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WO2025126019A1 - Molécules d'arn - Google Patents

Molécules d'arn Download PDF

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Publication number
WO2025126019A1
WO2025126019A1 PCT/IB2024/062402 IB2024062402W WO2025126019A1 WO 2025126019 A1 WO2025126019 A1 WO 2025126019A1 IB 2024062402 W IB2024062402 W IB 2024062402W WO 2025126019 A1 WO2025126019 A1 WO 2025126019A1
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WIPO (PCT)
Prior art keywords
aspects
rna
sequence
utr
methyl
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PCT/IB2024/062402
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English (en)
Inventor
Patricia Michele MCMONAGLE
Natalie Clare SILMON DE MONERRI
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Pfizer Corp Belgium
Pfizer Corp SRL
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Pfizer Corp Belgium
Pfizer Corp SRL
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Publication of WO2025126019A1 publication Critical patent/WO2025126019A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia which often progresses rapidly and is therefore difficult to treat (Stephens, D.S., FEMS Microbiol Rev, 2007. 31 (1 ): p. 3 to 14). The highest rates of invasive disease are in children under one year of age, then in children 1 to 4 years of age followed by a secondary peak in adolescent and young adults 15 to 25 years of age (Stephens, D.S., FEMS Microbiol Rev, 2007. 31 (1 ): p. 3-14; Gabutti, G., A. Stefanati, and P. Kuhdari, J Prev Med Hyg, 2015. 56(3): p. E1 16-20). N.
  • meningitidis is classified into twelve serogroups based on capsular polysaccharide structures.
  • Six of these serogroups (A, B, C, W, Y and X) are most commonly associated with clinical disease (McNeil, L.K., et al., Microbiology and Molecular Biology Reviews, 2013. 77(2): p. 234-252; Rosenstein, N.E., et al., New England Journal of Medicine, 2001. 344(18): p. 1378- 1388).
  • Disease caused by serogroups A, C, Y and W can be prevented with capsular polysaccharide-based quadrivalent conjugate vaccines.
  • capsular polysaccharide-based vaccines based on serogroup B have failed because of poor immunogenicity possibly due to its structural homology to a human neural antigen (Finne, J., M. Leinonen, and P.H. Makela, Lancet, 1983. 2(8346): p. 355-7; Bruge, J., et al., Vaccine, 2004. 22(9-10): p. 1087-96; Holst, J., et al., Vaccine, 2009. 27 Suppl 2: p. B3-12).
  • fHbp human factor H binding protein
  • fHbp is an ideal target because it is expressed by >97% of MnB strains and can stimulate a strong antibody response capable of killing serogroup B strains (Fletcher, L.D., et al., Infect Immun, 2004. 72(4): p. 2088-100).
  • fHbp can be categorized into two immunologically distinct subfamilies, A and B.
  • fHbp is utilized by meningococci to evade complement-mediated bactericidal activity by binding to human complement factor H (hCFH), a negative regulator of the alternative complement pathway (Schneider, M.C., et aL, J Immunol, 2006.
  • fHbp specifically binds to hCFH and not CFH from mice or most primates (Granoff, D.M., J.A. Welsch, and S. Ram, Infect Immun, 2009. 77(2): p. 764-9) making the development of rodent models challenging.
  • fHbp elicits protective antibody responses in mice, rabbits, rhesus macaques, and humans which is determined by complement-mediated bactericidal activity (Masignani, V., et al., J Exp Med, 2003. 197(6): p. 789-99; Jiang, H.Q., et al., Vaccine, 2010. 28(37): p.
  • RNA molecules e.g., an immunogenic RNA polynucleotide encoding an amino acid sequence, e.g., an immunogenic antigen, comprising a Neisseria meningitidis protein, an immunogenic variant thereof, or an immunogenic fragment of the Neisseria meningitidis protein or the immunogenic variant thereof, e.g., an antigenic peptide or protein such as fHbp as disclosed herein.
  • the immunogenic antigen comprises an epitope of a Neisseria meningitidis protein for inducing an immune response against Neisseria meningitidis in the subject.
  • RNA polynucleotide encoding an immunogenic antigen is administered to provide (following expression of the polynucleotide by appropriate target cells) antigen for induction, e.g., stimulation, priming, and/or expansion, of an immune response, e.g., antibodies and/or immune effector cells.
  • an immune response e.g., antibodies and/or immune effector cells.
  • the immune response to be induced according to the present disclosure is a B cell-mediated immune response, e.g., an antibody-mediated immune response. Additionally or alternatively, the immune response to be induced according to the present disclosure may be a T cell-mediated immune response.
  • the immune response is an anti- Neisseria meningitidis immune response.
  • RNA molecules comprising RNA (as the active principle) that may be translated into a protein in a recipient’s cells.
  • the RNA molecules may contain one or more structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (e.g., 5' cap, 5' UTR, 3' UTR, poly-A-tail).
  • the RNA molecules contain all of these elements. It is contemplated that in some aspects, 1 , 2, 3, or more of the foregoing structural elements can be excluded from the RNA molecules.
  • each uridine of the RNA molecule disclosed herein is replaced by N1 - methylpseudouridine (UJ) (e.g., modified RNA; modRNA).
  • UJ N1 - methylpseudouridine
  • the RNA molecules described herein may be formulated with, encapsulated in, or complexed with lipids and/or proteins to generate RNA particles (e.g., lipid nanoparticles (LNPs)) for administration.
  • the RNA molecules described herein are formulated with, encapsulated in, or complexed with lipids to generate RNA-lipid nanoparticles (e.g., RNA-LNPs) for administration.
  • the RNA molecules described herein are formulated with, encapsulated in, or complexed with proteins for administration. In one aspect, the RNA molecules described herein are formulated with, encapsulated in, complexed with lipids and proteins for administration. If a combination of different RNA molecules is used, the RNA molecules may be formulated together or formulated separately with lipids and/or proteins to generate RNA particles for administration.
  • RNA molecules and RNA-LNPs that include at least one open reading frame (ORF) encoding a Neisseria meningitidis antigen.
  • the Neisseria meningitidis antigen is a Neisseria meningitidis polypeptide.
  • the Neisseria meningitidis polypeptide is from serogroup B.
  • the Neisseria meningitidis serogroup B polypeptide is factor H binding protein (fHbp).
  • the serogroup B fHbp is from the A and/or B subfamily strains.
  • the fHbp is A05.
  • the fHbp is B01.
  • the Neisseria meningitidis polypeptide is full-length, truncated, or a fragment or variant thereof. In some aspects, the Neisseria meningitidis polypeptide comprises at least one mutation.
  • the fHbp encoded by the ORF is fused to a secretion signal. In some aspects the secretion signal is located at the N-terminal end of the fHbp. In some aspects, the fHbp encoded by the ORF is fused to an anchor protein, or fragment thereof.
  • the anchor protein is influenza neuraminidase, or a fragment thereof. In some aspects, such anchor protein is fused at the N-terminal end of the fHbp.
  • RNA molecules that encode Neisseria meningitidis polypeptides that localize in the cellular membrane, localize in the Golgi and/or are anchored in the membrane and are secreted.
  • RNA molecules and RNA-LNPs that include at least one ORF encoding a Neisseria meningitidis polypeptide of Table 1 .
  • the Neisseria meningitidis polypeptide has at least, at most, exactly, or between (inclusive or exclusive) any two of 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99% or higher identity to any of the amino acid sequences of Table 1 , for example, any of SEQ ID NOs: 3, 5, 7, and 9.
  • Neisseria meningitidis polypeptide consists of any of the amino acid sequences of Table 1 , for example, any of SEQ ID NOs: 3, 5, 7, and 9.
  • RNA molecules and RNA-LNPs comprising at least one ORF comprising an RNA nucleic acid sequence of Table 1 .
  • the RNA molecule comprises a nucleic acid sequence that has at least, at most, exactly, or between (inclusive or exclusive) any two of 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99% or higher identity to any of the nucleic acid sequences of Table 1 , for example, any of SEQ ID NOs: 4, 6, 8, and 10.
  • each uridine of any of SEQ ID NOs: 4, 6, 8, and 10 is replaced by N1 - methylpseudouridine (4») (e.g., modified RNA; modRNA).
  • RNA molecules and RNA-LNPs that include a 5' untranslated region (5' UTR) and/or a 3' untranslated region (3' UTR).
  • the RNA molecule includes a 5' untranslated region (5' UTR).
  • the 5' UTR comprises a sequence comprising SEQ ID NO: 1.
  • the 5' UTR comprises a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99% or higher identity to SEQ ID NO: 1 .
  • the 5' UTR consists of SEQ ID NO: 1 .
  • the RNA molecules and RNA-LNPs include a 3' untranslated region (3' UTR).
  • the 3' UTR comprises a sequence comprising SEQ ID NO: 2.
  • the 3' UTR comprises a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 90%, 91 %, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99% or higher identity to SEQ ID NO: 2.
  • the 3' UTR consists of SEQ ID NO: 2.
  • the present disclosure further provides for RNA molecules and RNA-LNPs that include a 5' cap moiety.
  • the 5' cap moiety is (3'OMe) - m2 7 ’ 3 ' °Gppp (mi 2 ' °)ApG.
  • the present disclosure further provides for RNA molecules and RNA- LNPs that include a 3' poly-A tail.
  • the RNA molecule includes a 5' UTR and 3' UTR. In some aspects, the RNA molecule includes a 5' cap, 5' UTR, and 3' UTR. In some aspects, the RNA molecule includes a 5' cap, 5' UTR, 3' UTR, and poly-A tail. In some aspects, the RNA molecule includes a 5' UTR, 3' UTR, and poly-A tail. In some aspects, 1 , 2, 3, or more of the foregoing elements can be excluded from the RNA molecule. In some aspects, each uridine of any of the 5' UTR, 3' UTR, and poly-A tail is replaced by N1 - methylpseudouridine (4») (e.g., modified RNA; modRNA).
  • N1 - methylpseudouridine 4»
  • RNA molecules that include at least one open reading frame that was generated from codon-optimized DNA.
  • the open reading frame comprises a G/C content of at least, at most, exactly, or between (inclusive or exclusive) any two of 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, or 75%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, is or is about 50% to 75%, or is or is or about 55% to 70%.
  • the G/C content is or is about 58%, is or is about 66%, or is or is about 62%.
  • the present disclosure further provides RNA molecules comprising stabilized RNA.
  • the present disclosure further provides for RNA molecules that include RNA having at least one modified nucleotide (e.g., modified RNA; modRNA).
  • the modified nucleotide is pseudouridine, N1 -methylpseudouridine, N1 - ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2- thio-1 -methyl-1 -deaza-pseudouridine, 2-thio-1 -methyl-pseudouridine, 2-thio-5-aza- uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio- 1 -methyl- pseudouridine, 4-thio-ps
  • the modified nucleotide is N1 -methylpseudouridine (4»). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified nucleotides can be excluded from the RNA molecule.
  • the present disclosure further provides for RNA molecules that are messenger- RNA (mRNA) or self-replicating RNA. In some aspects, the RNA is a mRNA.
  • the present disclosure further provides for immunogenic compositions including the RNA molecules described herein.
  • the RNA molecules may be formulated in, encapsulated in, complex with, bound to or adsorbed on a lipid nanoparticle (LNP) (e.g., Neisseria meningitidis RNA-LNPs) in such immunogenic compositions.
  • LNP lipid nanoparticle
  • the lipid nanoparticle includes at least one of a cationic lipid, a polymer conjugated lipid (e.g., a PEGylated lipid), and at least one structural lipid (e.g., a neutral lipid and a steroid or steroid analog).
  • 1 , 2, 3, or more of the foregoing lipids can be excluded from the lipid nanoparticle.
  • the lipid nanoparticle includes a cationic lipid.
  • the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl)bis(2- hexyldecanoate) (ALC-0315).
  • the lipid nanoparticle includes a polymer conjugated lipid.
  • the lipid nanoparticle includes a PEGylated lipid, also referred to as a PEG-lipid.
  • the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide, glycol-lipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2- dimyristyloxlpropyl-3-amine (P
  • the PEGylated lipid is 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide (ALC-0159).
  • the lipid nanoparticle includes at least one structural lipid, such as a neutral lipid.
  • the neutral lipid is 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal
  • the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the lipid nanoparticle includes a second structural lipid, such as a steroid or steroid analog.
  • the steroid or steroid analog is cholesterol.
  • the lipid nanoparticle has a mean diameter of about 1 to about 500 nm, e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 1 10 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm,
  • the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA molecule/polynucleotide encoding a Neisseria meningitidis polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.01 , 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably of or of about 0.01 to 0.09 mg/mL, encapsulated in LNPs with a lipid composition comprising a cationic lipid at a concentration of or of about 0.8 to 0.95 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.80, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91 , 0.92, 0.93, 0.94, or 0.95 mg/mL), a PEGylated
  • the liquid composition further comprises a buffer composition comprising a first buffer at a concentration of or of about 0.1 to 0.3 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.10, 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 mg/mL), a second buffer at a concentration of or of about 1.25 to 1.4 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 .25, 1 .26, 1 .27, 1 .28, 1 .29, 1 .30, 1 .31 , 1 .32, 1 .33, 1 .34, 1 .35, 1 .36, 1 .37, 1 .38, 1 .39, or
  • 1 , 2, 3, 4, 5, or more of the foregoing elements can be excluded from the liquid RNA-LNP composition. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing element concentrations can be excluded from the liquid RNA- LNP composition.
  • the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding a Neisseria meningitidis polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.01 , 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably of or of about 0.01 to 0.09 mg/mL, encapsulated in LNPs with a lipid composition comprising ((4-hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of or of about 0.8 to 0.95 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.80, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88
  • the liquid composition further comprises a Tris buffer composition comprising tromethamine at a concentration of or of about 0.1 to 0.3 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 mg/mL) and Tris hydrochloride (HCI) at a concentration of or of about 1 .25 to 1 .4 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 .25, 1 .26, 1 .27, 1 .28, 1 .29, 1 .30, 1 .31 , 1 .32, 1 .33, 1 .34, 1 .35, 1 .36, 1 .
  • RNA-LNP composition 1. 2. 3, 4, 5, or more of the foregoing elements can be excluded from the liquid RNA- LNP composition. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing element concentrations can be excluded from the liquid RNA-LNP composition.
  • the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding a Neisseria meningitidis polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.01 , 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably of or of about 0.01 to 0.09 mg/mL, encapsulated in a LNP, and further comprising of or of about 5 to 15 mM Tris buffer(e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 mM) and of or of about 200 to 400 mM sucrose (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,
  • RNA-LNP composition 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
  • 1 , 2, 3, or more of the foregoing elements can be excluded from the liquid RNA-LNP composition.
  • 1 , 2, 3, 4, 5, or more of the foregoing element concentrations can be excluded from the liquid RNA-LNP composition.
  • the RNA-LNP immunogenic composition is a lyophilized (reconstituted) RNA-LNP composition comprising an RNA molecule/polynucleotide encoding a Neisseria meningitidis polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.01 , 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably of or of about 0.01 to 0.09 mg/mL, encapsulated in LNPs with a lipid composition comprising a cationic lipid at a concentration of or of about 0.8 to 0.95 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.80, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91 , 0.92, 0.93, 0.94, or 0.95 mg/mL),
  • a second buffer at a concentration of or of about 0.5 and 0.65 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.50, 0.51 ,
  • a stabilizing agent at a concentration of or of about 35 to 50 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/mL), and a salt diluent at a concentration of or of about 5 to 15 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 mg/mL) for reconstitution.
  • the lyophilized compositions are reconstituted in or in about 0.6 to 0.75 mL of the salt diluent (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.60, 0.61 , 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71 , 0.72, 0.73, 0.74, or 0.75 mL).
  • Concentrations in the lyophilized RNA- LNP composition are determined post-reconstitution.
  • 1 , 2, 3, 4, 5, or more of the foregoing elements can be excluded from the lyophilized RNA-LNP composition.
  • 1 , 2, 3, 4, 5, or more of the foregoing element concentrations can be excluded from the lyophilized RNA-LNP composition.
  • a lyophilized (reconstituted) RNA-LNP composition comprises an RNA polynucleotide encoding a Neisseria meningitidis polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between (inclusive or exclusive) any two of 0.01 , 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably of or of about 0.01 to 0.09 mg/mL, encapsulated in LNPs with a lipid composition of ALC-0315 at a concentration of or of about 0.8 to 0.95 mg/mL (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.80, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91 , 0.92, 0.93, 0.94, or 0.95 mg/mL), ALC-0159 at a concentration of or of about 0.05 to 0.15 mg
  • the lyophilized compositions are reconstituted in or in about 0.6 to 0.75 mL of sodium chloride (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 0.60, 0.61 , 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71 , 0.72, 0.73, 0.74, or 0.75 mL).
