mtb antigen, e.g., two, three, four or more variants of an Mtb antigen or of a fragment of the Mtb antigen. In some embodiments, two or more of such Mtb antigens or variants are present as a fusion protein.

[0310] In some embodiments, the peptide or polypeptide encoded by the RNA described herein may consist of the one or more variants of an Mtb antigen or of a fragment of the Mtb antigen, or may comprise the one or more variants of an Mtb antigen or of a fragment of the Mtb antigen and may comprise additional sequences such as secretion signals, extended-PK groups, tags and any other sequences. In some embodiments, the additional sequences are fused to the one or more variants of an Mtb antigen or of a fragment of the Mtb antigen, in some embodiments, separated by a linker. In these embodiments, the one or more variants of an Mtb antigen or of a fragment of the Mtb antigen may be considered the pharmaceutically active peptide or polypeptide even if additional sequences support the function or effect of the one or more variants of an Mtb antigen or of a fragment of the Mtb antigen. According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" means a peptide or polypeptide that can be used in the treatment of an individual where the expression of the peptide or polypeptide would be of benefit, e.g., in ameliorating the symptoms of a disease. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide has a positive or advantageous effect on the condition or disease state of an individual when administered to the individual in a therapeutically effective amount. A pharmaceutically active peptide or polypeptide may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease. The term "pharmaceutically active peptide or polypeptide" includes entire peptides or polypeptides, and can also refer to pharmaceutically active fragments thereof, such as the variant of an Mtb antigen or of a fragment of the Mtb antigen

[0311] According to certain embodiments, a "signal peptide" (or signal sequence) is fused, either directly or through a linker, to the N-terminus of a chimeric protein described herein.

[0312] In some embodiments, an open reading frame of the RNA described herein encodes a polypeptide that includes a signal sequence, e.g., that is functional in mammalian cells.

[0313] In some embodiments, a utilized signal sequence is "intrinsic" in that it is, in nature, associated with (e.g., linked to) the full-length antigen or antigen fragment at the N-terminus of the chimeric protein.

[0314] In some embodiments, a utilized signal sequence is non-native to the encoded polypeptide - e.g., is not naturally part of a full-length antigen or antigen fragment whose sequences are included in the encoded chimeric protein. In some embodiments, signal peptides are sequences, which are typically characterized by a length of about 15 to 30 amino acids.

[0315] In many embodiments, signal peptides are positioned at the N-terminus of an encoded chimeric protein as described herein, without being limited thereto. In some embodiments, signal peptides preferably allow the transport of the polypeptide encoded by RNAs of the present disclosure with which they are associated into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[0316] In some embodiments, a signal sequence such as MRVMAPRTULLLSGALALTETWAGS [SEQ ID NO: 5], or a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized. In some embodiments, a signal peptide is selected from those included in the Table 5 below and / or those encoded by the sequences in Table 6 below or a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto:

[0317] Table 5: Exemplary signal sequences

[0318] Table 6: Exemplary nucleotide sequences encoding signal sequences Furthermore, a secretory sequence, e.g., a sequence comprising the amino acid sequence selected from SEQ ID NOs: 5 to 25 or encoded by the nucleotide sequence selected from SEQ ID NOs: 26 to 39, may be fused to the N- terminus of the antigenic peptide or polypeptide. Mtb antigens, immunogenic variants thereof, or immunogenic fragments of the Mtb antigens or the immunogenic variants thereof may be fused to an extended-PK group, which increases circulation half-life. Non-limiting examples of extended-PK groups are described herein. It should be understood that other PK groups that increase the circulation half-life of peptides or polypeptides such as Mtb antigens, immunogenic variants thereof, or immunogenic fragments of the Mtb antigens or the immunogenic variants thereof are also applicable to the present disclosure. In certain embodiments, the extended-PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin, or recombinant serum albumin).

[0319] As used herein, the term "PK" is an acronym for "pharmacokinetic" and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. As used herein, an "extended-PK group" refers to a protein, peptide, or moiety that increases the circulation half-life of a biologically active molecule when fused to or administered together with the biologically active molecule. Examples of an extended-PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended-PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul; 16(7):903-15 which is herein incorporated by reference in its entirety. As used herein, an "extended-PK" polypeptide refers to a polypeptide moiety such as an Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or the immunogenic variant thereof in combination with an extended- PK group. In some embodiments, the extended-PK polypeptide is a fusion protein in which a polypeptide moiety is linked or fused to an extended-PK group.

[0320] In certain embodiments, the serum half-life of an extended-PK polypeptide is increased relative to the polypeptide alone (i.e., the polypeptide not fused to an extended-PK group). In certain embodiments, the serum half-life of the extended-PK polypeptide is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer relative to the serum half-life of the polypeptide alone. In certain embodiments, the serum half-life of the extended-PK polypeptide is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50- fold greater than the serum half-life of the polypeptide alone. In certain embodiments, the serum half-life of the extended-PK polypeptide is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0321] As used herein, "half-life" refers to the time taken for the serum or plasma concentration of a compound such as a peptide or polypeptide to reduce by 50%, in vivo, for example due to degradation and / or clearance or sequestration by natural mechanisms. An extended-PK polypeptide suitable for use herein is stabilized in vivo and its half-life increased by, e.g., fusion to serum albumin (e.g., human serum albumin (HSA) or mouse serum albumin (MSA)), which resist degradation and / or clearance or sequestration. The half-life can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982). In certain embodiments, the extended-PK group includes serum albumin, or fragments thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "albumin"). Polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.

[0322] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein such as a therapeutic protein, in particular an Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or the immunogenic variant thereof. The albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding an albumin. The therapeutic protein and albumin, once part of the albumin fusion protein, may each be referred to as a "portion", "region" or "moiety" of the albumin fusion protein (e.g., a "therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin). In some embodiments, an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, e.g. a liver cell, and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and / or assembly into multimeric proteins. An albumin fusion protein is preferably encoded by RNA in a non-processed form which in particular has a signal peptide at its N-terminus and following secretion by a cell is preferably present in the processed form wherein in particular the signal peptide has been cleaved off. In a most preferred embodiment, the "processed form of an albumin fusion protein" refers to an albumin fusion protein product which has undergone N-terminal signal peptide cleavage, herein also referred to as a "mature albumin fusion protein".

[0323] In preferred embodiments, albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

[0324] As used herein, "albumin" refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules. The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.

[0325] In certain embodiments, the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in US 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0326] The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms, "albumin and "serum albumin" are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof). As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fusion state.

[0327] The albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin. For instance, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0328] Generally speaking, an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.

[0329] According to the disclosure, albumin may be naturally occurring albumin or a fragment or variant thereof. Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.

[0330] Preferably, the albumin fusion protein comprises albumin as the N-terminal portion, and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion, and a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N- and C-termini are the same therapeutic proteins. In another preferred embodiment, the therapeutic proteins fused at the N- and C-termini are different therapeutic proteins. In some embodiments, the different therapeutic proteins are both Mtb antigens, immunogenic variants thereof, or immunogenic fragments of the Mtb antigens or the immunogenic variants thereof.

[0331] In some embodiments, the therapeutic protein(s) is (are) joined to the albumin through (a) peptide linker(s). A peptide linker between the fused portions may provide greater physical separation between the moieties and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor. The peptide linker may consist of amino acids such that it is flexible or more rigid. The linker sequence may be cleavable by a protease or chemically.

[0332] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain. In certain embodiments, an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain. This includes, but is not limited to, polypeptides comprising the entire CHI, hinge, CH2, and / or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derived from an immunoglobulin of any species and / or any subtype, including, but not limited to, a human IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fc domain encompasses native Fc and Fc variant molecules. As set forth herein, it will be understood by one of ordinary skill in the art that any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcyR binding).

[0333] The Fc domains of a polypeptide described herein may be derived from different immunoglobulin molecules. For example, an Fc domain of a polypeptide may comprise a CH2 and / or CH3 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.

[0334] In certain embodiments, an extended-PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "Fc domain"). The Fc domain does not contain a variable region that binds to antigen. Fc domains suitable for use in the present disclosure may be obtained from a number of different sources. In certain embodiments, an Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgGl constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.

[0335] Moreover, the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgGl, IgG2, IgG3, and IgG4.

[0336] A variety of Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and / or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc domain sequences (e.g. hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art recognized techniques.

[0337] In certain embodiments, the extended-PK group is a serum albumin binding protein such as those described in US2005 / 0287153, US2007 / 0003549, US2007 / 0178082, US2007 / 0269422, US2010 / 0113339, W02009 / 083804, and W02009 / 133208, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a serum immunoglobulin binding protein such as those disclosed in US2007 / 0178082, US2014 / 0220017, and US2017 / 0145062, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a fibronectin (Fn)- based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012 / 0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012 / 0094909. A non-limiting example of a Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin.

[0338] In certain aspects, the extended-PK polypeptide, suitable for use according to the disclosure, can employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended-PK moiety and a polypeptide moiety, e.g., an Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or the immunogenic variant thereof) in a linear amino acid sequence of a polypeptide chain. For example, peptide linkers may be used to connect an Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or the immunogenic variant thereof to a HSA domain.

[0339] In the following, embodiments of vaccine RNAs are described, wherein certain terms used when describing elements thereof have the following meanings: cap: 5'-cap structure, e.g., selected from the group consisting of m27'2'°G(5')ppSp(5')G (in particular its DI diastereomer), m27'3 OG(5')ppp(5')G, and m27'3'-°Gppp(mi2''0)ApG. hAg-Kozak: 5'-UTR sequence of the human alpha-globin mRNA with an optimized 'Kozak sequence' to increase translational efficiency.

[0340] Antigen: Sequences encoding the respective vaccine antigen(s) / epitope(s), i.e., one or more Mtb antigens, immunogenic variants thereof, or immunogenic fragments of the Mtb antigens or the immunogenic variants thereof. Glycine-serine linker (GS): Sequences coding for short peptide linkers predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.

[0341] FI element: The 3'-UTR is a combination of two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.

[0342] A30L70: A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells.