  • Concentrations in the lyophilized RNA-LNP composition are determined postreconstitution.
  • 1 , 2, 3, 4, 5, or more of the foregoing elements can be excluded from the lyophilized RNA-LNP composition.
  • 1 , 2, 3, 4, 5, or more of the foregoing element concentrations can be excluded from the lyophilized RNA-LNP composition.
  • RNA molecules, RNA-LNPs and immunogenic compositions that may be administered to a subject at a dose per administration of at least, at most, exactly, or between (inclusive or exclusive) any two of 1 pg, 15 pg, 30 pg, 45 pg, 60 pg, 75 pg, 90 pg, 100 pg or higher of Neisseria meningitidis RNA encapsulated in LNP.
  • 1 , 2, 3, 4, 5, or more of the foregoing concentrations of Neisseria meningitidis RNA encapsulated in LNP can be excluded.
  • RNA molecules, RNA-LNPs and immunogenic compositions that may be administered in a single dose.
  • the present disclosure further provides for RNA molecules, RNA-LNPs and immunogenic compositions that may be administered twice (e.g., Day 0 and on or about Day 7, Day 0 and on or about Day 14, Day 0 and on or about Day 21 , Day 0 and on or about Day 28, Day 0 and on or about Day 60, Day 0 and on or about Day 90, Day 0 and on or about Day 120, Day 0 and on or about Day 150, Day 0 and on or about Day 180, Day 0 and on or about 1 month later, Day 0 and on or about 2 months later, Day 0 and on or about 3 months later, Day 0 and on or about 6 months later, Day 0 and on or about 9 months later, Day 0 and on or about 12 months later, Day 0 and on or about 18 months later, Day 0 and on or about 2 years later, Day 0 and on or about 5 years later, or Day 0 and on on
  • the present disclosure further provides for RNA molecules, RNA-LNPs and immunogenic compositions that may be administered twice at Day 0 and on or about 2 months later.
  • the present disclosure further provides for RNA molecules, RNA-LNPs and immunogenic compositions that may be administered twice at Day 0 and on or about 6 months later.
  • the present disclosure further provides for RNA molecules, RNA-LNPs and immunogenic compositions that may be administered three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more times.
  • periodic boosters at intervals of 1 - 5 years may be desirable to maintain protective levels of the antibodies.
  • the present disclosure further provides for administration of at least one booster dose.
  • the present disclosure provides for a method of inducing an immune response against Neisseria meningitidis in a subject, including administering to the subject an effective amount of an RNA molecule, RNA-LNP and/or immunogenic composition described herein.
  • the present disclosure further provides for the use of an RNA molecule, RNA-LNP and/or immunogenic composition described herein in the manufacture of a medicament for use in inducing an immune response against Neisseria meningitidis in a subject.
  • the present disclosure provides for a method of preventing, treating, and/or ameliorating an infection, disease, or condition in a subject, including administering to a subject an effective amount of an RNA molecule, RNA-LNP and/or immunogenic composition described herein.
  • the present disclosure further provides for the use of an RNA molecule, RNA-LNP and/or immunogenic composition described herein in the manufacture of a medicament for use in preventing, treating, and/or ameliorating an infection, disease, or condition in a subject.
  • the infection, disease, or condition is associated with Neisseria meningitidis.
  • the infection, disease, or condition is meningococcal disease.
  • the infection, disease, or condition is bacterial meningitis.
  • the present disclosure provides for a method of preventing, treating, and/or ameliorating an infection, disease, or condition in a subject, including administering to a subject an effective amount of an RNA molecule and/or RNA-LNP that includes at least one open reading frame encoding a Neisseria meningitidis polypeptide or immunogenic composition described herein.
  • the subject is at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 months of age, or 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more years of age.
  • the subject is, is at least, is at most, or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older.
  • the subject is or is about 50 years of age or older.
  • 1 , 2, 3, 4, 5, or more of the foregoing age groups are not administered the RNA molecules and/or RNA-LNPs.
  • the subject is immunocompetent. In some aspects, the subject is immunocompromised.
  • the present disclosure provides for a method or use described herein, wherein the RNA molecule, RNA-LNP and/or immunogenic composition is administered as a vaccine.
  • the present disclosure provides a method or use described herein, wherein the RNA molecule, RNA-LNP and/or immunogenic composition is administered by intradermal, intramuscular, or intranasal injection.
  • any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “use of” any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
  • Use of the one or more compositions may be employed based on any of the methods described herein.
  • FIG. 1 shows the design of mRNA constructs encoding secreted Neisseria meningitidis fHbp.
  • FIG. 2 shows the expression of secreted fHbp-B01 -N47S and fHbp-A05-N46S from modRNA transcripts.
  • A Quantification of secreted fHbp-B01 -N47S and fHbp- A05-N46S in Expi293 culture supernatant over a 12-point titration using Octet biolayer interferometry.
  • B Anti-fHbp Western blot with detection by mAb MN86-994-1 1 -1 on transfected Expi293 cell culture supernatants (top) and cellular fractions (bottom).
  • FIG. 3 shows the structure of Type II Signal Anchor Protein Neuraminidase.
  • Neuraminidase is a type II signal anchor membrane protein that exists as a tetramer with a catalytic domain in the head region, a stalk region and transmembrane domain with a short cytoplasmic tail. The N-terminus of each monomer is located in the cytoplasm, with the C-terminus forming the globular head (McAuley, J.L., et al., Front Microbiol, 2019. 10: p. 39).
  • FIG. 4 shows the design of mRNA constructs encoding membrane-anchored fHbp antigens in the BMD576 backbone.
  • mRNA constructs were designed to encode fHbp-A05-N46S and fHbp-B01 -N47S antigens with an NA N-terminal membrane targeting sequence.
  • the cytoplasmic tail (CT) red
  • transmembrane domain (TMD) yellow
  • 7 amino acids of the of WSN/33 neuraminidase stalk region (SR) green
  • GOI N-terminus of fHbp
  • FIG. 5 shows the proportion of Expi293 Cells Expressing fHbp-A05-N46S and fHbp-B01 -N47S on the cell surface.
  • Expi293 cells were transfected with modRNA transcripts from BMD576 constructs encoding fHbp-A05-N46S and fHbp-B01 -N47S with the WSN/33 neuraminidase anchor. Protein expression on the cell surface was evaluated by flow cytometry 24 hours after transfection using mAb MN86-994-11 -1 and Human A05/B01 antisera for detection.
  • FIG. 6 shows the immunogenicity of fHBP modRNA in CD-1 mice. Serum collected from CD-1 mice following immunization at post-dose 2 (study week 6; 2- weeks post second immunization) or post-dose 3 (study week 10; 2-weeks post-third immunization) were evaluated in the SBA against a heterologous MenB fHbp subfamily A strain (A56, left panel) or a subfamily B strain (B44, right panel).
  • FIG. 7 shows the immunogenicity of fHBP modRNA in Swiss Webster mice. Serum collected from Swiss Webster mice following immunization at post-dose 2 (study week 6; 2-weeks post second immunization) or post-dose 3 (study week 10; 2- weeks post-third immunization) were evaluated in the SBA against a heterologous MenB fHbp subfamily A strain (A56, left panel) or a subfamily B strain (B44, right panel).
  • RNA molecule e.g., RNA polynucleotide
  • ORF open reading frame
  • the Neisseria meningitidis antigen is a fHbp polypeptide.
  • the Neisseria meningitidis polypeptide is a fHbp polypeptide from serogroup B.
  • the fHbp polypeptide from serogroup B is from the subfamily A strain.
  • the fHbp polypeptide from serogroup B is from the subfamily B strain.
  • the Neisseria meningitidis polypeptide comprises an amino acid sequence of Table 1.
  • the RNA molecules comprise an ORF comprising an RNA nucleic acid sequence of Table 1 .
  • the RNA molecule comprises at least one of a 5' cap, 5' UTR, 3' UTR and poly-A tail.
  • the present disclosure provides for an RNA molecule comprising modified nucleotides (e.g., modified RNA; modRNA).
  • the present disclosure provides for an immunogenic composition comprising any one of the RNA molecules encoding a Neisseria meningitidis polypeptide described herein complexed with, encapsulated in, and/or formulated with one or more lipids, and forming lipid nanoparticles (RNA-LNPs).
  • the present disclosure further provides for an immunogenic composition comprising any one of the RNA molecules comprising at least one RNA nucleic acid described herein complexed with, encapsulated in, and/or formulated with one or more lipids, and forming RNA-LNPs.
  • the present disclosure further provides for a method of preventing, treating and/or ameliorating an infection, disease and/or condition (e.g., meningococcal disease) in a subject via administering to a subject an effective amount of an RNA molecule, RNA- LNP and/or an immunogenic composition described herein.
  • an infection, disease and/or condition e.g., meningococcal disease
  • the present disclosure further provides for the use of the RNA molecule, RNA-LNP and/or an immunogenic compositions described herein as a vaccine.
  • the terms “about” and “approximately” and “substantially” are used according to their plain and ordinary meaning in the area of cell and molecular biology to indicate a deviation of ⁇ 10% of the value(s) to which it is attached. Therefore, in any disclosed aspect, the terms may be substituted with “within [a percentage] of” what is specified. In one non-limiting aspect, the percentage includes 0.1 , 0.5, 1 , 5, and 10 percent.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • essentially all is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some aspects, essentially all means equal to any one of, at least any one of, or between (inclusive or exclusive) any two of 95, 96, 97, 98, 99, or 100% of members of the group have that property.
  • compositions and methods for their use may “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
  • compositions and methods of the present disclosure that “comprise,” “have,” “include” or “contain” one or more elements possesses those one or more elements, but are not limited to possessing only those one or more elements.
  • an element of a composition or method of the present disclosure that “comprises,” “has,” “includes” or “contains” one or more features possesses those one or more features, but is not limited to possessing only those one or more features.
  • compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure.
  • the words “consisting of” (and any form of consisting of, such as “consist of” and “consists of”) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
  • inhibitors includes any measurable decrease (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of a 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease) or complete (e.g., 100%) inhibition to achieve a desired result.
  • the terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of a 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% increase) to achieve a desired result or production of a protein or molecule.
  • the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest.
  • a reference, standard, or control may be tested and/or determined substantially simultaneously and/or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and/or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.
  • isolated may refer to a nucleic acid and/or polypeptide that is substantially free of cellular material, bacterial material, viral material, and/or culture medium (when produced by recombinant DNA techniques) of their source of origin, and/or chemical precursors and/or other chemicals (when chemically synthesized).
  • an isolated compound refers to one that may be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel.
  • an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state and/or is altered or removed from the natural state through human intervention.
  • a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state, is “isolated.”
  • An isolated nucleic acid may exist in substantially purified form, or may exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered.
  • nucleic acid is a molecule comprising nucleic acid components and refers to DNA or RNA molecules. It may be used interchangeably with the term “polynucleotide.”
  • a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
  • Nucleic acids may also encompass modified nucleic acid molecules, such as base-modified, sugar-modified, backbone-modified, etc. DNA or RNA molecules.
  • Nucleic acids may exist in a variety of forms such as: isolated segments and recombinant vectors of incorporated sequences and/or recombinant polynucleotides encoding polypeptides, e.g., antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide; anti-sense nucleic acids for inhibiting expression of a polynucleotide; mRNA; saRNA; and complementary sequences of the foregoing described herein.
  • Nucleic acids may encode an epitope to which antibodies may bind.
  • epitope refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
  • an epitope is comprised of a plurality of chemical atoms and/or groups on an antigen.
  • such chemical atoms and/or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation.
  • such chemical atoms and/or groups are physically near to each other in space when the antigen adopts such a conformation.
  • at least some such chemical atoms and/or groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).
  • Nucleic acids may be single-stranded or double-stranded and may comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification of the polypeptide, transport, secretion, post-translational modification, and/or for therapeutic benefits such as targeting and/or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
  • polynucleotide refers to a nucleic acid molecule that may be recombinant and/or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (e.g., nucleic acids 100 residues or less in length) and recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences.
  • Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.
  • polynucleotide variants having substantial identity to the sequences disclosed herein, such as those comprising at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. In some aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 95% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 99% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • nucleic acid segments regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the nucleic acids may be any length.
  • nucleotides may be, for example, at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 1 1000, 12000, 13000, 14000, 15000 or more nucleotides in length, and/or may comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector.
  • nucleic acid fragment of almost any length may be employed, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • 1 , 2, 3, or more of the foregoing nucleic acid sequences can be excluded from the nucleic acid segments of the disclosure.
  • the term “gene” refers to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, and/or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or a substantially similar polypeptide.
  • a gene product may be a transcript.
  • a gene product may be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1 ) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.) (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • 1 , 2, 3, or more of the foregoing steps can be excluded from expression of nucleic acid sequences of the disclosure.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non- naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • DNA sequence The specific order of the monomers, e.g., the order of the bases linked to the sugar/phosphate-backbone, is called the DNA sequence.
  • DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g., by A/T-base-pairing and G/C-base-pairing.
  • DNA may contain all, or a majority of, deoxyribonucleotide residues.
  • deoxyribonucleotide means a nucleotide lacking a hydroxyl group at the 2' position of a [3-D-ribofuranosyl group.
  • DNA may or may not encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.
  • RNA means a nucleic acid molecule comprising nucleotides such as adenosine-monophosphate, uridine-monophosphate, guanosinemonophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone.
  • the backbone is formed by phosphodiester bonds between the sugar, e.g., ribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • RNA may be obtainable by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus and/or the mitochondria.
  • RNA messenger-RNA
  • Processing of the premature RNA comprises various posttranscriptional modifications such as splicing, 5' capping, polyadenylation, and/or export from the nucleus and/or the mitochondria.
  • Mature messenger RNA is processed and provides the nucleotide sequence that may be translated into an amino acid sequence of a peptide or protein.
  • a mature mRNA may comprise a 5' cap, a 5' UTR, an open reading frame, a 3' UTR and a poly-A tail sequence.
  • 1 , 2, 3, or more of the foregoing elements can be excluded from the mature mRNA of the disclosure.
  • RNA may contain all, or a majority of, ribonucleotide residues.
  • ribonucleotide means a nucleotide with a hydroxyl group at the 2' position of a [3-D-ribofuranosyl group.
  • RNA may be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein.
  • mRNA generally contains a 5' untranslated region (5' UTR), a polypeptide coding region, and a 3' untranslated region (3' UTR).
  • RNA may or may not encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, and modified RNA (modRNA).
  • nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s.
  • any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U” (e.g., chemically modified or not chemically modified).
  • An “isolated RNA” is defined as an RNA molecule that may be recombinant and/or has been isolated from total genomic nucleic acid.
  • An isolated RNA molecule and/or protein may exist in substantially purified form, or may exist in a non-native environment such as, for example, a host cell.
  • modified RNA refers to an RNA molecule having at least one addition, deletion, substitution, and/or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides, and/or to the 5' and/or 3' end(s) of RNA.
  • such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide.
  • a modified nucleotide may replace one or more uridine and/or cytidine nucleotides.
  • these replacements may occur for every instance of uridine and/or cytidine in the RNA sequence, or may occur for only select uridine and/or cytidine nucleotides.
  • Such alterations to the standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides and/or deoxynucleotides.
  • at least one uridine nucleotide may be replaced with N1 -methylpseudouridine in an RNA sequence.
  • Other such altered nucleotides are known to those of skill in the art.
  • Such altered RNA molecules are considered analogs of naturally-occurring RNA.
  • the RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides.
  • the RNA may be replicon RNA (replicon), in particular self-replicating RNA, or selfamplifying RNA (saRNA).
  • polypeptide refers to a polymer of amino acid monomers, e.g., a molecule comprising at least two amino acid residues.
  • Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing polypeptides can be excluded from the polypeptides of the disclosure.
  • Polypeptides may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer.
  • a protein comprises one or more peptides or polypeptides, and may be folded into a 3-dimensional form, which may be required for the protein to exert its biological function.
  • wild type or ”WT or “native” refer to the endogenous version of a molecule that occurs naturally in an organism.
  • wild type versions of a protein or polypeptide are employed, however, in other aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response.
  • the terms described above may be used interchangeably.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity and/or function (recognizing that proteins or polypeptides may have multiple activities and/or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity and/or function yet retain a wild type activity and/or function in other respects, such as immunogenicity.
  • a protein is specifically mentioned herein, it is in general a reference to a native (wild type) or recombinant (modified) protein.
  • the protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, produced by solid-phase peptide synthesis (SPPS) (e.g., an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution), liquid phase peptide synthesis (e.g., sequential addition of monomer building blocks in a liquid phase), a combination of solid and liquid phase peptide synthesis, and/or other in vitro methods.
  • SPPS solid-phase peptide synthesis
  • Assembling nucleic acids by a ligase may also be used to promote intermolecular ligation of the 5' and 3' ends of polynucleotide chains through the formation of a phosphodiester bond, and the ligation product produced thereby can, e.g., encode a recombinant protein and/or be used to introduce a protein-encoding nucleic acid into an expression vector.
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro and/or that is a replication product of such a molecule.
  • fragment with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, e.g., 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 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%, or at least 99% of the amino acid residues from an amino acid sequence.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least, at most, exactly, or between (inclusive or exclusive) any two of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 70% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 80% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 85% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 90% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 95% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 97% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.
  • any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • chemical moieties e.g., carbohydrates, lipids, phosphate groups
  • Variant antigens/polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties, e.g., that enhance their immunogenicity, enhance their expression, and/or improve their stability and/or PK/PD properties in a subject.
  • Variant antigens/polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity are well known in the art.
  • PK/PD properties of a protein variant can be measured using art recognized techniques, e.g., by determining expression of antigens in a vaccinated subject over time and/or by looking at the durability of the induced immune response.
  • the stability of protein(s) encoded by a variant nucleic acid may be measured by assaying thermal stability and/or stability upon urea denaturation and/or may be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art.
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least, at most, exactly, or between (inclusive or exclusive) any two of 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid.
  • RNA may be used as a therapeutic modality to treat and/or prevent a number of conditions in mammals, including humans.
  • Methods described herein comprise administration of the RNA described herein to a mammal, such as a human.
  • RNA include an antigen-coding RNA vaccine to induce robust neutralizing antibodies and accompanying/concomitant T cell response to achieve protective immunization.
  • minimal vaccine doses are administered to induce robust neutralizing antibodies and accompanying/concomitant T cell response to achieve protective immunization.
  • the RNA administered is in vitro transcribed RNA.
  • Prevent or “prevention,” as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder, or condition has been delayed for a predefined period of time.
  • An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public.
  • an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • the term “vaccination” refers to the administration of an immunogenic composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent (e.g., a bacterium such as Neisseria meningitidis).
  • a disease-associated agent e.g., a bacterium such as Neisseria meningitidis.
  • vaccination may be administered before, during, and/or after exposure to a disease-associated agent, and in certain aspects, before, during, and/or shortly after exposure to the agent.
  • vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition.
  • vaccination generates an immune response to an infectious agent.
  • vaccination generates an immune response to a bacterium, such as Neisseria meningitidis.
  • An immune response refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism.
  • An immune response may be measured by assays that include, but are not limited to, assays measuring the presence and/or amount of antibodies that specifically recognize a protein and/or cell surface protein, assays measuring T cell activation and/or proliferation, and/or assays that measure modulation in terms of activity and/or expression of one or more cytokines.
  • the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
  • the two or more regimens may be administered simultaneously; in some aspects, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some aspects, such agents are administered in overlapping dosing regimens.
  • “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
  • combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some aspects, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
  • a dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses, each of which is separated in time from other doses.
  • individual doses are separated from one another by a time period of the same length; in some aspects, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.
  • a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (e.g., is a therapeutic dosing regimen). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing regimens can be excluded.
  • Meningococcal disease is a disease caused by infection from the Neisseria meningitidis bacteria. Meningococcal disease can case meningitis, which is an infection of the brain and spinal cord, and it can also result blood infections.
  • RNA molecules e.g., RNA polynucleotides
  • the present disclosure provides for RNA molecules (e.g., RNA polynucleotides) comprising at least one open reading frame encoding a Neisseria meningitidis polypeptide.
  • the present disclosure further provides for an immunogenic composition
  • an immunogenic composition comprising at least one RNA molecule encoding a Neisseria meningitidis polypeptide complexed with, encapsulated in, and/or formulated with one or more lipids, and forming lipid nanoparticles (LNPs).
  • Neisseria meningitidis is a Gram-negative encapsulated bacterium that can cause sepsis, meningitis, and death.
  • N meningitidis can be classified into at least 12 serogroups (including serogroups A, B, C, 29E, H, I, K, L, W-135, X, Y and Z) based on chemically and antigenically distinctive polysaccharide capsules. Strains from five of the serogroups (A, B, C, Y, and W135) are responsible for the majority of disease.
  • Meningococcal meningitis is a devastating disease that can kill children and young adults within hours despite the availability of antibiotics.
  • Cross-protective vaccines or compositions effective against diverse MnB isolates are needed as is determining real-world vaccine coverage against a panel of diverse or heterologous meningococcal strains (e.g., representing different geographical regions).
  • the RNA molecules disclosed herein comprise an open reading frame encoding a Neisseria meningitidis antigen.
  • the Neisseria meningitidis antigen is a Neisseria meningitidis polypeptide.
  • the Neisseria meningitidis polypeptide is a fHbp, or a fragment or a variant thereof.
  • the fHbp is from serogroup B.
  • the fHbp from serogroup B is from the subfamily A strain.
  • the fHbp from serogroup B is from the subfamily B strain.
  • the fHbp polypeptide is fused to at least one other polypeptide, such as a secretion signal, or an anchor protein, such as influenza neuraminidase (NA).
  • a secretion signal such as a secretion signal
  • an anchor protein such as influenza neuraminidase (NA).
  • the Neisseria meningitidis polypeptide is designed to avoid ER/golgi retention of polypeptides, leading to increased surface expression of the antigen.
  • the variant polypeptides are truncated to remove the ER retention portion (e.g., the anchor domain) and/or the cytoplasmic tail portion of the polypeptide.
  • the Neisseria meningitidis polypeptides are mutated (e.g., mutations in one or more phosphorylated acidic motif(s)) to reduce Neisseria meningitidis polypeptide localization to the ER/golgi/TGN.
  • the Neisseria meningitidis polypeptide comprises an additional sequence to aid secretion of the polypeptide.
  • the Neisseria meningitidis polypeptide is modified to reduce or eliminate glycosylation sites, such as mutating certain amino acids that are predicted to be glycosylated when expressed in mammals. For example, in one aspect an asparagine residue within the fHbp predicted to be glycosylated during mammalian expressed is mutated to a serine residue.
  • the Neisseria meningitidis polypeptide is a full-length polypeptide. In some aspects, the Neisseria meningitidis polypeptide is a truncated polypeptide. In some aspects, the Neisseria meningitidis polypeptide is a variant of a Neisseria meningitidis polypeptide. In some aspects, the Neisseria meningitidis polypeptide is a fragment of a Neisseria meningitidis polypeptide.
  • the RNA molecule encodes a N meningitidis polypeptide of Table 1 (see Example 8). In some aspects, the RNA molecule encodes a fHbp polypeptide comprising an amino acid sequence of any of SEQ ID NOs: 3, 5, 7, and 9, or fragment or variant thereof.
  • the RNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten or more polypeptides.
  • one RNA molecule may also encode more than one polypeptide of interest, such as an antigen, e.g., a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens.
  • Bicistronic or multicistronic RNAs may include more than one polypeptide of interest with intervening sequences between the polypeptides of interest comprising an internal ribosome entry site (IRES) sequence(s) that allow for internal translation initiation between the polypeptides of interest, and/or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide.
  • IRES sequences and 2A peptides may be used, in some aspects, to enhance expression of multiple proteins from the same vector.
  • IRES sequences and 2A peptides may be used, in some aspects, to enhance expression of multiple proteins from the same vector.
  • IRES sequences and 2A peptides may be used, in some aspects, to enhance expression of multiple proteins from the same vector.
  • IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.
  • the sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression and/or replication of the RNA, to provide additional stability and/or resistance to degradation, and/or to reduce immunogenicity, relative to an unmodified RNA molecule.
  • the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA.
  • one or more of the foregoing reasons for modification of the RNA molecule can be excluded.
  • the RNA molecule of the present disclosure comprises an open reading frame having at least one codon modified sequence.
  • a codon modified sequence relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type coding sequence.
  • a codon modified sequence may show improved resistance to degradation, improved stability, and/or improved translatability.
  • G/C content of a coding region (e.g., of a gene of interest sequence; open reading frame (ORF)) of an RNA is increased compared to the G/C content of the corresponding coding sequence of a wild type RNA encoding the gene of interest, wherein in some aspects, the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the wild type RNA.
  • This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)/C (cytidine) content are more stable than sequences having an increased A (adenosine)/U (uridine) content.
  • the most favorable codons for the stability may be determined (so-called alternative codon usage).
  • G/C content of a coding region of an RNA described herein is increased by at least, at most, exactly, or between (inclusive or exclusive) any two of 10%, 20%, 30%, 40%, 50%, 55%, or even more compared to the G/C content of a coding region of a wild type RNA.
  • the coding region of the N. meningitidis RNA described herein comprises a G/C content of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some aspects, the coding region of the N. meningitidis RNA described herein comprises a G/C content of or of about 50% to 75%, 55% to 70%, 50% to 60%, 60% to 70%, 70% to 80%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, or 75% to 80%. In some aspects, the coding region of the N.
  • meningitidis RNA described herein comprises a G/C content of or of about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, or 75%.
  • the coding region of the N. meningitidis RNA described herein comprises a G/C content of or of about 58%, 66% or 62%.
  • the RNA molecule includes from or from about 20 to 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1 ,000, from 30 to 1 ,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1 ,000, from 100 to 1 ,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1 ,000, from 500 to 1 ,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1 ,000 to 1 ,500, from 500
  • the RNA molecule includes at least 100 nucleotides.
  • the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 1 1 ,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; or between 7,000 and 25,000 nucleotides.
  • the RNA molecule has at least, at most, exactly, between (inclusive or exclusive) any two of, or about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1000, 1050, 1 100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,
  • RNA molecules of the present disclosure may be prepared by any method know in the art, including chemical synthesis and in vitro methods, such as RNA in vitro transcription. In some of the aspects, the RNA of the present disclosure is prepared using in vitro transcription.
  • the RNA molecule of the present disclosure is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/diafiltration.
  • the RNA molecule of the present disclosure is lyophilized to be temperature stable.
  • an RNA is or comprises messenger RNA (mRNA) that relates to an RNA transcript that encodes a polypeptide.
  • mRNA messenger RNA
  • an RNA disclosed herein comprises: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5' UTR); a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3' UTR); and/or a polyadenylate (poly-A) sequence.
  • an RNA disclosed herein comprises the following components in the 5' to 3' orientation: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5' UTR), a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3' UTR); and a poly-A sequence.
  • a 5' cap comprising a 5' cap disclosed herein
  • a 5' untranslated region comprising a cap proximal sequence
  • a sequence encoding a protein e.g., a polypeptide
  • 3' untranslated region 3' UTR
  • 1 , 2, 3, 4, 5, or more of the foregoing dosing regimens can be excluded.
  • an RNA disclosed herein further comprises a signal peptide.
  • signal peptides and amino acid and nucleic acid sequences encoding such peptides can be found in, e.g., WO 2018/170270, the disclosure of which is incorporated by reference herein in its entirety.
  • an RNA disclosed herein encodes an antigenic fusion protein.
  • the encoded antigen or antigens may include two or more proteins (e.g., protein and/or protein fragment) joined together.
  • the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to an antigen.
  • Antigenic fusion proteins retain the functional property from each original protein.
  • an RNA disclosed herein encodes fusion proteins that comprise an antigen linked to a scaffold moiety.
  • the RNA further encodes a linker located between at least one or each domain of the fusion protein.
  • the antigens disclosed herein are fused to a secretion signal.
  • the antigens disclosed herein are fused to an anchor protein, or fragment thereof.
  • the anchor protein is influenza neuraminidase, or a fragment thereof.
  • the secretion signal is fused at the C-terminal end of the antigen protein.
  • the secretion signal is fused at the N-terminal end of the antigen protein.
  • the anchor protein, or fragment thereof is fused at the C-terminal end of the antigen protein.
  • the anchor protein, or fragment thereof is fused at the N-terminal end of the antigen protein.
  • the RNA molecules are not chemically modified and comprise the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, and/or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, and/or dT).
  • the RNA molecules may comprise modified nucleobases that may be incorporated into modified nucleosides and nucleotides.
  • the RNA molecule may include one or more modified nucleotides.
  • modified nucleic acids refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) and/or cytidine (C) ribonucleosides and/or deoxyribonucleosides in at least one of their position, pattern, percent and/or population.
  • modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides and/or non-naturally occurring modified nucleotides and nucleosides.
  • modifications can include those at the sugar, backbone, and/or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
  • Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
  • Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891 ; PCT/US2014/070413;
  • RNA molecules of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally- occurring nucleotides and nucleosides, or any combination thereof.
  • RNA molecules in some aspects, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides.
  • a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
  • RNA molecules include, without limitation, those described herein, and include, but are expressly not limited to, those modifications that comprise chemical modifications.
  • RNA molecules may comprise modifications that are naturally- occurring or non-naturally-occurring, or the RNA molecule may comprise a combination of naturally-occurring and non-naturally-occurring modifications.
  • RNA molecules may comprise non-natural modified nucleotides introduced during synthesis and/or post-synthesis of the RNA molecules to achieve desired functions and/or properties.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
  • RNA molecules may include any useful modification, for example, of a sugar, a nucleobase, and/or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage and/or to the phosphodiester backbone).
  • an internucleoside linkage e.g., to a linking phosphate, to a phosphodiester linkage and/or to the phosphodiester backbone.
  • nucleic acid e.g., RNA nucleic acids, such as mRNA nucleic acids.
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • nucleotide refers to a nucleoside, including a phosphate group.
  • Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, and/or recombinantly, to include one or more modified and/or non-natural nucleosides.
  • Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
  • a modified nucleobase is a modified guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methylinosine (mi l), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine
  • RNA molecules are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • the RNA molecules may be partially or fully (e.g., uniformly) modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide e.g., purine and/or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in a polynucleotide of the present disclosure are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, and/or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C and/or A+G+C.
  • a polynucleotide can be uniformly modified with pseudouridine, meaning that all uridine residues in the RNA sequence are replaced with pseudouridine.
  • the RNA molecule may include phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.
  • the RNA molecules may include one or more structural and/or chemical modifications and/or alterations which impart useful properties to the polynucleotide including, in some aspects, reduced degradation in the cell or organism and/or lack of a substantial induction of the innate immune response of a cell into which the RNA molecule is introduced.
  • a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted and/or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides.
  • the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”.
  • the same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”.
  • the dinucleotide “CO” has been inserted, resulting in a structural modification to the polynucleotide.
  • a modified RNA molecule, introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • a modified RNA molecule, introduced into a cell or organism may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure.
  • the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below.
  • the RNA may include a 5' cap comprising a 7’-methylguanosine, and the first 1 , 2, or 3 5' ribonucleotides may be methylated at the 2’ position of the ribose.
  • the RNA molecule described herein includes a 5' cap which generally “caps” the 5' end of the RNA and stabilizes the RNA molecule.
  • the 5' cap moiety is a natural 5' cap.
  • a “natural 5' cap” is defined as a cap that includes 7-methylguanosine connected to the 5' end of an mRNA molecule through a 5' to 5' triphosphate linkage.
  • a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose.
  • a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'0MeG). In some aspects, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G). In some aspects, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3'0 methylation at a ribose (m7(3'OMeG)).
  • the 5' cap may be incorporated during RNA synthesis (e.g., co- transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping).
  • co-transcriptional capping with a cap disclosed herein improves the capping efficiency of an RNA compared to co- transcriptional capping with an appropriate reference 5' cap.
  • improving capping efficiency may increase the translation efficiency and/or translation rate of an RNA and/or increase expression of an encoded polypeptide.
  • capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.
  • an RNA described herein comprises a 5' cap or a 5' cap analog, e.g., a Cap 0, a Cap 1 or a Cap 2.
  • a provided RNA does not have uncapped 5'-triphosphates.