[0343] In some embodiments, vaccine RNA described herein has one of the following structures: cap-hAg-Kozak-Antigen(s)-FI-A30L70 cap-hAg-Kozak-sec-Antigen(s)-FI-A30L70

[0344] In some embodiments, vaccine antigen described herein has the structure: sec-Antigen

[0345] In some embodiments, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO: 104. In some embodiments, sec of the encoded vaccine antigen / epitope comprises an amino acid sequence selected from SEQ ID NOs: 5 to 25 or encoded by the nucleotide sequence selected from SEQ ID NOs: 26 to 39. In some embodiments, FI comprises the nucleotide sequence of SEQ ID NO: 105. In some embodiments, A30L70 comprises the nucleotide sequence of SEQ ID NO: 106.

[0346] In some embodiments, the sequence encoding the vaccine antigen / epitope comprises a modified nucleoside replacing (partially or completely, preferably completely) uridine, wherein the modified nucleoside is selected from the group consisting of pseudouridine (ip), Nl-methyl-pseudouridine (mli ), and 5-methyl-uridine.

[0347] In some embodiments, the sequence encoding the vaccine antigen / epitope is codon-optimized.

[0348] In some embodiments, the G / C content of the sequence encoding the vaccine antigen / epitope is increased compared to the wild type coding sequence.

[0349] In some embodiments, the RNA (in particular, mRNA) described herein comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104; a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105; and a poly-A sequence comprising the nucleotide sequence of SEQ ID NO: 106.

[0350] In some embodiments, the RNA (in particular, mRNA) described herein comprises: m27'3'0Gppp(mi2'0) ApG as capping structure at the 5’-end of the mRNA; a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104; a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105; and a poly-A sequence comprising the nucleotide sequence of SEQ ID NO: 106.

[0351] In some embodiments, the RNA is unmodified. In some embodiments, the RNA is modified. In some embodiments, the RNA comprises Nl-methyl-pseudouridine (mlip) in place of at least one uridine (e.g., in place of each uridine). In some embodiments, the RNA (in particular, mRNA) described herein comprises: m27'3‘0Gppp( i2'0) ApG as capping structure at the 5'-end of the mRNA; a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104; a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105; and a poly-A sequence comprising the nucleotide sequence of SEQ ID NO: 106; and Nl-methyl-pseudouridine (mlip) in place of at least one uridine (e.g., in place of each uridine).

[0352] In some embodiments, RNA (in particular, mRNA) described herein (e.g., contained in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) may be presented as a product containing the vaccine RNA as active substance and other ingredients comprising: ALC- 0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide), l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol.

[0353] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated as a liquid, a solid, or a combination thereof.

[0354] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated for injection.

[0355] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated for intramuscular administration.

[0356] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated as a composition, e.g., a pharmaceutical composition.

[0357] In some embodiments, the particles are nanoparticles, such as lipid nanoparticles (LNPs).

[0358] In some embodiments, the composition, in particular the pharmaceutical composition, is a vaccine.

[0359] In some embodiments, the composition, in particular the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0360] In some embodiments, the RNA and / or the composition, in particular the pharmaceutical composition, is / are a component of a kit.

[0361] In some embodiments, the kit further comprises instructions for use of the RNA for inducing an immune response against Mycobacterium tuberculosis in a subject.

[0362] In some embodiments, the kit further comprises instructions for use of the RNA for therapeutically or prophylactically treating a Mycobacterium tuberculosis infection in a subject.

[0363] In some embodiments, the subject is a human.

[0364] In some embodiments, the RNA (in particular, mRNA), e.g., RNA encoding vaccine antigen, described in the present disclosure is non-immunogenic. RNA encoding an immunostimulant may be administered according to the present disclosure to provide an adjuvant effect. The RNA encoding an immunostimulant may be standard RNA or non- immunogenic RNA. RNA delivery

[0365] RNA described herein may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNA, or delivery mediated by delivery vehicles.

[0366] Some aspects of the disclosure involve the targeted delivery of the RNA disclosed herein to certain cells or tissues. In some embodiments, after administration of the RNA (in particular, mRNA) compositions / formulations described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA (in particular, mRNA) is translated by the target cell to produce the encoded peptide or polypeptide. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a cell in the lung. In some embodiments, the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. In some embodiments, the target cell is a cell in the lymph nodes. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. Thus, RNA (in particular, mRNA) compositions / formulations described herein may be used for delivering RNA to such target cell. The "lymphatic system" is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph. The primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and the bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, which include lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response. Lipid-based RNA delivery systems have an inherent preference to the liver, where, depending on the composition of the RNA delivery systems used, RNA expression in the liver can be obtained. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ for RNA expression is liver and the target tissue is liver tissue. The delivery to such target tissue is preferred, in particular, if presence of RNA or of the encoded peptide or polypeptide in this organ or tissue is desired and / or if it is desired to express large amounts of the encoded peptide or polypeptide and / or if systemic presence of the encoded peptide or polypeptide, in particular in significant amounts, is desired or required.

[0367] Delivery vehicles

[0368] To overcome the barriers to safe and effective RNA delivery, RNA may be administered with one or more delivery vehicles that protect the RNA from degradation, maximize delivery to on-target cells and minimize exposure to off- target cells. Such RNA delivery vehicles may complex or encapsulate RNA and include a range of materials, including polymers and lipids. In some embodiments, such RNA delivery vehicles may form particles with RNA.

[0369] RNA, in particular mRNA, described herein may be present in particles comprising (i) the RNA, and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged RNA are involved in particle formation. This results in complexation and spontaneous formation of RNA particles.

[0370] Different types of RNA containing particles have been described previously to be suitable for delivery of RNA in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA physically protects RNA from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape. In some embodiments, an agent to be delivered to a subject, e.g., a nucleic acid, a polypeptide, a small molecule, and the like, is encapsulated in a particle. In some embodiments, the RNA of the present disclosure is formulated in (e.g., encapsulated in) a particle, as further described herein. In some embodiments, a particle is a nucleic acid particle wherein the nucleic acid particle comprises a nucleic acid (e.g., DNA and / or RNA), and a cationic lipid, a cationically ionizable lipid, or a cationic polymer.

[0371] A "nucleic acid particle," as used herein, refers to a particle that encompasses or contains a nucleic acid, and, is part of a composition (e.g., a pharmaceutical composition) comprising multiple nucleic acid particles, that is useful for (i) enhancing nucleic acid stability, e.g., during storage, (ii) improving biodistribution of the nucleic acid or delivering a nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like), and / or (iii) facilitating cell uptake of the nucleic acid. As described herein, a nucleic acid particle may be formed from i) at least one cationic or cationically ionizable lipid or lipid-like material; ii) at least one cationic polymer such as polyethyleneimine, protamine, or a mixture thereof (i.e., a mixture of i) and ii)), and iii) a nucleic acid. Nucleic acid particles described herein include lipid nanoparticles (LNP), lipoplexes (LPX), liposomes, and polyplexes (PLX).

[0372] Electrostatic interactions between positively charged molecules such as cationic polymers and cationic lipids and negatively charged nucleic acids are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. The characteristics of a particle (e.g., nanoparticle) are determined, at least in part, from the components used to form the particle and the process used to prepare the particle. A description of the different types of particles and their structures is provided in ACS Nano 2021, 15, 11, 16982-17015.

[0373] In some embodiments, a nucleic acid particle described herein is a nanoparticle. As used in the present disclosure, "nanoparticle" refers to a particle having an average diameter suitable for parenteral administration and is less than 1000 nm in diameter. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 120 nm, about 50 nm to about 100 nm, or about 60 nm to about 90 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 40 nm to about 120 nm. The term "average diameter" or "mean diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using an appropriate algorithm (e.g., the so-called cumulant algorithm for monodisperse samples), which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter," "mean diameter," "diameter," or "size" for particles is used synonymously with this value of the Z-average.

[0374] A composition comprising nucleic acid particles can be characterized by its polydispersity index, that is, the relative uniformity of particles within a given composition. For example, compositions described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less of said nanoparticles. In some embodiments, a composition comprising nucleic acid particles, as described herein, may exhibit a PDI less than about 0.3. By way of example, a composition comprising nucleic acid particles described herein can exhibit a PDI in a range of about 0.1 to about 0.3, or about 0.2 to about 0.3. The polydispersity index of a given composition can be calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter." Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles.

[0375] Nucleic acid particles described herein can be characterized by an "N / P ratio," which is the molar ratio of cationic (nitrogen) groups (the "N" in N / P) in the cationic lipid or polymer to the anionic (phosphate) groups (the "P" in N / P) in RNA. It is understood that a cationic group is one that is either in permanently cationic form (e.g., N+), or one that is ionizable to become cationic (e.g., under certain pH conditions). Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1, e.g., an N / P ratio of about 4 is intended to mean about 4:1. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio greater than or equal to 1, greater than or equal to 2, or greater than or equal to 4. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio that is less than 24, less than 18, or less than 12. In some embodiments, a nucleic acid particle (e.g., an RNA LNP) described herein has an N / P ratio that is from about 2 to about 24, about 4 to about 18, about 4 to about 12, or about 4 to about 8. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., an RNA LNP) described herein is about 6.

[0376] Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. As used herein, an "ionizable" lipid, e.g., a "cationically ionizable" lipid or "ionizable" polymer, e.g., a "cationically ionizable" polymer is a lipid or polymer that may be, in some embodiments, neutral at physiological pH, but is capable of becoming cationic (i.e., becoming positively charged) at acidic pH.

[0377] The present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like material, and / or at least one cationic polymer which associate with the nucleic acid to form nucleic acid particles (e.g., RNA nanoparticles) and compositions comprising such particles. The nucleic acid particles (e.g., RNA nanoparticles) may comprise nucleic acid which is complexed by different non-covalent interactions (e.g., electrostatic, hydrogen bonding, pi-stacking, van der Waals, etc.) to the particle. In some embodiments, the particles described herein are not viral particles, in particular, they are not infectious viral particles, i.e., they are not able to virally infect cells.

[0378] In a nucleic acid particle (e.g., RNA nanoparticle) composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle formulation. In that case, each individual nucleic acid particle formulation will comprise one nucleic acid species. In some embodiments, a composition comprises more than one individual nucleic acid particle (e.g., RNA nanoparticle) formulation. Respective pharmaceutical compositions are referred to as "mixed particulate formulations." Such mixed particulate formulations may be obtainable by forming, separately, individual nucleic acid particle formulations, and mixing these to produce a formulation comprising a mixed population of nucleic acid-containing particles. Alternatively, different nucleic acid species may be formulated together as a "combined particulate formulation." Such formulations may be obtainable by mixing a combined formulation of different nucleic acid species with a particle-forming agent, to produce particles that comprise more than one nucleic acid species.