  • the 5' end of the RNA is capped with a modified ribonucleotide.
  • the 5' cap moiety is a 5' cap analog.
  • an RNA may be capped with a 5' cap analog.
  • Cap structures include, but are not limited to, 7 mG(5')ppp(5')NipN2p (Cap 0), 7 rnG(5')ppp(5')Ni m pNp (Cap 1 ), and 7 rnG(5')ppp(5')Ni m pN2 m p (Cap 2).
  • 1 , 2, 3, 4, 5, or more of the foregoing cap structures can be excluded from the RNA molecules disclosed herein.
  • an RNA described herein comprises a Cap 0.
  • Cap 0 is a N7-methyl guanosine
  • a Cap 0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G).
  • a Cap 0 structure is connected to an RNA via a 5' to 5'-triphosphate linkage and is also referred to herein as m7G, m7Gppp, and/or m7G(5')ppp(5').
  • -A 5' cap may be methylated with the structure 7 mG(5')ppp(5')NipN2p (Cap 0) or a derivative thereof, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5'-end of an mRNA.
  • An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyl-transferase and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated Cap 0 structures.
  • VCE Vaccinia Virus Capping Enzyme
  • Cap 0 structures play an important role in maintaining the stability and translational efficacy of the RNA molecule. In the cell, the Cap 0 structure is essential for efficient translation of the mRNA that carries the cap.
  • an RNA described herein comprises a Cap 1 , e.g., as described herein.
  • the 5' cap of the RNA molecule may be further modified on the 2'0 position by a 2'-O-methyltransferase, which results in the generation of a Cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy.
  • a Cap 1 structure comprises a guanosine nucleoside methylated at the 7- position of guanine (m7G) and a 2'0 methylated first nucleotide in an RNA (2'OMeNi).
  • a Cap 1 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as rn7GpppN m , wherein N m denotes any nucleotide with a 2'0 methylation, 7 rnG(5')ppp(5')Ni m pNp, m7Gppp(2'OMeN-i), and/or m7G(5')ppp(5')(2'OMeNi).
  • Ni is chosen from A, C, G, or U. In some aspects, Ni is A. In some aspects, Ni is C. In some aspects, Ni is G. In some aspects, Ni is U.
  • a Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and one or more additional modifications, e.g., methylation on a ribose, and a 2'0 methylated first nucleotide in an RNA.
  • a Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine, a 3'0 methylation at a ribose (m7(3'OMeG)), and a 2'0 methylated first nucleotide in an RNA (2'0MeNi).
  • a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising A3U4G5.
  • N1 and N2 are each independently chosen from: A, C, G, or U.
  • N1 is A and N2 is G.
  • 1 , 2, 3, 4, 5, or more of the foregoing cap proximal sequences can be excluded from the 5' UTR of the RNA molecules disclosed herein.
  • the 5' UTR is functionally linked to the ORF, e.g., associated with the ORF such that it may exert a function, e.g., increasing, enhancing, stabilizing, and/or prolonging protein production from an RNA molecule and/or increasing protein expression and/or total protein production from an RNA molecule, compared to a reference RNA molecule comprising a reference 5' UTR or an RNA molecule lacking a 5' UTR.
  • 1 , 2, 3, 4, 5, or more of the foregoing 5' UTR functions can be excluded.
  • Exemplary 5' UTRs include 5' UTRs derived from Xenopus or human alpha globin or beta globin, human cytochrome b-245 a, hydroxysteroid (17b) dehydrogenase, Tobacco etch virus, the CMV immediate-early 1 (IE1 ) gene, TEV, HSP705', c-Jun, or a homolog, fragment, or variant of any of the foregoing.
  • IE1 immediate-early 1
  • the 5' UTR is a fragment, homolog or variant of a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract), the 5' UTR derived from ribosomal protein Large 32 (L32) gene, the 5' UTR derived from the 5' UTR of an hydroxysteroid (17p) dehydrogenase 4 gene (HSD17B4), or the 5' UTR derived from the 5' UTR of ATP5A1.
  • 5' UTRs are derived from SEQ ID NOs: 1 -1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application WQ201 3/143700, the disclosure of which is incorporated herein by reference in its entirety, or a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing sequences.
  • the sequence GGGAUCCUACC (SEQ ID NO: 12) may also be used.
  • 1 , 2, 3, 4, 5, or more of the foregoing 5' UTR sequences may be excluded from the RNA molecules disclosed herein.
  • an RNA disclosed herein comprises a hAg 5' UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a human alpha globin 5' UTR provided in SEQ ID NO: 14. In some aspects, an RNA disclosed herein comprises a hAg 5' UTR provided in SEQ ID NO: 14.
  • Natural or wild type 3' UTRs comprise stretches of adenosines and uridines. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Class III ARES do not contain an AUUUA motif. Most proteins binding to AREs are known to destabilize the molecule.
  • cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.
  • a 3' UTR may have one or more AU-rich sequences removed. Alternatively the AU-rich sequences may remain in the 3' UTR.
  • a 3' UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g., a poly-A tail.
  • a 3' UTR of the mRNA is not translated into an amino acid sequence.
  • an RNA disclosed herein comprises a 3' UTR comprising an F element and/or an I element.
  • a 3' UTR and/or a proximal sequence thereto comprises a restriction site.
  • a restriction site is a BamHI site.
  • a restriction site is a Xhol site.
  • a 3' UTR is a heterologous UTR, e.g., is a UTR found in nature associated with a different ORF.
  • a 3' UTR is a synthetic UTR, e.g., does not occur in nature.
  • the 3' UTR is functionally linked to the ORF, e.g., associated with the ORF such that it may exert a function, e.g., increasing, enhancing, stabilizing, and/or prolonging protein production from an RNA molecule and/or increasing protein expression and/or total protein production from an RNA molecule, compared to a reference RNA molecule comprising a reference 3' UTR or an RNA molecule lacking a 3' UTR.
  • 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR functions may be excluded.
  • Exemplary 3' UTRs include 3' UTRs derived from an albumin gene, an a-globin gene, a [3-globin gene, a ribosomal protein gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (1 ) gene, or from a homolog, fragment, or variant of a 3' UTR of a gene comprising an albumin gene, an a-globin gene, a [3- globin gene, a ribosomal protein gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and/or a collagen alpha gene, such as a collagen alpha 1 (1 ) gene according to SEQ ID NOs: 1369-1390 of the patent application WO2013/143700, the disclosure of which is incorporated herein by reference in its entirety, or a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of
  • the 3' UTR comprises a sequence of a transcript including NM-000661.4, NM_001024921 .2, NM_000967.3, NM_001033853.1 , NMJD00968.3, NM_000969.3, NM_001024662.1 , NM_000970.3, NM_000971 .3, NMJD00972.2, NM_000975.3, NM_001 199802.1 , NM_000976.3, NM_ 000977.3, NM_033251 .2, NMJ 01243130.1 , NM_001243131 , NM_000978.3, NM_000979.3, NM_001270490.1 , NMJD00980.3, NM_000981.3, NM_000982.3, NM_000983.3, NM_000984.5, NM_000985.4, NM_001035006.2, NM_001 199340.1 , NM_001 199341 .1 , NMJD01
  • the 3' UTR comprises a sequence from the 3' UTR region of a gene encoding a ribosomal protein, e.g., ribosomal protein L9 (RPL9), ribosomal protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosomal protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomal protein L7a (RPL7A), ribosomal protein L1 1 (RPL1 1 ), ribosomal protein L12 (RPL12), ribosomal protein L13 (RPL13), ribosomal protein L23 (RPL23), ribosomal protein L18 (RPL18), ribosomal protein L18a (RPL18A), ribosomal protein L19 (RPL19), ribosomal protein L21 (RPL21 ), ribosomal protein L22 (RPL9 (
  • 5' UTRs that are heterologous and/or synthetic may be used with any desired 3' UTR sequence, and vice versa.
  • a heterologous 5' UTR may be used with a synthetic and/or heterologous 3' UTR.
  • an RNA disclosed herein comprises a 3' UTR having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a 3' UTR provided in SEQ ID NO: 15. In some aspects, an RNA disclosed herein comprises a 3' UTR provided in SEQ ID NO: 15.
  • an RNA disclosed herein comprises a 3' UTR having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a 3' UTR provided in any one of the SEQ ID NOs: 3 to 8 as disclosed in WO 2016/091391 A1 , which is incorporated by reference.
  • 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR sequences may be excluded from the RNA molecules disclosed herein.
  • the 5' and 3' UTRs may be operably linked to an open reading frame (ORF), which may be a sequence of codons that is capable of being translated into a polypeptide of interest.
  • An open reading frame may be a sequence of several DNA or RNA nucleotide triplets, which may be translated into a peptide or protein.
  • An ORF may begin with a start codon, e.g., a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG or AUG), at its 5' end and a subsequent region, which usually exhibits a length that is a multiple of 3 nucleotides.
  • An open reading frame may terminate with at least one stop codon, including but not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof.
  • an open reading frame may terminate with one, two, three, four or more stop codons, including but not limited to TAATAA, TAATAG, TAATGA, TAGTGA, TAGTAA, TAGTAG, TGATGA), TGATAG, TGATAA or UAAUAA, UAAUAG, UAAUGA, UAGUGA, UAGUAA, UAGUAG, UGAUGA, UGAUAG, UGAUAA, or any combination thereof.
  • an open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, e.g., in a vector or an mRNA.
  • An open reading frame may also be termed “(protein) coding region” or “coding sequence”.
  • the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames.
  • the ORF encodes a non-structural viral gene.
  • the ORF further includes one or more subgenomic promoters.
  • the RNA molecule includes a subgenomic promoter operably linked to the ORF.
  • a first RNA molecule does not include an ORF encoding any polypeptide of interest, whereas a second RNA molecule includes an ORF encoding a polypeptide of interest.
  • the first RNA molecule does not include a subgenomic promoter.
  • an RNA molecule comprising at least one open reading frame encoding a N. meningitidis polypeptide.
  • an RNA molecule comprises at least one open reading frame encoding a fHbp polypeptide.
  • RNA molecules described herein may include a gene of interest.
  • the gene of interest encodes a polypeptide of interest.
  • polypeptides of interest include, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties, those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery, or combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing polypeptides of interest may be excluded.
  • sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GENBANK®.
  • the RNA molecules include a coding region for a gene of interest.
  • a gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof.
  • an antigenic polypeptide comprises one epitope from an antigen.
  • an antigenic polypeptide comprises a plurality of distinct epitopes from an antigen.
  • an antigenic polypeptide comprising a plurality of distinct epitopes from an antigen is polyepitopic.
  • an antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide.
  • 1 , 2, 3, 4, 5, or more of the foregoing antigenic polypeptides may be excluded.
  • an antigen may refer to a substance, which is capable of being recognized by the immune system, e.g., by the adaptive immune system, and which is capable of eliciting an antigen-specific immune response, e.g., by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • An antigen may be or may comprise a peptide or protein, which may be presented by the MHC to T cells.
  • An antigen may be the product of translation of a provided nucleic acid molecule, e.g., an RNA molecule comprising at least one coding sequence as described herein.
  • fragments, variants and derivatives of an antigen such as a peptide or a protein, comprising at least one epitope are understood as antigens.
  • an RNA encoding a gene of interest is expressed in cells of a subject treated to provide a gene of interest, e.g., an antigen.
  • the RNA is transiently expressed in cells of the subject.
  • expression of a gene of interest, e.g., an antigen is at the cell surface.
  • a gene of interest, e.g., an antigen is expressed and presented in the context of MHC.
  • expression of a gene of interest, e.g., an antigen is into the extracellular space, e.g., the antigen is secreted.
  • the RNA molecules include a coding region for a gene of interest, e.g., an antigen. In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen, that is derived from a pathogen associated with an infectious disease. In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen, that is derived from N. meningitidis.
  • the RNA molecule encodes a fHbp comprising the sequence of any one of SEQ ID NOs: 3, 5, 7, and 9, or a fragment or variant thereof.
  • RNA molecules disclosed herein comprise a poly-adenylate (poly-A) sequence, e.g., as described herein.
  • a poly-A sequence is situated downstream of a 3' UTR, e.g., adjacent to a 3' UTR.
  • a “poly-A tail” or “poly-A sequence” refers to a stretch of consecutive adenine residues, e.g., of up to or up to about 400 adenosine nucleotides, e.g., from or from about 20 to about 400, preferably from or from about 50 to about 400, more preferably from or from about 50 to about 300, even more preferably from or from about 50 to about 250, most preferably from or from about 60 to about 250 adenosine nucleotides, which may be attached to the 3' end of the RNA molecule.
  • Poly-A sequences are known to those of skill in the art and may follow the 3' UTR in the RNA molecules described herein.
  • the poly-A tail may increase the stability, half-life, and/or translational efficiency of the RNA molecule.
  • 3'-end processing is a nuclear co-transcriptional process that promotes transport of mRNAs from the nucleus to the cytoplasm and affects the stability and the translation of mRNAs. Formation of this 3' end occurs in a two-step reaction directed by the cleavage/polyadenylation machinery and depends on the presence of two sequence elements in mRNA precursors (pre-m RNAs); a hexanucleotide polyadenylation signal and a downstream G/U-rich sequence. In a first step, pre-m RNAs are cleaved between these two elements to a free 3' hydroxyl. In a second step, the newly formed 3' end is extended by polyadenylation or addition of a poly-A sequence.
  • Polyadenylation refers to the addition of a poly-A sequence to an RNA molecule, e.g., to a premature mRNA. Polyadenylation may be induced by a so-called polyadenylation signal. This signal may be located within a stretch of nucleotides close to or at the 3'-end of an RNA molecule to be polyadenylated. A polyadenylation signal may also be comprised by the 3' UTR of the artificial nucleic acid molecule.
  • a polyadenylation signal typically comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA, though other sequences, preferably hexamer sequences, are also conceivable.
  • Polyadenylation typically occurs during processing of a pre-mRNA (also called premature-mRNA).
  • RNA maturation comprises the step of polyadenylation.
  • Poly-A tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly-T tract into the DNA template or by post-transcriptional addition using poly-A polymerase.
  • the term may relate to polyadenylation of RNA as a cellular process or to polyadenylation carried out by enzymatic reaction in vitro with a suitable enzyme, such as E. coll poly-A polymerase, or by chemical synthesis.
  • Such a random sequence may be at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the poly-A sequence is flanked on the 3' side by a poly-C sequence and/or a histone stem-loop sequence.
  • the poly-A sequence can be flanked on the 5' side by a 3' UTR element derived from, e.g., a human albumin or globin gene.
  • the poly-A sequence is separated from the polyadenylation signal by a nucleotide sequence comprising or consisting of at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 1 15, 120, 125, 130, 135, 140, 145, or 150 nucleotides, wherein the nucleotide sequence does preferably not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides.
  • N A or U
  • AA(U/T)AAA or A(U/T)(U/T)AAA may be recognized by most animal and bacterial cellsystems, for example, by the polyadenylation-factors, such as cleavage/polyadenylation specificity factor (CPSF) cooperating with CstF, PAP, PAB2, CFI and/or CFII.
  • CPSF cleavage/polyadenylation specificity factor
  • the polyadenylation signal (e.g., the consensus sequence NNUANA) is located less than or less than about 50 nucleotides, e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, downstream of the 3'-end of the 3' UTR element as defined herein such that transcription of an RNA molecule will result in a premature- RNA containing the polyadenylation signal downstream of its 3' UTR and subsequent attachment of a poly-A sequence to the premature-RNA.
  • a resulting RNA may comprise a 3' UTR, which comprises at least one poly-A sequence, and wherein the 3' UTR is followed by an additional poly-A sequence.
  • the poly-A sequence may be of any length.
  • the poly-A tail may be 5 to 300 nucleotides in length.
  • the RNA molecule includes a poly-A tail that comprises, consists essentially of, or consists of a sequence of or of about 25 to about 400 adenosine nucleotides, a sequence of or of about 50 to about 400 adenosine nucleotides, a sequence of or of about 50 to about 300 adenosine nucleotides, a sequence of or of about 50 to about 250 adenosine nucleotides, a sequence of or of about 60 to about 250 adenosine nucleotides, or a sequence of or of about 40 to about 100 adenosine nucleotides.
  • the poly-A tail comprises, consists essentially of, or consists of at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,
  • consists essentially of means that most nucleotides in the poly-A sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A sequence are adenosine nucleotides, but permits remaining nucleotides to be nucleotides other than adenosine nucleotides, such as uridine, guanosine, and/or cytosine.