[0379] Lipid Nanopartides (LNPs)

[0380] In some embodiments, a particle described herein is a lipid nanoparticle (LNP). LNPs have emerged as particularly useful vehicles for delivery of nucleic acids, for example as described in Theranostics, 2022 Oct 24;12(17):7509- 7531. It is understood that a LNP is structurally distinct from other nanoparticles previously used for nucleic acid delivery, such as a liposome, or a lipoplex. LNPs, as described herein, typically do not comprise a bilayer (unilamellar), or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous compartment. Moreover, LNPs, as described herein, typically do not comprise a central aqueous core or compartment. LNPs as described herein typically comprise nucleic acids (e.g., DNA or RNA such as mRNA) and lipids forming a disordered, non-lamellar phase. LNPs as described herein may be considered as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. See, e.g., ACS Nano 2021, 15, 11, 16982-17015; Aldosari, eta / ., Pharmaceutics, 2021, 13, 206. LNPs described herein generally comprise four categories of lipids in addition to a nucleic acid agent (e.g., DNA or RNA such as mRNA): a cationic or cationically ionizable lipid (typically a cationically ionizable lipid), a polymer- conjugated lipid, a helper lipid, and a steroid. A person of skill in the art will understand that various combinations of these four categories of lipids can be used to prepare lipid nanoparticles for use in delivering nucleic acid agents.

[0381] (i) Cationic or cationically ionizable lipids

[0382] As described generally herein, a nucleic acid particle comprises a nucleic acid and a cationic or a cationically ionizable lipid. In some embodiments, a cationic or cationically ionizable lipid useful for incorporation into a nucleic acid particle are those lipids having a polar head group and an aliphatic tail. In some embodiments, a cationic lipid is one where the polar head group has a permanently positive charge (for example, comprising a quaternary ammonium group). In some embodiments, a cationically ionizable lipid is a lipid wherein, at a given pH and in the context of an LNP, the lipid becomes positively charged, such as at below physiological pH (e.g., below pH about 7.4) or neutral pH (e.g., a pH around 7 to 7.5), or in some embodiments, at a pH of less than 7 (e.g., less than 6). In some embodiments, a cationically ionizable lipid is one comprising polar head group that comprises one or more a tertiary amine groups (or secondary or primary amine group) that can become positively charged. LNPs typically comprise cationically ionizable lipids.

[0383] In some embodiments, a lipid nanoparticle comprises about 30 mol% to about 60 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 35 mol% to about 55 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 40 mol% to about 50 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 50 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of a cationic or cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises 47.5 mol% of a cationic or cationically ionizable lipid.

[0384] Suitable cationic or cationically ionizable lipids are readily identified by those of skill in the art. In some embodiments, a cationic lipid or cationically ionizable lipid is one provided in WO 2010 / 144740 or WO 2012 / 016184, which are incorporated herein by reference in their entirety. For example, in some embodiments, a cationic lipid is selected from N-(2,3-dioleyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride (DOTAP), N,N- dioleyl-N,N-dimethylammonium chloride (DODAC); 3-(N-(N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC- Chol), and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). In some embodiments, a cationically ionizable lipid is selected from l,2-dioleoyl-3-dimethylammonium propane (DODAP); N,N-dimethyl-(2,3-dioleoyloxypropyl)amine (DODMA); and 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l- (9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (DLin-MC3-DMA).

[0385] In some embodiments, a cationically ionizable lipid is a lipid described in WO 2017 / 075531 or WO 2018 / 081480, each of which is incorporated by reference herein in its entirety. In some embodiments, a cationically ionizable lipid is a lipid represented by formula CL-I:

[0386] CL-I or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-I: one of L1or L2is -O(C=O)-, - (C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, - OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, - C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1 and G2are each independently unsubstituted Ci-Cn alkylene or C1-C12 alkenylene; G3is C1-C24 alkylene, Ci- C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; Rais H or C1-C12 alkyl; R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl; R3is H, OR5, CN, -C(=O)OR4, -0C(=O)R4or -NR5C(=O)R4; R4is Ci- C12 alkyl; R5is H or Ci-Ce alkyl; and x is 0, 1 or 2.

[0387] In some embodiments, a cationically ionizable lipid is ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2- hexyldeca noate) (ALC-0315) or ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) (ALC-

[0388] ALC-0315 ALC-0366

[0389] In some embodiments, a lipid nanoparticle comprises about 40 mol% to about 50 mol% of a cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of a cationically ionizable lipid. In some embodiments, a lipid nanoparticle comprises 47.5 mol% of a cationically ionizable lipid.

[0390] In some embodiments, a cationic lipid is one described in WO 2017 / 049245, which is incorporated by reference in its entirety. In some embodiments, a cationic lipid is represented by formula CL-II or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-II: Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR", and -R"M'R'; R2and R3 are independently selected from the group consisting of H, CI-M alkyl, C2-14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3- 6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQR2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nNR2, -C(O)OR, OC(O)R, -CX3, -CX2H, -CXH2, -CN, -NR2, -C(O)NR2, -NRC(O)R, -NRS(O)2R, -NRC(O)NR2, -NRC(S)NR2, -NRRS, -O(CH2)nOR, -NRC(=NR9)NR2, -NRC(=CHR9)NR2, -OC(O)NR2, - NRC(O)OR, -N(OR)C(O)R, -N(OR)S(O)ZR, -N(OR)C(O)OR, -N(OR)C(O)NR2, -N(OR)C(S)NR2, N(OR)C(=NR9)NR2, - N(OR)C(=CHR9)NR2, -C(=NR9)NR2, -C(=NR9)R, -C(O)NROR, and -CRNR2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R5is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)- , -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7is selected from the group consisting of C1-3 alkyl, C2.3 alkenyl, and H; Rsis selected from the group consisting of C3-6 carbocycle and heterocycle; R9is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)ZNR2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1.3 alkyl, C2- 3 alkenyl, and H; each R' is independently selected from the group consisting of Ci-is alkyl, C2-18 alkenyl, -R*YR", - YR", and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3- 6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0391] In some embodiments, a cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate) (SM-102):

[0392] SM-102

[0393] In some embodiments, a cationically ionizable lipid is a lipid described in WO 2015 / 095340, which is incorporated by reference herein in its entirety. In some embodiments, a cationic lipid is represented by formula CL-III or a pharmaceutically acceptable salt thereof, wherein, as related to formula CL-III: n and p are each, independently, 0, 1 or 2; Li is -OC(O)-, -C(O)O- or a bond; R1is heterocyclyl, heterocyclyl-Ci-s-alkyl or heterocyclyl- Ci-s-alkoxyl, each of which may be optionally substituted with 1, 2, 3, 4 or 5 groups, independently selected from halogen, formidamidine, Ci-s-alkyl, Cs-rcycloaikyl, Ca-z-cycloalkyl-Ci-s-alkyl, heterocyclyl, -[(Ci-C4)alkylene]v- N(R')R", -O-[(Ci-C4)alkylene]v-N(R')R" or -N(H)-[(Ci-C4)alkylene]v-N(R')R", where said (Ci-C4)alkylene is optionally substituted with one or more R groups; v is 0, 1, 2, 3 or 4; R is hydrogen or -Ci-g-alkyl or when v is 0 R is absent; R' and R", are each, independently, hydrogen, -Ci-s-alkyl; or R' and R" combine with the nitrogen to which they are bound, and optionally including another heteroatom selected from N, O and S, to form a 5-8 membered heterocycle or heteroaryl, optionally substituted with an -Ci-s-alkyl, hydroxy or cycloalkyl-Ci-s-;

[0394] Rzand R3are each, independently, C7-22 alkyl, C12-22 alkenyl, C3-8 cycloalkyl optionally substituted with 1, 2, or 3 Ci-

[0395] In some embodiments, a cationically ionizable lipid is represented by

[0396] In some embodiments, a cationic lipid is one described in WO 2018 / 087753, which is incorporated herein by reference in its entirety. In some embodiments, a cationic lipid is represented by formula CL-IV: or a pharmaceutically acceptable salt thereof, wherein, as applied for formula CL-IV: Y is O or NH; T is C or S; W is a bond, 0, NH or S; R1is selected from the group consisting of: (a) NR4R5, wherein R4and R5are each independently a C1-C4 alkyl; or R4and R5together with the nitrogen to which they are attached form a 5 or 6 membered heterocyclic or heteroaromatic ring, optionally containing one or more additional heteroatoms selected from the group consisting of 0, N and S; or NR4R5represent a guanidine group (-NHC(=NH)NH2); (b) the side chain of a natural or unnatural amino add; and (c) a 5 or 6 membered heterocyclic or heteroaromatic ring containing one or more heteroatoms selected from the group consisting of 0, N and S; R2and R3are selected from the group consisting of: (a) C10-C22 alkyl; (b) C10-C22 alkenyl; (c) C10-C22 alkynyl; (d) C4-C10 alkylene-Z-C4-C22 alkyl; and (e) C4-C10 alkylene-Z-C4-C22 alkenyl; Z is -0-C(=0)-, -C(=0)-0- or -0-; n is 0, 1, 2, 3, 4, 5 or 6; m is 0 or 1; p is 0 or

[0397] 1; and z is 0 or 2.

[0398] In some embodiments, a cationically ionizable lipid is selected from:

[0399] In some embodiments, a cationically ionizable lipid is one described in WO 2022 / 081750, which is incorporated herein by reference in its entirety.

[0400] In some embodiments, a cationically ionizable lipid is represented by formula CL-V-1: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula CL-V-1: each R1and each R2is independently selected from the group consisting of H, an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted CrCe heterocycloalkyl, optionally substituted Q-CB alkylcycloalkyl, optionally substituted C,- C6aryl, optionally substituted C3-C6heteroaryl, optionally substituted CrCs aryloxy, optionally substituted C7- C10 arylalkyl, optionally substituted C5-C10 heteroaryl alkyl group, optionally substituted amine; or R1and R2can together form a 3-7 membered heterocycloalkyl or heteroaryl ring; each R3, R4, R13and R14is independently selected from the group consisting of an optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl; each R5, R6, R7, R8, R9, R10, R15, and R16is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C1-C22 alkyl, optionally substituted C2-C22 alkenyl, optionally substituted C2-C22 alkynyl; each of w, x, y, and z is independently an integer from 0-10; each Q is independently an atom selected from 0, NH, NR1, and S; each of m is an integer from 0 to 8, preferably 0, 1, or 2; and each of L1and L2is independently selected from the group consisting of -C(=O)-; -0C(=O)-; -0C(=O)O-; - C(=O)O-; -C(=O)O(CR5R6R7)-; -NH-C(=O)-; -C(=O)NH-; -SO-; - SO2-; -SO3-; -NSO2-; -SO2N-; -NH((Ci-C8)alkyl)-; - N((Ci-C8)alkyl)2-; -NH((C6)aryl)-; -N((C6)aryl)2-; -NHC(=O)NH-; -NHC(=O)O-; -OC(=O)NH-; -NHC(=O)NR1-; - NHC(=O)O-; -OC(=O)NR1-; -C(=O)R1-; -CO((Ci-C8)alkyl)-; -CO((C6)aryl)-; -CO2((Ci-C8)alkyl)-; - CO2((C6)aryl)-; - SO2((Ci-C8)alkyl)-; and -SO2((C6)aryl)-.

[0401] In some embodiments, a cationically ionizable lipid is represented by formula CL-V-2: or a pharmaceutically acceptable salt thereof, wherein, as applied to CL-V-2: each R1’, R1, R2, R11, and R12is independently selected from the group consisting of H, an optionally substituted Ci- C22alkyl, optionally substituted C2-C22alkenyl, optionally substituted C2-C22alkynyl, optionally substituted C3- Cs cycloalkyl, optionally substituted C4-Ce heterocycloalkyl, optionally substituted C4-C8alkylcycloalkyl, optionally substituted OCs aryl, optionally substituted C3-C6heteroaryl, optionally substituted C4-C8aryloxy, optionally substituted C7-C10 arylalkyl, optionally substituted C5-C10 heteroarylalkyl group, optionally substituted amine; or R1and R2can together form cycloalkyl or heterocycloalkyl ring; if Q is S or 0 the R1attached to the S or 0 is an electron pair; each R3and R4is independently selected from the group consisting of an optionally substituted Cr C22alkyl, optionally substituted C2-C22alkenyl, optionally substituted C2-C22alkynyl; each R5, R6, R7, R8, R9, and R10is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted Cr C22alkyl, optionally substituted C2-C22alkenyl, optionally substituted C2-C22alkynyl; each of x, y, and z is independently an integer from 0-10; G and Q are each independently an atom selected from CH, 0, N, and S; each of m and n is an integer from 0-8; and each of L1and L2is independently selected from the group consisting of - C(=O)-; -OC(=0)-; -OC(=O)O-; -C(=O)O-; -C(=O)O(CR1R2R3)-; -NH-C(=O)-; -C(=O)NH-; -SO-; -SO2-; -SO3-; - NSO2-; -SO2N-; -NH((Ci-C8)alkyl)-; -N((Ci-C8)alkyl)2-; -NH((C6)aryl)-; -N((Ce)aryl)2-; -NHC(=O)NH-; -NHC(=O)O-; -OC(=O)NH-; -NHC(=O)NR1-; -NHC(=O)O-; -OC(=O)NR1-; -C(=O)R1-; -CO((CrC8)alkyl)-; -CO((C6)aryl)-; -CO2((Ci- Cs)alkyl)-; -CO2((C6)aryl)-; -SO2((CrC8)alkyl)-; and -SO2((C6)aryl)-.

[0402] In some embodiments, a cationically ionizable lipid is selected from: di(heptadecan-9-yl) 3,3'-((2-(4- methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2-(l- methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMe-Pyr); bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin- l-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); bis(2-octyldodecyl) 3,3'-(((l-methylpiperidin-3- yl)methyl)azanediyl)dipropionate (BODD-C2C2-lMe-3PipD); bis(2-octyldodecyl) 3,3'-((2- (dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin- l-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l- yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr); and bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l- yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ).

[0403] In some embodiments, a lipid nanoparticle (LNP) comprises a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, ALC-0315, ALC-366, SM-102, HY-501, EA-405, HY- 405, DODMA, and Dlin-MC3-DMA. In some embodiments, a LNP comprises a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, ALC-0315, SM-102, HY-501, and DODMA. In some embodiments, a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-lMe-Pyr; ALC-0315; ALC-0366; SM-102; HY-501; EA-405; HY-405; DODMA; and Dlin-MC3-DMA. In some embodiments, a LNP comprises about 40 mol% to about 50 mol% (e.g., about 47.5 mol%) (relative to the total amount of lipids in a LNP) of a cationic or cationically ionizable lipid selected from the group consisting of: BHD-C2C2-PipZ; BODD-C2C2-lMe-Pyr; ALC-315, SM-102; HY-501; and DODMA.

[0404] (ii) Helper lipids

[0405] As described herein, lipid nanoparticles of the present disclosure comprise a helper lipid. In some embodiments, a helper lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a helper lipid is a phospholipid. In some embodiments, a helper lipid is or comprises l,2-distearoyl-szf-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), l,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), ceramides, and their derivatives. In some embodiments, a helper lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DSPE, and SM. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the helper lipid is DSPC.

[0406] Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO 2021 / 026358, WO 2017 / 075531, and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference.

[0407] In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a helper lipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 10 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 10 mol% of DSPC.

[0408] (Hi) Polymer-conjugated lipids

[0409] As described herein, LNPs of the present disclosure comprise a polymer-conjugated lipid. In some embodiments, a polymer-conjugated lipid is a lipid conjugated to polyethylene glycol (a "PEG-lipid"). In some embodiments, a PEG-lipid is selected from pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethylene glycol)-2,3- dimyristoylglycerol (PEG-DMG) (e.g., l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000- DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2',3'-di(tetradecanoyloxy)propyl-l-0-(to-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate, and 2,3-di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a number average molecular weight (Mn) of about 2000 g / mol.

[0410] In some embodiments, a PEG-lipid is DMG-PEG. In some embodiments, a PEG-lipid is PEG2000-DMG:

[0411] In some embodiments, a PEG-lipid is provided in WO 2021 / 026358, WO 2017 / 075531, or WO 2018 / 081480, each of which is incorporated by reference in its entirety.

[0412] In some embodiments, a PEG-lipid is a compound of Formula PCL-I: or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PGL-I, R8and R9are each independently C10-C30 aliphatic, optionally Interrupted by one or more ester bonds, and w is an integer from 30 to 60. In some embodiments, a compound of Formula PCL-I is 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159). In some embodiments, a compound of Formula PCL-I is: or a pharmaceutically acceptable salt thereof, where n' is an integer from about 45 to about 50.

[0413] In some embodiments, the PEG-lipid is represented by: wherein n has a mean value ranging from 30 to 60. In some embodiments, n is 50. In one embodiment, the PEG- conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(o)-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol- 2,3-bis(tetradecyloxy) propylcarbamate (2000).

[0414] In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, ALC-159, PEG2000-C-DMA PEG-S-DMG, PEG-cer, and combinations thereof. In some embodiments, a PEG-lipid is ALC-0159 or PEG2000-DMG. In some embodiments, a PEG-lipid is ALC-0159. In some embodiments, a PEG-lipid is PEG2000- DMG.

[0415] In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion.

[0416] In some embodiments, a polymer-conjugated lipid is one described in WO 2024 / 028325, which is incorporated herein by reference in its entirety. In some embodiments, a polymer-conjugated lipid is represented by formula or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PCL-II: X2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester; Y is -CH2-, -(0-12)2-, or -(0-12)3-; z is 2 to 24; and n is 1 to 100. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)-; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; (vi) when X1is -0- then X2is -C(O)-; (vii) when X1is -C(S)- then X2is -O-; (viii) when X1is -0- then X2is -C(S)-; (ix) when X1is -C(O)- then X2is -S-; or (x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or CI-B alkyl. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is - NR1-; (ii) when X1is -NR1- then X2is -C(O)-; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; or (vi) when X1is -0- then X2is -C(O)-; wherein R1is hydrogen or CI-B alkyl.

[0417] In some embodiments, a poiymer-conjugated lipid comprises monomers of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the poiymer-conjugated lipid is or comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, a poiymer-conjugated lipid comprises monomers of unit PCL-II-1:

[0418] In some embodiments, a poiymer-conjugated lipid comprises, 5 to 50, 5 to 25 or 10 to 25 monomers of PCL-II-1. In some embodiments, a poiymer-conjugated lipid comprises 14 to 17 monomers of PCL-II-1. In some embodiments, a poiymer-conjugated lipid comprises 8 to 14 monomers of PCL-II-1. In some embodiments, a poiymer-conjugated lipid is selected from the table below:

[0419]

[0420] In some embodiments, an LNP comprises an polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid, as described herein. In some embodiments, nucleic acid particles (e.g., DNA or RNA particles) described herein comprise a polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0421] In some embodiments, a lipid nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer-conjugated lipid.

[0422] In some embodiments, a lipid nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid.

[0423] In some embodiments, a lipid nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid.

[0424] In some embodiments, a lipid nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE- AEEA14-AC; VE-AEEA14-AC; ALC-0159 and PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer- conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC, VE-AEEA14-AC, and PEG2000-DMG. In some embodiments, a molar ratio of a cationic or cationically ionizable lipid to a polymer-conjugated lipid is from about 2:1 to about 8:1.

[0425] (iv) Steroids As described generally herein, lipid nanoparticles further comprise a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is p-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol. In some embodiments, a sterol is cholesterol. In some embodiments, a lipid nanoparticle comprises about 39 to about 49 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises about 40 to about 46 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises about 40 to about 44 mol% of a steroid.

[0426] In some embodiments, a lipid nanoparticle comprises: about 30 to about 60 mol% of a cationically ionizable lipid; about 18.5 to about 48.5 mol% of a steroid (e.g., cholesterol); about 0 to about 30 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 35 to about 55 mol% of a cationically ionizable lipid; about 30 to about 40 mol% of a steroid (e.g., cholesterol); about 5 to about 25 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 40 to about 50 mol% of a cationically ionizable lipid; about 30 to about 45 mol% of a steroid (e.g., cholesterol); about 5 to about 15 mol% of a helper lipid (e.g., DSPC); and about 1 to about 2.5 mol% of a polymer-conjugated lipid.