  • “consists of” means that all nucleotides in the poly-A sequence, i.e., 100% by number of nucleotides in the poly-A sequence, are adenosine nucleotides.
  • the RNA molecule includes or includes about 80 consecutive adenosine residues.
  • Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation.
  • a poly-A tail may be located within an RNA molecule or other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, e.g., an mRNA, e.g., by transcription of the vector.
  • the RNA molecule may not include a poly-A tail.
  • a DNA encoding a poly-A tail disclosed herein comprises a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 16.
  • the DNA encoding the poly-A tail comprises a sequence of SEQ ID NO: 16.
  • an RNA disclosed herein comprises a poly-A tail comprising a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to any of SEQ ID NOs: 17 to 20.
  • the poly-A tail comprises a sequence of any of SEQ ID NOs: 17 to 20 +/- 2 adenosine (A) nucleotides. In one aspect, the poly-A tail comprises a sequence of any of SEQ ID NOs: 17 to 20 +/- 1 adenosine (A) nucleotides. In one aspect, the poly- A tail comprises a sequence of any of SEQ ID NOs: 17 to 20.
  • the poly-A tail comprises a sequence of any of SEQ ID NOs: 21 and 22.
  • 1 , 2, 3, 4, 5, or more of the foregoing poly-A sequences may be excluded from the RNA molecules disclosed herein.
  • the RNA molecules additionally include a chain terminating nucleoside.
  • a chain terminating nucleoside may include those nucleosides deoxygenated at the 2’ and/or 3' positions of their sugar group.
  • Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxycytosine, 3' deoxyguanosine, 3' deoxythymine, and 2', 3' dideoxynucleosides, such as 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxycytosine, 2', 3' dideoxyguanosine, and 2', 3' dideoxythymine.
  • 1 , 2, 3, 4, 5, or more of the foregoing chain terminating nucleosides may be excluded from the RNA molecules disclosed herein.
  • incorporation of a chain terminating nucleotide into an mRNA, for example at the 3'-terminus, may result in stabilization of the mRNA, as described, for example, in International Patent Publication No. WO 2013/103659.
  • a stem loop may include 4, 5, 6,
  • a stem loop may be located in any region of an mRNA.
  • a stem loop may be located in, before, or after an untranslated region (a 5' UTR or a 3' UTR), a coding region, or a poly-A sequence or tail.
  • a stem loop may affect one or more function(s) of an mRNA, such as initiation of translation, translation efficiency, and/or transcriptional termination.
  • histone stem-loop sequences may be histone stem-loop sequences disclosed in WO 2012/019780, the disclosure of which is incorporated herein by reference in its entirety.
  • Other non-limiting examples of histone stem loop structures and nucleic acid sequences encoding such structures can be found in, e.g., WO 2016/091391 , the disclosure of which is incorporated by reference herein in its entirety.
  • the combination of a poly-A sequence or polyadenylation signal and at least one histone stem-loop acts synergistically to increase the protein expression beyond the level observed with either of the individual elements.
  • the synergistic effect of the combination of poly-A and at least one histone stem-loop does not depend on the order of the elements and/or the length of the poly-A sequence.
  • the RNA does not comprise a histone downstream element (HDE).
  • HDE histone downstream element
  • An HDE includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.
  • the histone stem-loop is generally derived from histone genes, and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure.
  • the unpaired loop region is typically unable to base pair with either of the stem loop elements.
  • Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and/or base composition of the paired region.
  • wobble base pairing non- Watson-Crick base pairing
  • the at least one histone stemloop sequence comprises a length of 15 to 45 nucleotides.
  • the RNA molecules include (e.g., within the 3' UTR) a poly(C) sequence.
  • the poly-C sequences has at least, at most, exactly, or between (inclusive or exclusive) any two of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, or 200 cytidines.
  • the poly-C sequences has or has about 30 cytidines.
  • the RNA molecules include an internal ribosome entry site (IRES) sequence or IRES-motif.
  • IRES sequence separates ORFs, e.g., if the RNA encodes two or more peptides or proteins. An IRES-sequence may therefore be useful if the RNA molecule is a bi- or multicistronic nucleic acid molecule.
  • the RNA does not comprise an intron. In some aspects, the RNA may instead or additionally include a microRNA binding site.
  • RNA molecules including a combination of the elements disclosed herein can include, without limitation, in 5'-to-3'-direction, the following:
  • the RNA molecule may be an saRNA.
  • Self-amplifying RNA refers to RNA with the ability to replicate itself.
  • Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest.
  • a self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNA molecules.
  • RNA molecules may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, and/or may be transcribed to provide further transcripts with the same sense as the delivered RNA that are translated to provide in situ expression of the antigen.
  • an encoded gene of interest e.g., a viral antigen
  • the overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNA molecules, and consequently, the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.
  • the self-amplifying RNA includes at least one or more genes including any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins, or combination thereof. In some aspects, 1 , 2, 3, or more of the foregoing genes may be excluded from the self-amplifying RNA molecules disclosed herein. In some aspects, the self-amplifying RNA may also include 5'- and 3'-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and/or may be under the control of an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • a self-amplifying RNA molecule described herein encodes (i) an RNA-dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a bacterial antigen.
  • the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus proteins nsP1 , nsP2, nsP3, nsP4, or any combination thereof.
  • 1 , 2, 3, or more of the foregoing alphavirus proteins may be excluded from the RNA molecules disclosed herein.
  • the self-amplifying RNA molecule may have two open reading frames.
  • the first (5') open reading frame may encode a replicase; the second (3') open reading frame may encode a polypeptide comprising an antigen of interest.
  • the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides.
  • the saRNA molecule further includes (1 ) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence.
  • the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.
  • the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence.
  • the polypeptides generated from the selfamplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.
  • the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes.
  • the selfamplifying RNA described herein may encode epitopes capable of eliciting either a helper T cell response or a cytotoxic T cell response or both.
  • the RNA disclosed herein is produced by in vitro transcription or chemical synthesis.
  • transcription relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.
  • transcription comprises “in vitro transcription” or “IVT,” which refers to the process whereby transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for use in various applications, including, e.g., production of protein or polypeptides.
  • IVT in vitro transcription
  • the methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R. 1972. In vitro transcription, Ann Rev Biochem, 41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001 . In vitro Transcription, Current Protocols in Cell Biology, 2:1 1 .6:1 1 .6.1 -1 1 .6.17; Beckert, B. And Masquida, B. 2010.
  • Cloning vectors may be applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term “vector.” According to specific aspects, the RNA used is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template.
  • IVT-RNA in vitro transcribed RNA
  • Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template, and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses, Methods 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012, each incorporated herein by reference).
  • plasmid DNA and polymerase chain reaction amplification see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template, and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P.
  • the promoter for controlling transcription may be any promoter for any RNA polymerase.
  • RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • Synthetic IVT RNA products may be translated in vitro or introduced directly into cells, where they may be translated.
  • expression or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.
  • Such synthetic RNA products include but are not limited to, e.g., mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, and the like.
  • An IVT reaction typically utilizes a DNA template (e.g., a linear DNA template) as described and/or utilized herein, ribonucleotides (e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase.
  • a DNA template e.g., a linear DNA template
  • ribonucleotides e.g., non-modified ribonucleotide triphosphates or modified ribonucleotide triphosphates
  • an mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.
  • an RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template.
  • the promoter for controlling transcription may be any promoter for any RNA polymerase.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • starting material for IVT may include linearized DNA template, nucleotides, Rnase inhibitor, pyrophosphatase, and/or a polymerase (e.g., a T7 RNA polymerase).
  • the nucleotides may be manufactured in house, may be obtained from a supplier, or may be synthesized.
  • the nucleotides may be, but are not limited to, those described herein including natural and unnatural (modified) nucleotides.
  • RNA polymerases or variants may be used, including, but not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides.
  • a phage RNA polymerase e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides.
  • 1 , 2, 3, 4, 5, or more of the foregoing RNA polymerases may be excluded from.
  • the IVT process is conducted in a bioreactor.
  • the bioreactor may comprise a mixer.
  • nucleotides may be added into the bioreactor throughout the IVT process.
  • one or more post-IVT agents are added into the IVT mixture comprising RNA in the bioreactor after the IVT process.
  • exemplary post-IVT agents may include DNAse I configured to digest the linearized DNA template and/or proteinase K configured to digest DNAse I and T7 RNA polymerase.
  • the post-IVT agents are incubated with the mixture in the bioreactor after IVT.
  • the bioreactor may contain at least, at most, exactly, or between (inclusive or exclusive) any two of 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150 ,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500 or more liters IVT mixture.
  • the IVT mixture may have an RNA concentration that is or is not at least, at most, exactly, or between (inclusive or exclusive) any two of 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg/mL or more RNA.
  • the IVT mixture may include residual spermidine, residual DNA, residual proteins, peptides, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg 2+ , Na + , Ca 2+ ), RNA fragments, residual nucleotides, free phosphates, or any combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing can be excluded from the IVT mixture.
  • Isolation and/or purification of the nucleic acids described herein may include, but is not limited to, phenol/chloroform extraction and/or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid clean-up, quality assurance and quality control.
  • alcohol ethanol, isopropanol
  • purification procedures include AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis.
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis.
  • Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing purification may be excluded.
  • purified when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant.
  • a “contaminant” is any substance that makes another unfit, impure or inferior.
  • a purified nucleic acid e.g., DNA and RNA
  • a purified nucleic acid is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment and/or purification method.
  • the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid).
  • the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid).
  • a composition such as a pharmaceutical composition comprises more than one individual LNP formulation.
  • Respective pharmaceutical compositions are referred to as mixed LNP formulations.
  • Mixed LNP formulations according to the invention are obtainable by forming, separately, individual LNP formulations, as described above, followed by a step of mixing of the individual LNP formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing LNPs is obtainable. Individual LNP populations may be together in one container, comprising a mixed population of individual LNP formulations.
  • a “polymeric material,” as used herein, is given its ordinary meaning, e.g., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some aspects, such repeat units may all be identical; alternatively, in some cases, there may be more than one type of repeat unit present within the polymeric material.
  • a polymeric material is biologically derived, e.g., a biopolymer such as a protein.
  • additional moieties may also be present in the polymeric material, for example targeting moieties such as those described herein.
  • a polymeric material for use in accordance with the present disclosure is biocompatible.
  • Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations.
  • a biocompatible material is biodegradable, e.g., is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • a polymeric material may be or comprise protamine or polyalkylene imine, in particular protamine.
  • protamine is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (e.g., fish).
  • protamine is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.
  • protamine refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and/or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides that are artificial and designed for specific purposes and cannot be isolated from native or biological sources.
  • Cationic materials contemplated for use herein include those which are able to electrostatically bind nucleic acid.
  • cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid may be associated, e.g., by forming complexes with the nucleic acid and/or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and/or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.
  • lipid refers to a group of organic compounds that are characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids as well as sterol-containing metabolites such as cholesterol, and prenol lipids.
  • fatty acids include, but are not limited to, fatty esters and fatty amides.
  • glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine).
  • sphingolipids include, but are not limited to, ceramides phosphosphingolipids (e.g., sphingomyelins, phosphocholine), and glycosphingolipids (e.g., cerebrosides, gangliosides).
  • sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives. In some aspects, 1 , 2, 3, 4, 5, or more of the lipids may be excluded from the LNPs of the present disclosure.
  • Cationic or cationically ionizable lipids or lipid-like materials refer to a lipid or lipid-like material capable of being positively charged and able to electrostatically bind nucleic acid.
  • a “cationic lipid” or “cationic lipid-like material” refers to a lipid or lipid-like material having a net positive charge.
  • Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction.
  • cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl, or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • Exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
  • Cationic lipids may encapsulate negatively charged RNA.
  • a cationic lipid may comprise from or from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle.
  • DORIE 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1 - propanamium trifluoroacetate
  • DOSPA 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinolenyloxy-N,N-dimethylaminopropane
  • DOGS dioctadecylamidoglycyl spermine
  • CLinDMA 2-[5'- (cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'- o
  • the cationic lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some aspects, R 8 and R 9 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some aspects, w has a mean value ranging from 43 to 53. In other aspects, the average w is or is about 45. In other different embodiments, the average w is or is about 49.
  • the lipid nanoparticles comprise a polymer conjugated lipid.
  • the lipid nanoparticle comprises 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159), having the formula:
  • the PEG-lipid is or is not present in the LNP in an amount from or from about 1 to about 10 mole percent (mol %) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %), relative to the total lipid content of the nanoparticle.
  • the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulations.
  • Representative neutral lipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, ceramides, sphingomyelins, dihydro-sphingomyelins, cephalins, and cerebrosides.
  • Exemplary phospholipids include, for example, phosphatidylcholines, e.g., diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoy
  • the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), having the formula:
  • the LNPs comprise a neutral lipid
  • the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and/or SM.
  • 1 , 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure.
  • the steroid or steroid analogue is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing steroid or steroid analogues may be excluded from the LNPs of the present disclosure.
  • the steroid or steroid analogue is cholesterol.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing cholesterol derivatives may be excluded from the LNPs of the present disclosure.
  • the cholesterol has the formula:
  • the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some aspects, the molar ratio of the cationic lipid to the neutral lipid ranges from or from about 2:1 to about 8:1 , or from or from about 10:0 to about 1 :9, about 4:1 to about 1 :2, or about 3:1 to about 1 :1 .
  • the non-cationic lipid may comprise from or from about 0 mol % to about 90 mol %, from or from about 0 mol % to about 80 mol %, from or from about 0 mol % to about 70 mol %, from or from about 0 mol % to about 60 mol %, or from or from about 0 mol % to about 50 mol %, of the total lipid present in the particle.
  • neutral lipid e.g., one or more phospholipids and/or cholesterol
  • the non-cationic lipid e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol)
  • RNA molecule described herein may be analyzed and characterized using various methods. Analysis may be performed before and/or after capping. Alternatively, analysis may be performed before and/or after poly-A capture-based affinity purification. In another aspect, analysis may be performed before and/or after additional purification steps, e.g., anion exchange chromatography and the like.
  • RNA template quality may be determined using a Bioanalyzer chip-based electrophoresis system. In other aspects, RNA template purity is analyzed using analytical reverse phase HPLC. Capping efficiency may be analyzed using, e.g., total nuclease digestion followed by MS/MS quantitation of the dinucleotide cap species vs. uncapped GTP species.
  • In vitro efficacy may be analyzed by, e.g., transfecting an RNA molecule into a human cell line.
  • Protein expression of the polypeptide of interest may be quantified using methods such as ELISA and/or flow cytometry.
  • Immunogenicity may be analyzed by, e.g., transfecting RNA molecules into cell lines that indicate innate immune stimulation, e.g., PBMCs.
  • Cytokine induction may be analyzed using, e.g., methods such as ELISA to quantify a cytokine, e.g., Interferon- a.
  • Biodistribution may be analyzed by, e.g., bioluminescence measurements. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing analytic methods may be excluded.
  • an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, elevated expression of a gene of interest (e.g., an antigen); increased duration of expression (e.g., prolonged expression) of a gene of interest (e.g., an antigen); elevated expression and increased duration of expression (e.g., prolonged expression) of a gene of interest (e.g., an antigen); decreased interaction with IFIT 1 of an RNA polynucleotide; and/or increased translation of an RNA polynucleotide; is observed relative to an appropriate reference.
  • 1 , 2, 3, 4, 5, or more of the foregoing characteristics may not be observed after administration of a composition or medical preparation comprising an RNA molecule of the present disclosure.
  • a reference comprises an organism administered an otherwise similar RNA polynucleotide without a m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2 cap. In some aspects, a reference comprises an organism administered an otherwise similar RNA polynucleotide without a cap proximal sequence disclosed herein. In some aspects, a reference comprises an organism administered an otherwise similar RNA polynucleotide with a self-hybridizing sequence.
  • elevated expression is determined at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some aspects, elevated expression is determined at least 24 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some aspects, elevated expression is determined at least 48 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some aspects, elevated expression is determined at least 72 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.
  • elevated expression is determined at least 96 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some aspects, elevated expression is determined at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.
  • expression of a gene of interest is elevated or elevated about 2-fold to about 50-fold.
  • expression of a gene of interest is elevated or elevated about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10- fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50- fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40- fold to about 50-fold, or about 45-fold to about 50-fold.
  • expression of a gene of interest is or is not elevated at least, at most, exactly, or between (inclusive or exclusive) of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21 -fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30- fold, 31 -fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41 -fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, or 50-fold, or any range or value derivable therein.