[0427] In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin- l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE- AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% di(heptadecan-9-yl) 3,3'-((2-(4- methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG. In some embodiments, a lipid nanopartide comprises: about 47.5 mol% of ALC-0315; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises: about 47.5 mol% of ALC-366; about 10 mol% of DSPC; about 40.7 mol% of cholesterol; and about 1.8 mol% of ALC-159. In some embodiments, a lipid nanoparticle comprises about 50 mol% of SM-102; about 1.5 mol% of PEG2000-DMG; about 10 mol% of DSPC; and about 38.5 mol% of cholesterol. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2- yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMe-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% VE- AEEA14-AC. In some embodiments, a lipid nanoparticle comprises: 47.5 mol% bis(2-octyldodecyl) 3,3'-((2-(l- methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMe-Pyr); 10 mol% DSPC; 40.7 mol% cholesterol; and 1.8 mol% PEG2000-DMG.

[0428] (v) Manufacturing

[0429] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Publication Nos. 2016 / 0009637, 2015 / 0273068, 2014 / 0200257, 2013 / 0338210, 2013 / 0245107, 2013 / 0123338, 2013 / 0017223, 2012 / 0183581, 2012 / 0027803, 2011 / 0311583, 2011 / 0216622, 2011 / 0117125, 2007 / 0042031, 2006 / 0083780, 2005 / 017054, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, WO 2022 / 016089, WO 2022 / 081752, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

[0430] For example, in some embodiments, cationically ionizable lipids, helper lipids, and steroids are solubilized in an organic solvent such as ethanol, at a predetermined weight or molar ratios / percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles are prepared at a total lipid to nucleic acid (e.g., RNA) weight ratio of approximately 10:1 to 50:1. In some embodiments, such nucleic acid (e.g., RNA) can be diluted to 0.1 to 1.0 mg / mL (e.g., 0.4 mg / mL) in an acidic buffer, such as citrate or acetate having a pH of between about 4 to about 6. In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNAs) can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids, is combined with, e.g., injected into or continuously mixed with, an aqueous solution comprising nucleic acids.

[0431] In some embodiments, lipid and nucleic acid (e.g., RNA) solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationic lipid, a helper lipid, cholesterol, a conjugated lipid, and any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid (e.g., RNA) solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids (e.g,, RNAs). The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid (e.g., RNA).

[0432] In some embodiments, a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0433] Liposomes

[0434] In some embodiments, a nucleic acid particle is a liposome, wherein the liposome comprises a cationic lipid and a nucleic acid. Liposomes are lipid-based particles that comprise a bilayer (uni-lamellar) or a concentric series of multiple bilayers (multi-lamellar) separated by aqueous compartments, enclosing a central aqueous core that encapsulates the agent for delivery (e.g., a nucleic acid such as RNA). Different types of liposomes are described, including e.g., small and large unilamellar vesicles, multilamellar vesicles, multivesicular liposomes. Many suitable methods are known for manufacturing liposomes (see e.g., Shah S, et al., Adv Drug Deliv Rev. 2020; 154-155: 102- 122), including e.g., solvent evaporation or lipid film hydration, solvent dispersion or reverse phase evaporation, optionally followed by processes to manipulate the size of the liposomes, such as e.g., sonication, homogenization and extrusion. Examples of liposomes that may be suitable for nucleic acid (e.g., RNA) delivery are described in PCT App. Pub. No. WO 2012 / 006378, WO 2013 / 006825, WO 2019 / 077053 and WO 2022 / 069632, each of which is incorporated herein by reference in its entirety.

[0435] In some embodiments, liposomes may be formed from one or more lipids selected from neutral lipids, phospholipids, cholesterol, and / or cationic lipids. In some embodiments, liposomes may comprise one or more phospholipids and optionally cholesterol. Suitable phospholipids for forming liposomes include DSPC, DPPC, DMPC, DOPC, DOPE, and DSPE. In some embodiments, a cationic lipid for use in a liposome is selected from 1,2-dioleoyl- 3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleoyloxypropyl)amine (DODMA), N-(2,3- dioleyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride (DOTAP), 4-(dimethylamino)butanoic acid, and (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (Dlin-MC3-DMA). In some embodiments a cationic lipid for use in a liposome is selected from DOTMA and DOTAP. In some embodiments a cationic lipid is DOTMA.

[0436] In some embodiments, a liposome may further comprise an additional lipid. In some embodiments, an additional lipid is a neutral lipid. As used herein, a "neutral lipid" refers to a lipid having a net charge of zero. Examples of suitable neutral lipids include, but are not limited to, l,2-di-(9Z-octadecenoyl)-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In specific embodiments, the second lipid is DOPE, cholesterol and / or DOPC.

[0437] Lipoplexes (LPX) In some embodiments, a nucleic acid particle is a lipoplex, wherein the lipoplex comprises a cationic lipid and a nucleic acid. Lipoplex particles (LPX) may be prepared by mixing liposomes with nucleic acid (e.g., RNA, where lipoplex particles comprising RNA are referred to as "RNA lipoplex particles"). RNA LPX particles typically form spontaneously from electrostatic interactions between positively charged liposomes and negatively charged RNA, and typically have a multilamellar structure. LPX (e.g., RNA LPX) typically comprise one or more cationic lipids and optionally one or more additional lipids. Examples of lipoplexes that are suitable for nucleic acid (e.g., RNA) delivery, as well as methods of manufacture, are described in PCT App. Pub. No. WO 2019 / 077053 and WO 2022 / 069632, each of which is incorporated herein by reference in its entirety.

[0438] In some embodiments, a cationic lipid for use in a LPX is selected from DODAP, DODMA, DOTMA, DDAB, DOTAP, and Dlin-MC3-DMA. In some embodiments a cationic lipid for use in a LPX is selected from DOTMA and DOTAP. In some embodiments a cationic lipid for use in a LPX is DOTMA. In some embodiments, a LPX further comprises an additional lipid. In some embodiments, an additional lipid is a neutral lipid. As used herein, a "neutral lipid" refers to a lipid having a net charge of zero. Examples of suitable neutral lipids include, but are not limited to, DOPE, DOPC, diacylphosphatidyl choline, diacylphosphatidyl ethanol amine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In specific embodiments, the second lipid is DOPE, cholesterol and / or DOPC.

[0439] In some embodiments, LPX may be manufactured by first preparing liposomes by injecting a solution of the lipids (e.g., DOTMA and DOPE) in ethanol into water or a suitable aqueous phase to form a liposome colloid. LPX may then be prepared by mixing the liposome colloid with a solution comprising nucleic acid (e.g., RNA). In one embodiment, RNA LPX particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1:9, from about 4:1 to 1:2, from about 3:1 to about 1:1, or about 2:1. In one embodiment, the ratio of positive charges (e.g., in DOTMA) to negative charges (e.g., in the RNA), in the RNA LPX particles, is from about 1:2 to 1.9:2, or about 1.3:2.0. RNA LPX particles may have an average diameter that ranges from about 200 to about 800 nm, such as from about 300 nm to about 500 nm.

[0440] Polymer-based particles (Polvplexes) and other delivery systems

[0441] In some embodiments, a nucleic acid particle described herein is a polymer-based particle (i.e., a polyplex, PLX). In some embodiments, a nucleic acid particle is a polyplex particle, and comprises a cationic polymer and a nucleic acid. Examples of polyplex particles that are suitable for nucleic acid (e.g., RNA) delivery are described in PCT App. Pub. No. WO 2021 / 001417, which is incorporated herein by reference in its entirety. Nucleic acid polyplex particles typically form spontaneously from electrostatic interactions between positively charged cationic polymer (e.g., PEI) and negatively charged nucleic acid (e.g., RNA). In some embodiments a polyplex particle may further comprise one or more lipids, in which case it may be referred to as a lipopolyplex (LPLX). In some embodiments, a cationic polymer is a polycationic polymer, e.g., a polymer having one or more cationic or cationically ionizable groups. In some embodiments, one or more cationic or ionizable groups comprise a nitrogen atom. Cationic polymers useful for preparing complexes described herein can be homopolymers, heteropolymers, or block-co-polymers.

[0442] In some embodiments, a cationic polymer is poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, a cationic polymer is a homopolymer. It is understood that a cationic polymer described herein can be linear or branched. In some embodiments, a cationic polymer is linear. In some embodiments, a cationic polymer is poly(ethylenimine).

[0443] In some embodiments, a cationic polymer is a heteropolymer (e.g., a linear heteropolymer) comprising copolymers of one or more of poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof. In some embodiments, a cationic polymer is a heteropolymer comprising poly(ethylenimine) and poly(propylenimine).

[0444] In some embodiments, a cationic polymer has between 250 and 2000 repeating monomer units (such as between 1500 and 2000 repeating monomer units). In some embodiments, a cationic polymer is a polymer described herein, having a number average molecular weight (Mn) of about 600 Daltons (Da) to about 400,000 Da (such as about 20,000 to about 120,000 Da).

[0445] In some embodiments, a complex comprises a cationic polymer and a nucleic acid, wherein the cationic polymer is or comprises a polyamine derivative (e.g., a carboxylated polyamine derivative). Suitable polyamine derivatives for delivery of nucleic acids, such as RNA, are described in WO 2014 / 053245 and WO 2014 / 056590, both of which are incorporated herein by reference in their entirety.

[0446] In some embodiments, a polyamine derivative comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms, and each of said hydrophobic linker may comprise from 1 to 3 heteroatoms selected from O, N, and S; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, and may comprise from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.

[0447] In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions is a polyalkylenimine (e.g., polyethylenimine) derivative having one or more carboxyalkyl substituents comprising from 6 to 40 carbon atoms, and one or more hydrophobic substituents selected from hydrocarbon substituents having at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, wherein each of said hydrophobic substituents may be or may comprise an alkyl group and / or each of said hydrophobic substituents may be or may comprise an aryl group.

[0448] In some embodiments, the polyamine derivative has (i) a linear polyethylenimine moiety of from 2 to 500 kDa (in terms of number average molecular weight), or (ii) a branched polyethylenimine moiety of from 0.5 to 200 kDa (in terms of number average molecular weight); and the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and / or alkylarylalkyls. Other suitable polymers include, for example, Viromer® and jetPEI® (Polyplus). Other suitable polymers or lipidoids useful for delivery of nucleic acids, such as RNA, are described in WO 2014 / 207231, WO 2016 / 097377 and WO 2024 / 042236, all of which are incorporated herein by reference in their entirety. Other delivery systems suitable for nucleic acid (e.g., RNA) delivery, which are based on oligosaccharide compounds, are described in WO 2023 / 067121, WO 2023 / 067123, WO 2023 / 067124, WO 2023 / 067125, and WO 2023 / 067126, all of which are incorporated herein by reference in their entirety.

[0449] Doses

[0450] The term "dose" as used herein refers in general to a "dose amount" which relates to the amount of RNA administered per administration, i.e., per dosing.

[0451] In some embodiments, administration of RNA of the present disclosure may be performed by single administration or boosted by multiple administrations.

[0452] In some embodiments, an amount the RNA described herein from 0.1 pg to 300 pg, 0.5 pg to 200 pg, or 1 pg to 100 pg, such as about 1 pg, about 3 pg, about 10 pg, about 30 pg, about 50 pg, or about 100 pg may be administered per dose.

[0453] In some embodiments, a regimen described herein includes at least one dose. In some embodiments, a regimen includes a first dose and at least one subsequent dose. In some embodiments, a regimen includes a first dose and two subsequent doses. In some embodiments, the first dose is the same amount as at least one subsequent dose. In some embodiments, the first dose is the same amount as all subsequent doses. In some embodiments, the first dose is a different amount as at least one subsequent dose. In some embodiments, the first dose is a different amount than all subsequent doses. In some embodiments, a regimen comprises two doses. In some embodiments, a regimen consists of two doses. In some embodiments, a regimen comprises three doses. In some embodiments, a regimen consists of three doses.

[0454] In one embodiment, the disclosure envisions administration of a single dose. In one embodiment, the disclosure envisions administration of a priming dose followed by one or more booster doses. The booster dose or the first booster dose may be administered 7 to 90 days, 14 to 60 days, or 30 to 60 days following administration of the priming dose. For example, the booster dose or the first booster dose may be administered about 56 days following administration of the priming dose. The second booster dose may be administered 120 to 270 days, or 150 to 210 days following administration of the priming dose. For example, the second booster dose may be administered about 180 days following administration of the priming dose.

[0455] In some embodiments, an amount of the RNA described herein of 60 pg or lower, 50 pg or lower, or 40 pg or lower may be administered per dose.

[0456] In some embodiments, an amount of the RNA described herein of at least 0.25 pg, at least 0.5 pg, at least 1 pg, at least 2 pg, at least 3 pg, at least 4 pg, at least 5 pg, at least 10 pg, at least 20 pg, at least 30 pg, or at least 40 pg may be administered per dose.

[0457] In some embodiments, an amount of the RNA described herein of 0.25 pg to 60 pg, 0.5 pg to 55 pg, 1 pg to 50 pg, 5 pg to 40 pg, or 10 pg to 30 pg may be administered per dose.

[0458] In some embodiments, a regimen administered to a subject may comprise a plurality of doses (e.g., at least two doses, at least three doses, or more). In some embodiments, a regimen administered to a subject may comprise a first dose and at least one further dose (e.g., a second or a second and a third dose), which doses are given at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, or more. In some embodiments, such doses may be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart. In some embodiments, doses may be administered days apart, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In some embodiments, doses may be administered about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeks apart, or about 1 to more than 6 weeks apart. In some embodiments, a minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more.

[0459] In some embodiments, a first dose and a second dose (and / or other subsequent doses) may be administered by intramuscular injection. In some embodiments, a first dose and a second dose (and / or other subsequent doses) may be administered in the deltoid muscle. In some embodiments, a first dose and a second dose (and / or other subsequent doses) may be administered in the same arm. In some embodiments, an mRNA composition described herein is administered (e.g., by intramuscular injection) as a series of three doses. In some embodiments, each dose is about 30 pg. In some embodiments, each dose is about 10 pg. In some embodiments, each dose is about 3 pg. In some embodiments, each dose is about 1 pg.

[0460] In some such embodiments, an mRNA composition described herein is administered to subjects of age 12 or older. In some such embodiments, an mRNA composition described herein is administered to subjects of age 5 to 11. In some such embodiments, an mRNA composition described herein is administered to subjects of age 2 to less than 5. In some such embodiments, an mRNA composition described herein is administered to subjects of age 12 or older and each dose is about 30 ug. In some such embodiments, an mRNA composition described herein is administered to subjects of age 5 to 11 and each dose is about 10 ug. In some such embodiments, an mRNA composition described herein is administered to subjects of age 2 to less than 5 and each dose is about 3 pg.

[0461] Compositions comprising nucleic acid

[0462] K composition comprising one or more RNAs described herein, e.g., in the form of RNA particles, may comprise salts, buffers, or other components as further described below.

[0463] In some embodiments, a salt for use in the compositions described herein comprises sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmolality agent for preconditioning RNA prior to mixing with lipids. In some embodiments, the compositions described herein may comprise alternative organic or inorganic salts. Alternative salts include, without limitation, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).

[0464] Generally, compositions for storing RNA particles such as for freezing RNA particles comprise low sodium chloride concentrations, or comprises a low ionic strength. In some embodiments, the sodium chloride is at a concentration from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 50 mM.

[0465] According to the present disclosure, the RNA particle compositions described herein have a pH suitable for the stability of the RNA particles and, in particular, for the stability of the RNA. Without wishing to be bound by theory, the use of a buffer system maintains the pH of the particle compositions described herein during manufacturing, storage and use of the compositions. In some embodiments of the present disclosure, the buffer system may comprise a solvent (in particular, water, such as deionized water, in particular water for injection) and a buffering substance. The buffering substance may be selected from 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)propane-l,3-diol (Tris), acetate, and histidine. In some embodiments, the buffering substance is HEPES. In some embodiments, the buffering substance is Tris.

[0466] Compositions (in particular, RNA compositions / formulations) described herein may also comprise a cryoprotectant and / or a surfactant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of RNA activity during storage, freezing, and / or lyophilization, for example to reduce or prevent aggregation, particle collapse, RNA degradation and / or other types of damage.

[0467] In some embodiments, the cryoprotectant is a carbohydrate. The term "carbohydrate", as used herein, refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

[0468] In some embodiments, the cryoprotectant is a monosaccharide. The term "monosaccharide", as used herein refers to a single carbohydrate unit {e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose and the like.

[0469] In some embodiments, the cryoprotectant is a disaccharide. The term "disaccharide", as used herein refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose and the like. In some embodiments, the cryoprotectant is sucrose.

[0470] The term "trisaccharide" means three sugars linked together to form one molecule. Examples of a trisaccharides include raffinose and melezitose.

[0471] In some embodiments, the cryoprotectant is an oligosaccharide. The term "oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. An oligosaccharide can be oxidized or reduced.

[0472] In an embodiment, the cryoprotectant is a cyclic oligosaccharide. The term "cyclic oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, p cyclodextrin, or y cyclodextrin.

[0473] Other exemplary cyclic oligosaccharide cryoprotectants include compounds which include a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. A cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms. The term "cyclodextrin moiety", as used herein refers to cyclodextrin (e.g., an a, p, or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer. A cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker. A cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.

[0474] Carbohydrate cryoprotectants, e.g., cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates. For example, in an embodiment, the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-p-cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified cyclodextrins).

[0475] An exemplary cryoprotectant is a polysaccharide. The term "polysaccharide", as used herein refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin and the like.

[0476] In some embodiments, RNA particle compositions may include sucrose. Without wishing to be bound by theory, sucrose functions to promote cryoprotection of the compositions, thereby preventing RNA (especially mRNA) particle aggregation and maintaining chemical and physical stability of the composition. In some embodiments, RNA particle compositions may include alternative cryoprotectants to sucrose. Alternative stabilizers include, without limitation, trehalose and glucose. In a specific embodiment, an alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose.

[0477] A preferred cryoprotectant is selected from the group consisting of sucrose, trehalose, glucose, and a combination thereof, such as a combination of sucrose and trehalose. In a preferred embodiment, the cryoprotectant is sucrose. Some embodiments of the present disclosure contemplate the use of a chelating agent in an RNA composition described herein. Chelating agents refer to chemical compounds that are capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), a salt of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, nitrilotriacetic acid, transdiaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid. In some embodiments, the chelating agent is EDTA or a salt of EDTA. In some embodiments, the chelating agent is EDTA disodium dihydrate. In some embodiments, the EDTA is at a concentration from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM or from about 0.25 mM to about 1 mM.

[0478] In an alternative embodiment, the RI A particle compositions described herein do not comprise a chelating agent.

[0479] Pharmaceutical compositions

[0480] The agents described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g., for use in treating or preventing a disease involving an antigen, in particular tuberculosis.

[0481] The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.

[0482] The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-di behenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0483] The pharmaceutical compositions of the present disclosure may be in a storable form (e.g., in a frozen or lyophilized / freeze-dried form) or in a "ready-to-use form" (i.e., in a form which can be immediately administered to a subject, e.g., without any processing such as diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed, or a freeze-dried pharmaceutical composition has to be reconstituted, e.g. by using a suitable solvent (e.g., deionized water, such as water for injection) or liquid (e.g., an aqueous solution).

[0484] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0485] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0486] The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In some embodiments relating to the treatment of a particular disease, the desired reaction may relate to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in some embodiments, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition, or symptoms thereof. An effective amount of the pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0487] The pharmaceutical compositions of the present disclosure may contain buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0488] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0489] The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants

[0490] The term "diluent" relates a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol, glycerol and water.

[0491] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy- propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0492] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0493] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0494] Routes of administration of pharmaceutical compositions

[0495] In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for intramuscular administration.

[0496] In some embodiments, intramuscular administration comprises administration into the upper arm, in particular into the musculus deltoideus. If more than one dose, e.g., three doses, of a pharmaceutical composition described herein is administered, the different administrations may be into the same arm. Use of compositions

[0497] Compositions described herein may be used in the therapeutic or prophylactic treatment of diseases wherein provision of one or more peptides or polypeptides, i.e., vaccine antigens, described herein to a subject results in a therapeutic or prophylactic effect. In some embodiments, the disease is infection with Mycobacterium tuberculosis. In some embodiments, the disease is tuberculosis.