  • elevated expression (e.g., increased duration of expression) of a gene of interest persists for at least, at most, exactly, or between (inclusive or exclusive) of 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours after administration of a composition or a medical preparation comprising an RNA polynucleotide.
  • elevated expression of a gene of interest persists for at least 24 hours after administration.
  • elevated expression of a gene of interest persists for at least 48 hours after administration.
  • elevated expression of a gene of interest (e.g., an antigen) persists for at least 72 hours after administration.
  • elevated expression of a gene of interest persists for at least 96 hours after administration. In some aspects, elevated expression of a gene of interest (e.g., an antigen) persists for at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.
  • the disclosure concerns evoking or inducing an immune response in a subject against a N. meningitidis protein, e.g., a wild type or variant fHbp.
  • the immune response may protect against and/or treat a subject exposed to, having, suspected of having, and/or at risk of developing an infection or related disease, particularly those related to N. meningitidis.
  • the immunogenic compositions of the disclosure are to prevent N. meningitidis infections by inoculation or vaccination of a subject.
  • the immunogenic compositions immunize the subject against N. meningitidis up to 1 year (e.g., for a single N. meningitidis season).
  • the immunogenic compositions immunize the subject against N. meningitidis for up to 2 years.
  • the immunogenic compositions immunize the subject against N. meningitidis for more than 2 years.
  • the immunogenic compositions immunize the subject against N. meningitidis for more than 3 years.
  • the immunogenic compositions immunize the subject against N. meningitidisfor more than 4 years.
  • the immunogenic compositions immunize the subject against N. meningitidis for 5-10 years, or longer.
  • the present disclosure includes the implementation of serological assays to evaluate whether and to what extent an immune response is induced or evoked by compositions of the disclosure.
  • immunoassays There are many types of immunoassays that may be implemented. Immunoassays encompassed by the present disclosure include, but are not limited to, those described in U.S. Patent 4,367,1 10 (double monoclonal antibody sandwich assay) and U.S. Patent 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
  • Immunoassays generally are binding assays.
  • the immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Immunohistochemical detection using tissue sections is also particularly useful.
  • antibodies or antigens are immobilized on a selected surface, such as a well in a polystyrene microtiter plate, dipstick, or column support. Then, a test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody may be detected.
  • Detection is generally achieved by the addition of another antibody, specific for the desired antigen or antibody, that is linked to a detectable label. This type of ELISA is known as a “sandwich ELISA.” Detection also may be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • Competition ELISAs are also possible implementations in which test samples compete for binding with known amounts of labeled antigens or antibodies.
  • the amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating, or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes.
  • Antigen or antibodies may also be linked to a solid support, such as in the form of plate, beads, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody.
  • a solid support such as in the form of plate, beads, dipstick, membrane, or column matrix
  • the sample to be analyzed is applied to the immobilized antigen or antibody.
  • a plate with either antigen or antibody one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely-adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein, and solutions of milk powder.
  • BSA bovine serum albumin
  • casein casein
  • solutions of milk powder The coating allows for blocking of nonspecific adsorption sites on
  • N. meningitidis polypeptides, proteins, and/or peptides in a variety of ways, including the detection of the presence of N. meningitidis to diagnose an infection.
  • a method of detecting the presence of infection involves the steps of obtaining a sample suspected of being infected by one or more N. meningitidis strains, such as a sample taken from an individual, for example, from one’s blood, saliva, tissues, bone, muscle, cartilage, and/or skin.
  • diagnostic assays utilizing the polypeptides, proteins, and/or peptides of the present disclosure may be carried out to detect the presence of N. meningitidis, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassay, western blot analysis and ELISA assays.
  • a method of diagnosing an infection wherein a sample suspected of being infected with, previously infected with, or infected with, N. meningitidis has added to it the polypeptide, protein, or peptide, in accordance with the present disclosure, and N. meningitidis is indicated by antibody binding to the polypeptides, proteins, and/or peptides, or polypeptides, proteins, and/or peptides binding to the antibodies in the sample.
  • Also contemplated is a method of testing a sample suspected of being infected with, previously infected with, or infected with, N. meningitidis has added to it the polypeptide, protein, or peptide, in accordance with the present disclosure, and N. meningitidis is indicated by antibody binding to the polypeptides, proteins, and/or peptides, or polypeptides, proteins, and/or peptides binding to the antibodies in the sample.
  • RNA molecules encoding N. meningitidis polypeptides, proteins, and/or peptides in accordance with the disclosure may be used for to treat, prevent, and/or reduce the severity of illness from infection due to N. meningitidis infection (e.g., active or passive immunization) and/or for use as research tools.
  • N. meningitidis infection e.g., active or passive immunization
  • any of the above-described polypeptides, proteins, and/or peptides may be labeled directly with a detectable label for identification and quantification of N. meningitidis.
  • Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads.
  • Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).
  • RNA molecules encoding N. meningitidis polypeptides, RNA-LNPs and compositions thereof confer protective immunity to a subject.
  • Protective immunity refers to a body’s ability to mount a specific immune response that protects the subject from developing a particular disease or condition that involves the agent against which there is an immune response.
  • An immunogenically effective amount is capable of conferring protective immunity to the subject.
  • the RNA molecules encoding N. meningitidis polypeptides, RNA-LNPs and compositions thereof of the present disclosure may be used to induce a balanced immune response against N. meningitidis comprising both cellular and humoral immunity, without many of the risks associated with attenuated bacterial vaccination.
  • a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T- lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
  • T- lymphocytes e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
  • immune response refers to the development of a humoral (antibody mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against an antigen. Such a response may be an active response or a passive response.
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to activate antigen-specific CD4 (+) T helper cells and/or CD8 (+) cytotoxic T cells.
  • the response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils and/or other components of innate immunity.
  • 1 , 2, 3, 4, 5, or more of the foregoing cell types may be excluded from an immune response.
  • active immunity refers to any immunity conferred upon a subject from the production of antibodies in response to the presence of an of an antigen, e.g., a N. meningitidis polypeptide encoded by an RNA molecule of the present disclosure.
  • passive immunity includes, but is not limited to, administration of activated immune effectors including cellular mediators and/or protein mediators (e.g., monoclonal and/or polyclonal antibodies) of an immune response.
  • a monoclonal or polyclonal antibody composition may be used in passive immunization to treat, prevent, and/or reduce the severity of illness caused by infection by organisms that carry the antigen recognized by the antibody.
  • An antibody composition may include antibodies that bind to a variety of antigens that may in turn be associated with various organisms.
  • the antibody component may be a polyclonal antiserum.
  • the antibody or antibodies are affinity purified from an animal or second subject that has been challenged with an antigen(s).
  • an antibody mixture may be used, which is a mixture of monoclonal and/or polyclonal antibodies to antigens present in the same, related, or different microbes or organisms, such as bacteria, including but not limited to N. meningitidis.
  • Passive immunity may be imparted to a patient or subject by administering to the patient immunoglobulins (Ig) and/or other immune factors obtained from a donor or other non-patient source having a known immunoreactivity.
  • an immunogenic composition of the present disclosure may be administered to a subject who then acts as a source or donor for globulin, produced in response to challenge with the immunogenic composition (“hyperimmune globulin”), that contains antibodies directed against a N. meningitidis or other organism.
  • hyperimmune globulin that contains antibodies directed against a N. meningitidis or other organism.
  • a subject thus treated would donate plasma from which hyperimmune globulin would then be obtained, via conventional plasma-fractionation methodology, and administered to another subject in order to impart resistance against and/or to treat N. meningitidis infection.
  • epitopes and “antigenic determinant” are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize.
  • B-cell epitopes may be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols (1996).
  • Antibodies that recognize the same epitope may be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • T cells recognize continuous epitopes of or of about nine amino acids for CD8 cells or of or of about 13-15 amino acids for CD4 cells.
  • T cells that recognize the epitope may be identified by in vitro assays that measure antigen-dependent proliferation, as determined by 3 H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) and/or by cytokine secretion.
  • the presence of a cell-mediated immunological response may be determined by proliferation assays (CD4 (+) T cells) and/or CTL (cytotoxic T lymphocyte) assays.
  • proliferation assays CD4 (+) T cells
  • CTL cytotoxic T lymphocyte
  • the relative contributions of humoral and cellular responses to the protective and/or therapeutic effect of an immunogenic composition may be distinguished by separately isolating IgG and T cells from an immunized syngeneic animal and measuring protective and/or therapeutic effect in a second subject.
  • antibody or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal or recipient, which proteins include IgG, IgD, IgE, IgA, IgM and related proteins. Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent.
  • immunological agent or “immunogen” or “antigen” are used interchangeably to describe a molecule capable of inducing an immunological response against itself on administration to a recipient, either alone, in conjunction with an adjuvant, and/or presented on a display vehicle.
  • RNA molecules and/or RNA-LNPs disclosed herein may be administered in a pharmaceutical composition or a medicament and may be administered in the form of any suitable pharmaceutical composition.
  • a pharmaceutical composition is for therapeutic and/or prophylactic treatment.
  • the disclosure relates to a composition for administration to a host.
  • the host is a human. In other aspects, the host is a non-human.
  • Formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing an active ingredient (e.g., RNA molecules and/or RNA-LNPs) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • an active ingredient e.g., RNA molecules and/or RNA-LNPs
  • a pharmaceutical composition or formulation in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1 % and 100%, e.g., between 0.5 and 50%, between 1 -30%, between 5-80%, at least 80% (w/w), or at least, at most, exactly, or between (inclusive or exclusive) any two of 0.1 %, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (w/w) active ingredient.
  • 0.1 % and 100% e.g., between 0.5 and 50%, between 1 -30%, between 5-80%, at least 80% (w/w), or at least, at most, exactly, or between (inclusive or exclusive) any two of 0.1 %, 0.15%,
  • RNA molecules and/or RNA-LNPs disclosed herein may be administered in a pharmaceutical composition which may be formulated into preparations in solid, semi-solid, liquid, lyophilized, frozen, and/or gaseous forms.
  • an RNA molecule and/or RNA-LNPs disclosed herein may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers, salts, buffers, preservatives, and optionally other therapeutic agents.
  • a pharmaceutical composition disclosed herein comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • pharmaceutical compositions do not include an adjuvant (e.g., they are adjuvant free).
  • excipient refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient.
  • excipients include without limitation, carriers, diluents (e.g., solvents, dispersion media, and/or other liquid vehicles, dispersion or suspension aids), granulating and/or dispersing agents, surface active agents, isotonic agents, thickening and/or emulsifying agents, preservatives, binders, lubricants and/or oil, coloring, sweetening and/or flavoring agents, stabilizers, antioxidants, antimicrobial and/or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelants, cryoprotectants, and/or bulking agents.
  • 1 , 2, 3, 4, 5, or more of the foregoing excipients may be excluded from the pharmaceutical compositions disclosed herein.
  • carrier refers to a component which may be natural, synthetic, organic, or inorganic, in which the active component is combined in order to facilitate, enhance and/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’s solution, Ringer’s lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.
  • the pharmaceutical composition of the present disclosure includes sodium chloride.
  • 1 , 2, 3, 4, 5, or more of the foregoing carriers may be excluded from the pharmaceutical compositions disclosed herein.
  • diluent relates a diluting and/or thinning agent.
  • the term “diluent” includes any one or more of fluid, liquid or solid suspension and/or mixing media.
  • suitable diluents for use in a pharmaceutical compositions of the present disclosure include, without limitation, ethanol, glycerol, saline, water, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing diluents may be excluded from the pharmaceutical compositions disclosed herein.
  • suitable granulating and/or dispersing agents include, without limitation, starches, pregelatinized starches, or microcrystalline starch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone), (providone), cross-linked polyvinylpyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, etc., and/or combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing granulating and/or dispersing agents may be excluded from the pharmaceutical compositions disclosed herein.
  • Suitable surface active agents for use in a pharmaceutical compositions of the present disclosure include, without limitation, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ®30]), PLUORINC®F 68, POLOXAMER®188, etc. and/or combinations thereof.
  • natural emulsifiers e.g.,
  • Suitable preservatives for use in a pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben, thimerosal, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisole, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing preservatives may be excluded from the pharmaceutical compositions disclosed herein.
  • Suitable antimicrobial and/or antifungal agents for use in a pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, benzethonium chloride, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing antimicrobial and/or antifungal agents may be excluded from the pharmaceutical compositions disclosed herein.
  • Suitable binders for use in a pharmaceutical compositions of the present disclosure include, without limitation, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing binders may be excluded from the pharmaceutical compositions disclosed herein.
  • Suitable lubricants and/or oil for use in a pharmaceutical compositions of the present disclosure include, without limitation, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, etc., and combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing lubricants and/or oils may be excluded from the pharmaceutical compositions disclosed herein.
  • antioxidants for use in a pharmaceutical compositions of the present disclosure include, without limitation, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing antioxidants may be excluded from the pharmaceutical compositions disclosed herein.
  • Suitable osmolality adjusting agents, pH adjusting agents, and buffers for use in a pharmaceutical compositions of the present disclosure include, without limitation, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCI), sodium malate, sodium carbonate, etc., and/or combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing osmolality adjusting agents may be excluded from the pharmaceutical compositions disclosed herein.
  • Suitable chelating agents for use in a pharmaceutical compositions of the present disclosure include, without limitation, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, etc., and combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing chelating agents may be excluded from the pharmaceutical compositions disclosed herein.
  • EDTA ethylenediaminetetraacetic acid
  • compositions can be formulated using one or more excipients (e.g., one or more carriers and/or diluents) to, e.g.
  • (1 ) increase stability; (2) increase cell transfection; (3) permit the sustained and/or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues and/or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo.
  • 1 , 2, 3, 4, 5, or more of the foregoing excipient purposes may be excluded.
  • Pharmaceutically acceptable excipients e.g., carriers and/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).
  • compositions may be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutical composition comprises an RNA molecule (e.g., polynucleotide) disclosed herein formulated with a lipid-based delivery system.
  • the composition includes a lipid-based delivery system (e.g., LNPs) (e.g., a lipid-based vaccine), which delivers a nucleic acid molecule to the interior of a cell, where it may then replicate, inhibit protein expression of interest, and/or express the encoded polypeptide of interest.
  • the delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen.
  • the composition comprises at least one RNA molecule encoding a N.
  • compositions comprising one or more lipids associated with a nucleic acid or a polypeptide/peptide (e.g., N. meningitidis RNA-LNPs).
  • the immunogenic composition including a lipid-based delivery system may further include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents, and/or excipients, in some cases.
  • the immunogenic composition including a lipid-based delivery system further includes a pharmaceutically acceptable vehicle.
  • each of a buffer, stabilizing agent, and optionally a salt may be included in the immunogenic composition including a lipid-based delivery system.
  • any one or more of a buffer, stabilizing agent, salt, surfactant, preservative, and excipient may be excluded from the immunogenic composition including a lipid-based delivery system.
  • the immunogenic composition including a lipid-based delivery system further comprises a stabilizing agent.
  • the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinylpyrolidone, glycine, or a combination thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing stabilizing agents may be excluded from the immunogenic compositions disclosed herein.
  • the stabilizing agent is a disaccharide, or sugar.
  • the stabilizing agent is sucrose.
  • the stabilizing agent is trehalose. In a further aspect, the stabilizing agent is a combination of sucrose and trehalose. In some aspects, the total concentration of the stabilizing agent(s) in the composition is or is about 5% to about 10% w/v. For example, the total concentration of the stabilizing agent may or may not be equal to at least, at most, exactly, or between (inclusive or exclusive) of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v or any range or value derivable therein.
  • the stabilizing agent concentration includes, but is not limited to, a concentration of or of about 10 mg/mL to about 400 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 150 mg/mL, about 100 mg/mL to about 140 mg/mL, about 100 mg/mL to about 130 mg/mL, about 100 mg/mL to about 120 mg/mL, about 100 mg/mL to about 1 10 mg/mL, or about 100 mg/mL to about 105 mg/mL.
  • the concentration of the stabilizing agent is or is not equal to at least, at most, exactly, or between (inclusive or exclusive) of 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, or more.
  • the mass amount of the stabilizing agent and the mass amount of the RNA are in a specific ratio. In one aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 5000. In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 2000. In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 1000. In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 500. In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 100. In another aspect, the ratio of the mass amount of the stabilizing agent and the pharmaceutical substance is no greater than 50.