[0498] The term "disease" (also referred to as "disorder" herein) refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.

[0499] The term "disease involving an antigen" refers to any disease which implicates an antigen, e.g. a disease which is characterized by the presence of an antigen. The disease involving an antigen can be an infectious disease. The antigen may be a disease-associated antigen, such as a bacterial antigen. In some embodiments, a disease involving an antigen is a disease involving cells comprising and / or expressing an antigen, and preferably presenting the antigen on the cell surface, e.g., in the context of MHC.

[0500] The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold). Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, hepatitis, sexually transmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis, HIV / acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), the bird flu, and influenza.

[0501] In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

[0502] The term "therapeutic treatment" relates to any treatment which improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0503] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably. The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal {e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other non-mammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease e.g., cancer, infectious diseases) but may or may not have the disease, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In some embodiments of the present disclosure, the "individual" or "subject" is a "patient". In some embodiments, the terms "individual" and "subject" relate to pregnant women and immunocompromised persons.

[0504] The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

[0505] RNA described herein may be administered to a subject for delivering the RNA to cells of the subject.

[0506] RNA described herein may be administered to a subject for delivering a therapeutic or prophylactic peptide or polypeptide (e.g., a pharmaceutically active peptide or polypeptide) to the subject, wherein the RNA encodes a therapeutic or prophylactic peptide or polypeptide.

[0507] RNA described herein may be administered to a subject for treating or preventing a disease in a subject, wherein delivering the RNA to cells of the subject is beneficial in treating or preventing the disease.

[0508] RNA described herein may be administered to a subject for treating or preventing a disease in a subject, wherein the RNA encodes a therapeutic or prophylactic peptide or polypeptide and wherein delivering the therapeutic or prophylactic peptide or polypeptide to the subject is beneficial in treating or preventing the disease.

[0509] In some embodiments of the disclosure, the aim is to induce an immune response by providing RNA described herein.

[0510] A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, RNA described herein is applicable for inducing or enhancing an immune response. RNA described herein is thus useful in a prophylactic and / or therapeutic treatment of a disease involving an antigen or epitope.

[0511] In some embodiments of the disclosure, the aim is to provide an immune response against cells comprising an antigen, e.g., Mtb antigen.

[0512] In some embodiments of the disclosure, the aim is to prophylactically or therapeutically treat tuberculosis by vaccination.

[0513] Due to the high degree of sequence conservation of the disclosed antigens between different Mycobacterium species, including Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium orygis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canetti and Mycobacterium pinnipedii, exposure of a subject to Mycobacterium tuberculosis antigens will result in a high degree of cross-reactivity with antigens of other Mycobacterium species. Therefore, a vaccine based on or directed at Mycobacterium tuberculosis antigens will elicit a robust immune response against other Mycobacterium species, in particular Mycobacterium bovis, Mycobacterium caprae, Mycobacterium orygis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canetti and Mycobacterium pinnipedii as well.

[0514] In some embodiments of the disclosure, the aim is to provide an immune response against a Mycobacterium selected from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium orygis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canetti and Mycobacterium pinnipedii and to prevent or treat tuberculosis.

[0515] In preferred embodiments of the disclosure, the aim is to provide an immune response against Mycobacterium tuberculosis. In some embodiments of the disclosure, the aim is to treat an infection with Mycobacterium tuberculosis.

[0516] In some embodiments of the disclosure, the aim is to prevent or treat disease symptoms caused by an infection with Mycobacterium tuberculosis.

[0517] In some embodiments of the disclosure, the aim is to provide protection against an infection with Mycobacterium tuberculosis by vaccination.

[0518] In some embodiments of the disclosure, the aim is to provide protection against an outbreak of disease in a subject infected with Mycobacterium tuberculosis. In some embodiments of the disclosure, the aim is to provide protection against symptoms of tuberculosis in a subjected infected with Mycobacterium tuberculosis.

[0519] In some embodiments, the RNA is present in a composition as described herein.

[0520] In some embodiments, the RNA is administered in a pharmaceutically effective amount.

[0521] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0522] In some embodiments, the subject treated had been exposed to Mycobacterium tuberculosis. In some embodiments, the subject treated had not been exposed to Mycobacterium tuberculosis.

[0523] In some embodiments, the treatments described herein involve pre- or post-exposure vaccination against Mycobacterium tuberculosis, or a combination thereof.

[0524] "Fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C- terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N- terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5’-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., at least 5, at least 6, at least 7, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30, up to 50, up to 80, up to 100, up to 150 or up to 200 consecutive amino acids of the amino acid sequence.

[0525] The phrase "full-length antigen or antigen fragment(s) representing a mycobacterium tuberculosis antigen or immunogenic variant thereof as used herein refers to the mycobacterium tuberculosis antigen or an immunogenic variant of the mycobacterium tuberculosis antigen, or one or more fragments of the mycobacterium tuberculosis antigen or an immunogenic variant of the mycobacterium tuberculosis antigen, wherein the fragments may or may not be overlapping. An immunogenic variant of a mycobacterium tuberculosis antigen or one or more fragments of a mycobacterium tuberculosis antigen or an immunogenic variant of a mycobacterium tuberculosis antigen are capable of inducing an immune response against the mycobacterium tuberculosis antigen when delivered to a subject, e.g. in the form of a protein or an RNA transcribed by a cell of the subject. In some embodiments, a fragment of a mycobacterium tuberculosis antigen or an immunogenic variant of a mycobacterium tuberculosis antigen comprises at least one epitope, e.g., at least one T cell epitope, of a mycobacterium tuberculosis antigen or an immunologically equivalent variant of said at least one epitope. In some embodiments, a fragment of a mycobacterium tuberculosis antigen or an immunogenic variant of a mycobacterium tuberculosis antigen comprises a fragment of, e.g., at least 5, at least 6, at least 7, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of said mycobacterium tuberculosis antigen or immunogenic variant of a mycobacterium tuberculosis antigen. The phrase "encoding at least one full-length antigen or antigen fragment representing at least one mycobacterium tuberculosis antigen or immunogenic variant thereof" with respect to RNA encompasses monocistronic and polycistronic RNAs.

[0526] If only one mycobacterium tuberculosis antigen or immunogenic variant thereof is represented, the RNA may encode the full length mycobacterium tuberculosis antigen or immunogenic variant thereof and / or may encode one or more fragments of the mycobacterium tuberculosis antigen or immunogenic variant thereof. If the RNA encodes a full length mycobacterium tuberculosis antigen or immunogenic variant thereof and at least one fragment of the mycobacterium tuberculosis antigen or immunogenic variant thereof, the full length mycobacterium tuberculosis antigen or immunogenic variant thereof and one or more of the at least one fragment of the mycobacterium tuberculosis antigen or immunogenic variant thereof may be encoded by different open reading frames located on the same or on different RNA molecules. If the RNA encodes more than one fragment of the mycobacterium tuberculosis antigen or immunogenic variant thereof, one or more of the more than one fragment of the mycobacterium tuberculosis antigen or immunogenic variant thereof may be encoded by different open reading frames located on the same or on different RNA molecules.

[0527] If more than one mycobacterium tuberculosis antigen or immunogenic variant thereof is represented, the RNA may either encode the full-length antigen of one or more mycobacterium tuberculosis antigen or immunogenic variant thereof, may encode one or more fragments of one or more mycobacterium tuberculosis antigen or immunogenic variant thereof, or a combination thereof. In some embodiments, the RNA encodes the full-length antigen of each of the more than one mycobacterium tuberculosis antigen or immunogenic variant thereof. In some embodiments, the RNA encodes one or more fragments of each of the more than one mycobacterium tuberculosis antigen or immunogenic variant thereof. In some embodiments, the RNA encodes the full-length antigen of some of the more than one mycobacterium tuberculosis antigen or immunogenic variant thereof and encodes one or more fragments of some of the more than one mycobacterium tuberculosis antigen or immunogenic variant thereof, wherein the RNA may encode the full-length antigen as well as one or more fragments of the same mycobacterium tuberculosis antigen or immunogenic variant thereof. The full-length antigens and / or fragments discussed above may be encoded by the same or different open reading frames located on the same or on different RNA molecules.

[0528] The term "chimeric protein" is used herein as a synonym for "fusion protein" and means a protein comprising two or more subunits, such as a full-length antigen, antigen fragment and / or other functional amino acid sequence. Preferably, the fusion protein is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence of a further subunit. Subunits may be interspersed by a polypeptide linker.

[0529] "Variant," as used herein and with reference to an amino acid sequence (peptide or polypeptide), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, such as from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.

[0530] By "wild type" or "WT" or "native" herein is meant an amino acid sequence that is found in nature, including allelic variations and / or naturally occurring mutations. A wild type amino acid sequence, peptide or polypeptide has an amino acid sequence that has not been intentionally modified by man.

[0531] The term "non-native" as used herein in conjunction with amino acid sequences is meant to refer to amino acid sequences not found in nature, i.e., that have been intentionally modified by man - either in sequence or in sequence context. In one embodiment, a non-native signal peptide sequence fused or operatively linked to a mycobacterium tuberculosis antigen denotes that said signal peptide in nature does not occur fused or operatively linked to to said mycobacterium tuberculosis antigen, either because said signal peptide can naturally be found fused or operatively linked only to other mycobacterium tuberculosis antigens or only in other organisms, such as mammals, e.g. human, other bacteria besides mycobacterium tuberculosis or viruses. Embodiments for such exogenous signal peptides are provided herein. In another embodiment, a non-native signal peptide has been mutated in a purposeful manner (e.g., by random mutagenesis and targeted selection or by guided mutagenesis techniques, including, e.g., sequence synthesis) in order to obtain certain functional properties or to eliminate certain functional properties, resulting in a signal peptide structurally and functionally distinct from a signal peptide found in nature fused or operatively linked to the mycobacterium tuberculosis antigen in question.

[0532] In some embodiments, the degree of similarity, such as identity between a given amino acid sequence and a reference amino acid sequence, will be given as being at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given, e.g., for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, such as sequence identity, can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS:: needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0533] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0534] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website {e.g., at blast.ncbi.nlm.nih.gov / Blast,cgi?PAGE_TYPE=BlastSearch8i.BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0535] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared {e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0536] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0537] Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and, e.g., at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0538] As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0539] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0540] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0541] According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.