  • the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 10. In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 1 . In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 0.5. In another aspect, the ratio of the mass amount of the stabilizing agent and the RNA is no greater than 0.1 . In another aspect, the stabilizing agent and RNA comprise a mass ratio of or of about 200 - 2000 of the stabilizing agent : 1 of the RNA.
  • the immunogenic composition including a lipid-based delivery system further comprises a buffer.
  • buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, trome
  • the buffer is a HEPES buffer, a Tris buffer, and/or a PBS buffer.
  • the buffer is Tris buffer.
  • the buffer is a HEPES buffer.
  • the buffer is a PBS buffer.
  • the buffer concentration may or may not be equal to at least, at most, exactly, or between (inclusive or exclusive) of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 1 1 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein.
  • the buffer may be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6.
  • the buffer may or may not be at least, at most, exactly, or between (inclusive or exclusive) of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.
  • the buffer is at pH 7.4.
  • the immunogenic composition including a lipid-based delivery system may further comprise a salt.
  • salts include but not limited to sodium salts and/or potassium salts.
  • the salt is a sodium salt.
  • the sodium salt is sodium chloride.
  • the salt is a potassium salt.
  • the potassium salt comprises potassium chloride.
  • any one or more of the foregoing salts may be excluded from the immunogenic compositions disclosed herein.
  • the concentration of the salts in the composition may be or be about 70 mM to about 140 mM.
  • the salt concentration may or may not be equal to at least, at most, exactly, or between (inclusive or exclusive) of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.
  • the salt concentration includes, but is not limited to, a concentration of or of about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 15 mg/mL.
  • the concentration of the salt is or is not equal to at least, at most, exactly, or between (inclusive or exclusive) of 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 1 1 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, or more.
  • the salt may be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6.
  • “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial and/or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • aqueous solvents e
  • Diluents include but are not limited to ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, corn rice, and potato; and celluloses such as microcrystalline cellulose.
  • the amount of diluent in the composition may range from or from about 10% to about 90% by weight of the total composition, e.g., from or from about 25% to about 75%, about 30% to about 60% by weight, or about 12% to about 60%.
  • Preservatives for use in the compositions disclosed herein include but are not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing excipients may be excluded from the immunogenic compositions disclosed herein.
  • a pharmaceutical composition comprises a N. meningitidis RNA molecule encoding a N. meningitidis polypeptide as disclosed herein that is complexed with, encapsulated in, and/or formulated with one or more lipids to form N. meningitidis RNA-LNPs.
  • RNA-LNP composition is a liquid.
  • the N. meningitidis RNA-LNP composition is frozen.
  • the N. meningitidis RNA-LNP composition is lyophilized.
  • a N. meningitidis RNA-LNP composition comprises a N. meningitidis RNA polynucleotide molecule encoding a N. meningitidis polypeptide as disclosed herein, encapsulated in LNPs with a lipid composition of a cationic lipid, a PEGylated lipid (e.g., PEG-lipid), and one or more structural lipids (e.g., a neutral lipid).
  • any one or more of the foregoing lipids may be excluded from the LNPs of the pharmaceutical compositions disclosed herein.
  • the N. meningitidis RNA-LNP composition further comprises one or more buffers and stabilizing agents, and optionally, salt diluents.
  • the N. meningitidis RNA-LNP composition comprises a cationic lipid, a PEGylated lipid, one or more structural lipids, one or more buffers, a stabilizing agent, and optionally, a salt diluent.
  • 1 , 2, 3, or more of the foregoing elements are excluded from the N. meningitidis RNA-LNP composition.
  • a N. meningitidis RNA-LNP composition comprises one or more buffers.
  • the one or more buffers may comprise any one or more buffering agents disclosed herein.
  • the composition comprises a Tris buffer comprising at least a first buffer and a second buffer.
  • the first buffer is tromethamine.
  • the second buffer is Tris hydrochloride (HCI).
  • the first buffer and second buffer of the Tris buffer are or are not included in the composition at a concentration of at least, at most, between (inclusive or exclusive) of, or exactly 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61 , 0.
  • the N. meningitidis RNA-LNP composition is a liquid composition comprising a T ris buffer.
  • the T ris buffer comprises a first buffer.
  • the first buffer is tromethamine.
  • the first buffer e.g., tromethamine
  • the first buffer is included in the liquid composition at a concentration of at least 0.1 , at least .05, at least 0.1 , at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL.
  • the first buffer e.g., tromethamine
  • the first buffer is included in the liquid composition at a concentration of between 0.05 and 0.15, between 0.15 and 0.25, between 0.25 and 0.35, between 0.35 and 0.45, between 0.45 and 0.55, between 0.55 and 0.65, between 0.65 and 0.75, between 0.75 and 0.85, or between 0.85 and 0.95.
  • the first buffer e.g., tromethamine
  • the first buffer is included in the liquid composition at a concentration of between 0.05 and 0.1 , between 0.1 and 0.15, between 0.15 and 0.2, between 0.2 and 0.25, between 0.25 and 0.3, between 0.3 and 0.35, between 0.35 and 0.4, between 0.4 and 0.45, between 0.45 and 0.5, between 0.5 and 0.55, between 0.55 and 0.6, between 0.6 and 0.65, between 0.65 and 0.7, between 0.7 and 0.75, between 0.75 and 0.8, between 0.8 and 0.85, between 0.85 and 0.9, between 0.9 and 0.95, or between 0.95 and 1 mg/mL.
  • the first buffer e.g., tromethamine
  • the first buffer is included in the liquid composition at a concentration of or of about 0.1 to 0.3 mg/mL. In specific aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of or of about 0.15 to 0.25 mg/mL.
  • the first buffer (e.g., tromethamine) is or is not included in the liquid composition at a concentration of or of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.1 , 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.3 mg/mL.
  • the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of or of about 0.20 mg/mL.
  • the N. meningitidis RNA-LNP composition is a liquid composition comprising a Tris buffer comprising a second buffer.
  • the second buffer comprises Tris HCI.
  • the second buffer (e.g., Tris HCI) is or is not included in the liquid composition at a concentration of at least, at most, between (inclusive or exclusive) of, or exactly 0.5, 0.55, 1 , 1 .01 , 1 .02, 1 .03, 1 .04, 1 .05, 1.06, 1.07, 1.08, 1.09, 1.1 , 1.1 1 , 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21 , 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31 , 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41 , 1.42, 1.43,
  • the second buffer (e.g., Tris HCI) is included in the liquid composition at a concentration of at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1 , at least 1 .05, at least 1 .10, at least 1 .15, at least 1 .20, at least 1 .25, at least 1 .30, at least 1 .35, at least 1 .40, at least 1 .45, or at least 1 .50 mg/mL.
  • Tris HCI Tris HCI
  • the second buffer (e.g., Tris HCI) is included in the liquid composition at a concentration of between 0.5 and 0.6, between 0.6 and 0.7, between 0.7 and 0.8, between 0.8 and 0.9, between 0.9 and 1 , between 1 and 1.10, between 1.10 and 1.20, between 1.20 and 1 .30, between 1 .30 and 1 .40, or between 1 .40 and 1 .50 mg/mL.
  • Tris HCI Tris HCI
  • the second buffer e.g., Tris HCI
  • the second buffer is included in the liquid composition at a concentration of or of about 1 .25 to 1 .40 mg/mL.
  • the second buffer e.g., Tris HCI
  • the second buffer is included in the liquid composition at a concentration of or of about 1 .30 to 1 .40 mg/mL.
  • the second buffer (e.g., Tris HCI) is or is not included in the liquid composition at a concentration of or of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 1 .25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31 , 1.32, 1.33, 1.34, or 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg/mL.
  • the second buffer e.g., Tris HCI
  • the N. meningitidis RNA-LNP composition is a lyophilized composition comprising a T ris buffer.
  • the T ris buffer comprises a first buffer.
  • the first buffer is tromethamine.
  • the first buffer (e.g., tromethamine) is or is not included in the lyophilized composition at a concentration, after reconstitution, of at least, at most, between (inclusive or exclusive) of, or exactly 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL.
  • the first buffer e.g., tromethamine
  • the first buffer is included in the lyophilized composition at a concentration, after reconstitution, of at least 0.01 , of at least 0.05, of at least 0.1 , of at least 0.15, of at least 0.2, of at least 0.25, of at least 0.3, of at least 0.35, of at least 0.4, of at least 0.45, or of at least 0.5 mg/mL.
  • the first buffer e.g., tromethamine (Tris base)
  • Tris base tromethamine
  • the first buffer is included in the lyophilized composition at a concentration, after reconstitution, of between 0.01 and 0.05, between 0.05 and 0.1 , between 0.1 and 0.15, between 0.15 and 0.2, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, or between 0.45 and 0.5 mg/mL.
  • Tris base Tris base
  • the first buffer e.g., tromethamine
  • the first buffer is included in the lyophilized composition at a concentration, after reconstitution, of or of about 0.01 and 0.15 mg/mL.
  • the first buffer e.g., tromethamine
  • the first buffer is included in the lyophilized composition at a concentration, after reconstitution, of or of about 0.01 and 0.10 mg/mL.
  • the first buffer e.g., tromethamine
  • the first buffer (e.g., tromethamine) is or is not included in the lyophilized composition at a concentration, after reconstitution, of or of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.1 1 , 0.12, 0.13, 0.14, or 0.15 mg/mL.
  • the first buffer (e.g., tromethamine) is included in the lyophilized composition at a concentration, after reconstitution, of or of about 0.09 mg/mL.
  • lyophilized compositions are reconstituted in a suitable carrier and/or diluent.
  • the carrier and/or diluent may comprise any one or more carriers and/or diluents disclosed herein.
  • the carrier and/or diluent comprises a salt diluent, such as sodium chloride (NaCI) (e.g., saline, e.g., physiological or normal saline).
  • NaCI sodium chloride
  • the sodium chloride may comprise 0.9% sodium chloride for injection.
  • the lyophilized compositions are or are not reconstituted in at least, at most, between (inclusive or exclusive) of, or exactly 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 , 0.32, 0.33,
  • the lyophilized compositions are reconstituted in at least 0.1 , at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1 mL of sodium chloride.
  • the lyophilized compositions are reconstituted in or in about 0.6 to 0.75 mL of sodium chloride/saline. In specific aspects, the lyophilized compositions are reconstituted in or in about 0.65 to 0.75 mL of sodium chloride/saline. In specific aspects, the lyophilized compositions are or are not reconstituted in or in at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.6, 0.61 , 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71 , 0.72, 0.73, 0,74, or 0.75 mL of sodium chloride/saline.
  • the salt diluent e.g., NaCI
  • the salt diluent is or is not included in the lyophilized composition at a concentration, after reconstitution, of at least, at most, between (inclusive or exclusive) of, or exactly 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5,
  • the salt diluent e.g., NaCI
  • the salt diluent is or is not included in the lyophilized composition at a concentration, after reconstitution, of in at least, at most, between (inclusive or exclusive) of, or exactly 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/mL.
  • the salt diluent e.g., NaCI
  • the salt diluent is included in the lyophilized composition at a concentration, after reconstitution, of at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, or at least 20 mg/mL.
  • the salt diluent e.g., NaCI
  • the salt diluent is included in the lyophilized composition at a concentration, after reconstitution, of between or between about 5 and 15 mg/mL.
  • the salt diluent e.g., NaCI
  • the salt diluent is or is not included in the lyophilized composition at a concentration, after reconstitution, of or of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 mg/mL.
  • the salt diluent is included in the lyophilized composition at a concentration, after reconstitution, of or of about 9 mg/mL.
  • the pH of the N. meningitidis RNA-LNP composition may or may not be at least, at most, exactly, or between (inclusive or exclusive) of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.
  • the N. meningitidis RNA-LNP composition is at a pH of at least 6.5, at least 7.0, at least 7.5, at least 8.0, or at least 8.5.
  • meningitidis RNA-LNP composition is at a pH between 6.0 and 7.5, between 6.5 and 7.5, between 7.0 and 8.0, between and 7.5 and 8.5. In specific aspects, the N. meningitidis RNA-LNP composition is between 7.0 and 8.0. In specific aspects, the N. meningitidis RNA-LNP composition is or is not at least, at most, exactly, between (inclusive or exclusive) any two of, or about pH 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In specific aspects, the N. meningitidis RNA-LNP composition is at or at about pH 7.4. In some aspects, sodium hydroxide buffer may be used for a buffer pH adjustment.
  • a N. meningitidis RNA-LNP composition comprises a N. meningitidis RNA polynucleotide encoding a N. meningitidis polypeptide as disclosed herein, encapsulated in LNPs with a lipid composition of an cationic lipid at a concentration of or of about 0.8 to 0.95 mg/mL, a PEGylated lipid at a concentration of or of about 0.05 to 0.15 mg/mL, a first structural lipid at a concentration of or of about 0.1 to 0.25 mg/mL, and a second structural lipid at a concentration of or of about 0.3 to 0.45 mg/mL.
  • one or more of the foregoing elements may be excluded from the N. meningitidis RNA-LNP composition.
  • a N. meningitidis RNA-LNP composition comprises a N. meningitidis RNA polynucleotide encoding a N. meningitidis polypeptide as disclosed herein, encapsulated in LNPs with a lipid composition of ALC-0315 at a concentration of or of about 0.8 to 0.95 mg/mL, ALC-0159 at a concentration of or of about 0.05 to 0.15 mg/mL, DSPC at a concentration of or of about 0.1 to 0.25 mg/mL, and cholesterol at a concentration of about 0.3 to 0.45 mg/mL.
  • one or more of the foregoing elements may be excluded from the N. meningitidis RNA-LNP composition.
  • the N. meningitidis RNA-LNP composition is a liquid N. meningitidis RNA-LNP composition
  • the liquid N. meningitidis RNA-LNP composition further comprises a buffer composition comprising a first buffer at a concentration of or of about 0.15 to 0.3 mg/mL, a second buffer at a concentration of or of about 1 .25 to 1 .4 mg/mL, and a stabilizing agent at a concentration of or of about 95 to 110 mg/mL.
  • the N. meningitidis RNA-LNP composition is a liquid N. meningitidis RNA-LNP composition, and the liquid N.
  • meningitidis RNA-LNP composition further comprises a Tris buffer composition comprising tromethamine at a concentration of or of about 0.1 to 0.3 mg/mL, Tris HCI at a concentration of or of about 1.25 to 1.4 mg/mL, and sucrose at a concentration of or of about 95 to 1 10 mg/mL.
  • Tris buffer composition comprising tromethamine at a concentration of or of about 0.1 to 0.3 mg/mL, Tris HCI at a concentration of or of about 1.25 to 1.4 mg/mL, and sucrose at a concentration of or of about 95 to 1 10 mg/mL.
  • sucrose at a concentration of or of about 95 to 1 10 mg/mL.
  • one or more of the foregoing elements may be excluded from the N. meningitidis RNA-LNP composition.
  • the N. meningitidis RNA-LNP composition is a lyophilized N. meningitidis RNA-LNP composition, and the lyophilized N. meningitidis RNA-LNP composition further comprises (after reconstitution) a first buffer at a concentration of or of about 0.01 and 0.15 mg/mL, a second buffer at a concentration of or of about 0.5 and 0.65 mg/mL, a stabilizing agent at a concentration of or of about 35 to 50 mg/mL, and a salt diluent at a concentration of between or between about 5 and 15 mg/mL.
  • one or more of the foregoing elements may be excluded from the N. meningitidis RNA-LNP composition.
  • the N. meningitidis RNA-LNP composition is a lyophilized N. meningitidis RNA-LNP composition, and the lyophilized N. meningitidis RNA-LNP composition further comprises (after reconstitution) a Tris buffer composition comprising tromethamine at a concentration of or of about 0.01 and 0.15 mg/mL, Tris HCI at a concentration of or of about 0.5 and 0.65 mg/mL, sucrose at a concentration of or of about 35 to 50 mg/mL, and sodium chloride (NaCI) at a concentration of or of about 5 to 15 mg/mL.
  • a Tris buffer composition comprising tromethamine at a concentration of or of about 0.01 and 0.15 mg/mL, Tris HCI at a concentration of or of about 0.5 and 0.65 mg/mL, sucrose at a concentration of or of about 35 to 50 mg/mL, and sodium chloride (NaCI) at a concentration of or of about 5 to 15 mg
  • a pharmaceutical composition described herein is an immunogenic composition for inducing an immune response.
  • an immunogenic composition is a vaccine.