[0542] In particular, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (in particular, mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0543] In this respect, an "open reading frame" or "ORF" is a continuous stretch of codons beginning with a start codon and ending with a stop codon.

[0544] The term "expression" as used herein includes the transcription and / or translation of a particular nucleotide sequence.

[0545] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.

[0546] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide. Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.

[0547] Terms such as "enhance" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level.

[0548] "Physiological pH" as used herein refers to a pH of about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0549] As used in the present disclosure, "% w / v" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (mL).

[0550] As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).

[0551] As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.

[0552] As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like material.

[0553] The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum 2 is taken over all the different kinds of ions (i) in solution.

[0554] According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions. Regarding the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low so as to prevent degradation of the nucleic acid. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between nucleotides such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 pM or less. In some embodiments, there are no or essentially no free divalent ions.

[0555] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0556] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used herein interchangeably. The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.

[0557] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0558] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.

[0559] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source.

[0560] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.

[0561] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given with respect to the EDTA disodium salt.

[0562] The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.

[0563] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.

[0564] According to the present disclosure, the term "peptide" refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having at least about 151 amino acids. "Peptides" and "polypeptides" are both protein molecules, although the terms "protein" and "polypeptide" are used herein usually as synonyms.[056...

Claims

Claims1. An RNA molecule comprising a nucleic acid sequence encoding an amino acid sequence comprising at least one variant of a Mycobacterium tuberculosis antigen or a fragment thereof, wherein the amino acid sequence of the at least one variant is characterized by one or more of the following modifications compared to the corresponding wildtype Mycobacterium tuberculosis antigen or fragment thereof: a) deletion or substitution of one or more amino acids subject to O-linked glycosylation in eukaryotic cells, b) deletion or substitution of an N-terminal cysteine residue, and / or c) addition of a non-native transmembrane domain or multimerization domain, optionally wherein the Mycobacterium tuberculosis antigen is LpqH or PstSl.

2. The RNA molecule of claim 1, wherein the variant is modified compared to the Mycobacterium tuberculosis antigen or fragment thereof in that the endogenous signal peptide has been replaced with a non-native signal peptide.

3. The RNA molecule of claim 1 or 2, wherein LpqH comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or an amino acid sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2.

4. The RNA molecule of claim 3, wherein the at least one variant of LpqH comprises a deletion at one or more amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2.

5. The RNA molecule of claim 3 or 4, wherein the at least one variant of LpqH comprises a deletion of an amino acid sequence corresponding to positions 23 to 48 of SEQ ID NO 1 or SEQ ID NO: 2.

6. The RNA molecule of claim 3 or 4, wherein the at least one variant of LpqH comprises a substitution at one or more amino acid positions corresponding to position 23, 24, 27, 28, 29, 31, 43, and / or 48 of SEQ ID NO: 1 or SEQ ID NO: 2.

7. The RNA molecule of any one of claims 3 to 6, wherein the N-terminal cysteine of LpqH is at an amino acid position corresponding to position 22 of SEQ ID NO: 1 or SEQ ID NO: 2.

8. The RNA molecule of any one of claims 3 to 7, wherein the endogenous signal peptide of LpqH is at the N- terminal end of the antigen, optionally wherein the endogenous signal peptide is at amino acid positions corresponding to positions 1 to 21 of SEQ ID NO: 1 or SEQ ID NO: 2.

9. The RNA molecule of any one of claims 1 to 8, wherein PstSl comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 4.

10. The RNA molecule of claim 9, wherein the at least one variant of PstSl comprises a deletion at one or more amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

11. The RNA molecule of claim 9 or 10, wherein the at least one variant of PstSl comprises a deletion of an amino acid sequence corresponding to positions 25 to 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

12. The RNA molecule of claim 9 or 10, wherein the at least one variant of PstSl comprises a substitution at one or more amino acid positions corresponding to position 26, 30, 32, 35, 41, 44 and / or 45 of SEQ ID NO: 3 or SEQ ID NO: 4.

13. The RNA molecule of any one of claims 9 to 12, wherein the N-terminal cysteine of PstSl is at an amino acid position corresponding to position 24 of SEQ ID NO: 3 or SEQ ID NO: 4.

14. The RNA molecule of any one of claims 9 to 13, wherein the endogenous signal peptide is at the N-terminal end of the antigen, optionally wherein the endogenous signal peptide is at amino acid positions corresponding to positions 1 to 23 of SEQ ID NO: 3 or SEQ ID NO: 4.

15. The RNA molecule of any one of claims 9 to 14, wherein the non-native signal peptide is a viral signal peptide.

16. The RNA molecule of claim 15, wherein the non-native signal peptide is an HSV-1 glycoprotein D signal peptide, a HSV-2 glycoprotein D signal peptide, a Japanese encephalitis PRM signal sequence or a VSVg protein signal sequence.

17. The RNA molecule of any one of claims 1 to 16, wherein a) the transmembrane domain is a transmembrane domain from Nipah virus (NiV) or a transmembrane domain from HSV, optionally a HSV-gDl-TM; or b) the multimerization domain is a ferritin domain, optionally a ferritin domain from Helicobacter pylori (HP-ferritin), or the multimerization domain is derived from a Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase, or the multimerization domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or 100% identity to the amino acid sequence of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, or 132.

18. The RNA molecule of any one of claims 1 to 17, wherein the transmembrane domain and / or multimerization domain is attached to the N-terminus of the at least one variant and / or the C-terminus of the at least one variant, optionally with a linker, further optionally wherein the linker is a glycine-serine linker (GS linker).

19. The RNA molecule of any one of claims 1 to 18, wherein the at least one variant of LpqH comprises the amino acid sequence of any one of SEQ ID NOs: 50, 56, 62, 66, 68, 70 or 72.

20. The RNA molecule of any one of claims 1 to 19, wherein the at least one variant of PstSl comprises the amino acid sequence of any one of SEQ ID NOs: 76, 82, 88, 92, 94, 96 or 98.

21. The RNA molecule of any one of claims 1 to 20, wherein the RNA molecule comprises: a) a 5' cap, optionally having a capl structure or comprising m27'3'‘0Gppp(miz'-°)ApG, b) a 5'-UTR, optionally comprising a modified human alpha-globin 5'-UTR or having the nucleotide sequence of SEQ ID NO: 104, or a nucleotide sequence having at least 98%, 96%, 94%, or 80% identity to the nucleotide sequence of SEQ ID NO: 104, c) a 3 -UTR, optionally comprising a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA or having thenucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 105, d) a polyA sequence, wherein the polyA sequence optionally is an interrupted sequence of A nucleotides, comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a nucleotide linker sequence of 10 nucleotides or comprises the nucleotide sequence of SEQ ID NO: 106, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 106, and / or e) modified nucleotides, nucleosides or nucleobases, optionally comprising modified uridines in place of at least one or in place of all uridines, wherein the modified uridines further optionally are Nl-methyl- pseudouridine.

22. The RNA molecule of any one of claims 1 to 22, wherein the coding sequence of the RNA molecule is codon- optimized and / or is characterized in that its G / C content is decreased compared to the parental sequence.

23. A protein encoded by the RNA molecule of any one of claims 1 to 22.

24. A DNA molecule encoding the RNA molecule of any one of claims 1 to 22.

25. A pharmaceutical composition comprising one or more RNA molecules of any one of claims 1 to 22.

26. The pharmaceutical composition of claim 25, wherein the one or more RNA molecule is formulated in a lipid formulation, optionally wherein the one or more RNA molecule is formulated in lipid nanoparticles or liposomes.

27. The pharmaceutical composition of claim 25 or 26, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants, and wherein the adjuvants optionally comprise a) an RNA encoding one or more immunomodulating molecules, optionally wherein the one or more immunomodulating molecules comprise cytokines, and / or b) one or more immunity inducing or immunomodulating moieties, optionally comprising a peptidoglycan moiety.

28. The pharmaceutical composition of any one of claims 25 to 27, wherein the one or more RNA molecules are formulated for intramuscular administration.

29. A kit comprising one or more pharmaceutical compositions of any one of claims 25 to 28.

30. The kit of claim 29, comprising two or more pharmaceutical compositions which comprise the same or different RNA molecules of any one of claims 1 to 22 in separate vials and optionally comprising instructions for use of the one or more pharmaceutical composition for treating or preventing tuberculosis.

31. An RNA molecule of any one of claims 1 to 22, a protein of claim 23, a DNA molecule of claim 24, a pharmaceutical composition of any one of claims 25 to 28 or a kit of claims 29 or 30 for use as a medicament.

32. The RNA molecule, protein, DNA molecule, pharmaceutical composition or kit for use of claim 31, wherein the use comprises treating or preventing tuberculosis in a subject.

33. A method for treating or preventing tuberculosis in a subject, wherein the method comprises administering the RNA molecule of any one of claims 1 to 22, the protein of claim 23, the DNA of claim 24 or the pharmaceutical composition of any one of claims 25 to 28 to the subject.

34. Use of the RNA molecule of any one of claims 1 to 22, the protein of claim 23, the DNA of claim 24 or the pharmaceutical composition of any one of claims 25 to 28 for the manufacture of a medicament for treating or preventing tuberculosis in a subject.

35. The RNA molecule, protein, DNA molecule, pharmaceutical composition or kit for use of claim 32, the method of claim 33 or the use of claim 34, wherein the tuberculosis is caused by an infection with a Mycobacterium, optionally selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium orygis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium canetti and Mycobacterium pinnipedii.

36. The RNA molecule of any one of claims 17 to 22, wherein the multimerization domain is a Mycobacterium tuberculosis 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, Mycobacterium bovis 6,7-dimethyl-8- ribityllumazine synthase or fragment thereof, Mycobacterium bovis BCG 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, or Mycobacterium leprae 6,7-dimethyl-8-ribityllumazine synthase or fragment thereof, optionally wherein the Mycobacterium 6,7-dimethyl-8-ribityllumazine synthase is encoded by a Mycobacterium RibH gene.

37. The RNA molecule of any one of claims 17 to 22 or 36, wherein the multimerization domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, at least 99% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 125, 126, 127, 128, 129, 130, 131, or 132.

Images