  • the compositions described herein include at least one isolated nucleic acid or polypeptide molecule as described herein.
  • the immunogenic compositions comprise nucleic acids, and the immunogenic compositions are nucleic acid vaccines.
  • the immunogenic compositions comprise RNA (e.g., mRNA, saRNA), and vaccines are RNA vaccines.
  • the immunogenic compositions comprise DNA, and vaccines are DNA vaccines.
  • the immunogenic compositions comprise a polypeptide, and vaccines are polypeptide vaccines.
  • Conditions and/or diseases that may be treated with the nucleic acid and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, and/or improper production of protein or nucleic acids.
  • the composition is substantially free of one or more impurities or contaminants and, for instance, includes nucleic acid or polypeptide molecules that are equal to at least, at most, exactly, or between (inclusive or exclusive) of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure, or at least 99% pure.
  • the present disclosure includes methods for preventing, treating and/or ameliorating an infection, disease or condition in a subject, including administering to a subject an effective amount of an RNA molecule that includes at least one open reading frame encoding a polypeptide or composition described herein.
  • the disclosure contemplates vaccines for use in both active and passive immunization aspects.
  • Immunogenic compositions proposed to be suitable for use as a vaccine, may be prepared from RNA molecules encoding polypeptide(s), such as fHbp.
  • immunogenic compositions are lyophilized for more ready formulation into a desired vehicle.
  • vaccines that contain nucleic acid and/or peptide or polypeptide as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
  • vaccines are formulated with a combination of substances, as described in U.S. Patents 6,793,923 and 6,733,754, which are incorporated herein by reference. In some aspects, one or more of the foregoing elements may be excluded from a vaccine.
  • Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of or of about 0.5% to about 10%. In some aspects, suppositories may be formed from mixtures containing the active ingredient in the range of or of about 1% to about 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing excipients may be excluded from an oral formulation. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain or contain about 10% to about 95% of active ingredient.
  • vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including the capacity of the individual’s immune system to synthesize antibodies and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations and/or other administrations.
  • Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application within a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.
  • the vaccine it will be desirable to have one administration of the vaccine. In some aspects, it will be desirable to have multiple administrations of the vaccine, e.g., 2, 3, 4, 5, 6, or more administrations.
  • the vaccinations may be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , or 12 week intervals, including all ranges there between. In some aspects, vaccinations may be at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 month intervals, including all ranges there between.
  • Periodic boosters at intervals of 1 to 5 years may be desirable to maintain protective levels of the antibodies. i. CARRIERS
  • a pharmaceutically acceptable carrier may include the liquid or non-liquid basis of a composition.
  • the carrier may be water, such as pyrogen-free water, isotonic saline or buffered (aqueous) solutions, e.g., phosphate, citrate buffered solutions.
  • Water and/or a buffer such as an aqueous buffer, may be used, containing a sodium salt, a calcium salt, and and/or a potassium salt.
  • the sodium, calcium and/or potassium salts may occur in the form of their halogenides, e.g., chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
  • sodium salts include, but are not limited to, NaCI, Nal, NaBr, Na2CO3, NaHCOa, NaaSC , NaaHPC , Na2HPO4-2H2O
  • potassium salts include, but are not limited to, KCI, KI, KBr, K2CO3, KHCO3, K2SO4, KH2PO4
  • calcium salts include, but are not limited to, CaCh, Cah, CaBr2, CaCOa, CaSC , Ca(OH)2.
  • Examples of further carriers may include sugars, such as, for example, lactose, glucose, trehalose and sucrose; starches, such as, for example, com starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
  • sugars such as, for example, lactose, glucose, trehalose and sucrose
  • starches such as, for example,
  • further carriers may include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington’s Pharmaceutical Sciences. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing carriers may be excluded. ii. ADJUVANTS
  • Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. A number of adjuvants may be used to enhance an antibody response. Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances.
  • Specific adjuvants that may be used include Freund’s adjuvant, oil such as MONTANIDE® ISA51 , IL1 , IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, alphainterferon, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts, such as aluminum hydroxide or other aluminum compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), lipopeptides (e.g., Pam3Cys), or RIBI, which contains three components extracted from bacteria (MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS)) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used.
  • MDP compounds such as thur-MDP and nor
  • Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as or as about 0.05 to about 0.1 % (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, or 0.1 %) solution in phosphate buffered saline, admixture with synthetic polymers of sugars (CARBOPOL®) used as an or as an about 0.25% solution, and/or aggregation of the protein in the vaccine by heat treatment with temperatures ranging between or between about 70 °C to about 101 °C for a 30-second to 2-minute period, respectively.
  • agents such as aluminum hydroxide or phosphate (alum), commonly used as or as about 0.05 to about 0.1 % (e.g., at least, at most, exactly, or between (inclusive or exclusive
  • Fab pepsin-treated antibodies to albumin
  • mixture with bacterial cells e.g., C. parvurri
  • endotoxins or lipopolysaccharide components of Gram-negative bacteria emulsion in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsion with a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used as a block substitute
  • FLUOSOL-DA® perfluorocarbon
  • 1 , 2, 3, 4, 5, or more of the foregoing adjuvants may be excluded.
  • BRM biologic response modifiers
  • BRMs have been shown to upregulate T cell immunity and/or downregulate suppressor cell activity.
  • BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low- dose Cyclophosphamide (CYP; 300 mg/m 2 ) (Johnson/ Mead, NJ) and cytokines such as y-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.
  • compositions and related methods of the present disclosure may also be used in combination with the administration of one or more other therapeutic and/or prophylactic agents.
  • these include, but are not limited to, the administration of traditional therapies, e.g., anti-bacterial agents.
  • the administration of one or more therapies to treat one or more symptoms of N. meningitidis infection including, but not limited to, steroids including corticosteroids, anti-inflammatories including acetaminophen or ibuprofen, pain-relief agents, creams or lotions to relieve itching, cool compresses, or various combinations thereof.
  • 1 , 2, 3, 4, 5, or more of the foregoing therapeutic agents may be excluded.
  • Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of the composition of the invention and each active agent in its own separate pharmaceutical dosage formulation.
  • a composition of the invention and the other active agent can be administered to the patient together in a single dosage composition such as an injection or tablet or capsule, or each agent administered in separate oral dosage formulations.
  • the compounds of the invention and one or more additional active agents can be administered at essentially the same time, e.g., concurrently, or at separately staggered times, e.g., sequentially; combination therapy is understood to include all these regimens.
  • a vaccine and/or therapy is used in conjunction with anti-bacterial treatment.
  • the vaccine and/or therapy may precede or follow treatment with another agent by intervals ranging from minutes to weeks.
  • the other agents and/or vaccines are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and immunogenic composition would still be able to exert an advantageously combined effect on the subject.
  • a pharmaceutical composition described herein may be administered intravenously, intraarterially, subcutaneously, intraderN1 -mally, intranasally, or intramuscularly.
  • the N. meningitidis RNA molecules and/or RNA- LNP compositions are administered intramuscularly.
  • the N. meningitidis RNA molecules and/or RNA-LNP compositions are administered intradermally.
  • the N. meningitidis RNA molecules and/or RNA-LNP compositions are administered intranasally.
  • the pharmaceutical composition is formulated for local administration and/or systemic administration.
  • Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration.
  • parenteral administration refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection.
  • the pharmaceutical composition is formulated for intramuscular, intradermal, or intranasal administration.
  • the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration. In some aspects, 1 , 2, 3, or more of the foregoing administration routes may be excluded.
  • a pharmaceutical composition within the scope of this disclosure may be in the form of a solid or liquid and may be frozen or lyophilized.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid, or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition when intended for oral administration, is in either solid or liquid form, where semi-solid, semiliquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch, lactose, or dextrins
  • disintegrating agents such as alginic acid, sodium alginate, PRIMOJEL®, corn starch and the like
  • lubricants such as magnesium stearate or STEROTEX®
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl salicylate, or orange flavoring
  • a coloring agent such as peppermint, methyl salicylate, or orange flavoring
  • the pharmaceutical composition when in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil. In some aspects, 1 , 2, 3, or more of the foregoing elements may be excluded from a solid composition.
  • parenteral preparation may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
  • physiological saline is the adjuvant.
  • an injectable pharmaceutical composition is sterile.
  • a liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a compound such that a suitable dosage will be obtained.
  • compositions within the scope of the disclosure are administered in a therapeutically and/or prophylactically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic and/or prophylactic agent employed; the metabolic stability and length of action of the therapeutic and/or prophylactic agent; the individual parameters of the patient, including the age, body weight, general health, gender, and diet of the patient; the mode, time, and/or duration of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • 1 , 2, 3, 4, 5, or more of the factors may be excluded from determining a therapeutically and/or prophylactically effective amount. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters.
  • compositions e.g., N. meningitidis RNA-LNP compositions
  • compositions e.g., N.
  • meningitidis RNA- LNP compositions may or may not be administered at dosage levels sufficient to deliver at least, at most, exactly, or between (inclusive or exclusive) any two of 0.0001 , 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14,
  • compositions e.g., pharmaceutical compositions comprising N. meningitidis RNA molecules and/or N. meningitidis RNA-LNPs
  • methods, kits and reagents for prevention and/or treatment of N. meningitidis in humans and other mammals are provided herein.
  • N. meningitidis RNA compositions may be used as therapeutic and/or prophylactic agents. They may be used in medicine to prevent and/or treat infectious disease.
  • the N. meningitidis RNA compositions are used to provide prophylactic protection from N. meningitidis of any serotype, genotype, strain, or isolate. It is envisioned that there may be situations where persons are at risk for infection with more than one strain of N. meningitidis.
  • N. meningitidis RNA compositions e.g., N.
  • N. meningitidis RNA-LNP compositions are particularly amenable to combination vaccination approaches due to a number of factors including, but not limited to, speed of manufacture, ability to rapidly tailor vaccines to accommodate perceived geographical threat, and the like.
  • the N. meningitidis RNA compositions e.g., N. meningitidis RNA- LNP compositions
  • the N. meningitidis RNA compositions utilize the human body to produce the antigenic protein
  • the N. meningitidis RNA compositions e.g., N. meningitidis RNA-LNP compositions
  • a combination N. meningitidis RNA composition can be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first N. meningitidis and further includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second N. meningitidis.
  • the N. meningitidis RNA compositions of the disclosure may be used to prime immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.
  • PBMCs peripheral blood mononuclear cells
  • RNA-LNPs after administration of a N. meningitidis RNA molecule described herein, e.g., formulated as RNA-LNPs, at least a portion of the RNA is delivered to a target cell. In some aspects, at least a portion of the RNA is delivered to the cytosol of the target cell. In some aspects, the RNA is translated by the target cell to produce the polypeptide or protein it encodes. In some aspects, the target cell is a spleen cell. In some aspects, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some aspects, the target cell is a dendritic cell and/or macrophage.
  • RNA molecules such as RNA-LNPs described herein may be used for delivering RNA to such target cell. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject comprising the administration of the RNA-particles described herein to the subject.
  • the RNA is delivered to the cytosol of the target cell. In some aspects, the RNA is translated by the target cell to produce the polypeptide or protein encoded by the RNA.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • 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, may be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleic acid compositions described herein are characterized by (e.g., when administered to a subject) sustained expression of an encoded polypeptide.
  • compositions are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from such human and, in some aspects, such expression persists for a period of time that is at least 36 hours or longer, including, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.
  • nucleic acid compositions described herein are characterized by (e.g., when administered to a subject) an induced and/or boosted immune response as a function of antigen production in the cell.
  • Increased antigen production may be demonstrated by, e.g., increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), and/or altered antigen specific immune response of the host cell.
  • the disclosure relates to a method of treating and/or preventing and/or reducing the severity of a N. meningitidis infection and/or illness caused by N. meningitidis.
  • the method includes administering to the subject an effective amount of an RNA molecule, RNA-LNP and/or composition as described herein.
  • the disclosure relates to a method of treating and/or preventing and/or reducing the severity of an infectious disease in a subject by, for example, inducing an immune response to an infectious disease in the subject.
  • the method includes administering a priming composition that includes an effective amount of an RNA molecule, RNA-LNP and/or composition described herein, and administering a booster composition including an effective amount of an RNA molecule, RNA-LNP and/or composition.
  • the composition elicits an immune response including an antibody response.
  • the composition elicits an immune response including a T cell response and/or a B cell response.
  • an immune response comprises a T cell response and a B cell response.
  • the composition elicits a neutralizing immune response.
  • a neutralizing immune response is an immune response that is a neutralizing antibody response and/or an effective neutralizing T cell response.
  • a neutralizing antibody response produces a level of antibodies that meet or exceed a seroprotection threshold.
  • the composition elicits an effective T cell response.
  • An effective T cell response is a response which produces a baseline level of infectious disease-activated and/or infectious diseasespecific T cells including CD8+ and CD4+ T helper type 1 cells.
  • the effective T cells comprises a high proportion of CD8+ T cells and/or CD4+ T cells, relative to a baseline level (in a naive subject). In some embodiments these T cells are differentiated towards an early- differentiated memory phenotype with co-expression of CD27 and CD28.
  • an effective amount of a N. meningitidis RNA-LNP composition comprising at least one N. meningitidis RNA molecule having an open reading frame encoding at least one N. meningitidis antigenic polypeptide results in a 2-fold to 200-fold (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 1 10-, 120-, 130-, 140-, 150-, 160-, 170-, 180-, 190-, or 200-fold) increase in serum neutralizing antibodies against N.
  • N. meningitidis relative to a traditional composition against N. meningitidis (e.g., the standard of care dose of a recombinant or purified N. meningitidis protein vaccine, a live attenuated or inactivated N. meningitidis vaccine, or a N. meningitidis VLP vaccine).
  • a traditional composition against N. meningitidis e.g., the standard of care dose of a recombinant or purified N. meningitidis protein vaccine, a live attenuated or inactivated N. meningitidis vaccine, or a N. meningitidis VLP vaccine.
  • the methods disclosed herein may involve administering to the subject a N. meningitidis RNA-LNP composition comprising at least one N. meningitidis RNA molecule having an open reading frame encoding at least one N. meningitidis antigenic polypeptide, thereby inducing in the subject an immune response specific to N. meningitidis antigenic polypeptide, wherein the immune response in the subject is equivalent to an immune response in a subject administered with a traditional composition against the N.
  • meningitid that is or is not at least, at most, in between (inclusive or exclusive) any two of, or exactly 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 times the dosage level relative to the RNA composition.
  • methods of the disclosure relate to prognosing, diagnosing, testing, monitoring, and/or treating a subject suspected of having had an infectious disease (e.g., N. meningitidis), having an infectious disease (e.g., N. meningitidis), at risk of having an infectious disease (e.g., N. meningitidis), and/or having symptoms of an infectious disease (e.g., N. meningitidis).
  • the subject may have one or more symptoms of an infectious disease (e.g., N. meningitidis).
  • the subject may be tested for one or more antigenic polypeptides or proteins (or antigenic portions thereof) from an infectious disease (e.g., N.
  • the subject having had an infectious disease (e.g., N. meningitidis), having an infectious disease (e.g., N. meningitidis), at risk of having an infectious disease (e.g., N. meningitidis), and/or having symptoms of an infectious disease (e.g., N.
  • meningitidis is prognosed, diagnosed, monitored, and/or treated with or for the infectious disease (e.g., N. meningitidis) based on measurement, assaying, or detection in a sample (e.g., blood, saliva, tissues, bone, muscle, cartilage, and/or skin) from the subject one or more antigenic polypeptides or proteins (or antigenic portions thereof) from an infectious disease (e.g., N. meningitidis) by one or more diagnostic tests (e.g., PCR testing to detect N.
  • the infectious disease e.g., N. meningitidis
  • a sample e.g., blood, saliva, tissues, bone, muscle, cartilage, and/or skin
  • diagnostic tests e.g., PCR testing to detect N.
  • the present disclosure further provides a kit comprising the RNA molecule, RNA-LNP, and/or composition.
  • the RNA molecule, RNA-LNP and/or composition described herein is administered to a subject that is or is less than about 1 years old, or is or is about 1 years old to about 10 years old, or is or is about 10 years old to about 20 years old, or is or is about 20 years old to about 50 years old, or is or is about 60 years old to about 70 years old, or older.
  • the subject is or is not at least, at most, exactly, or between (inclusive or exclusive) any two of less than 1 year of age, greater than 1 year of age, greater than 5 years of age, greater than 10 years of age, greater than 20 years of age, greater than 30 years of age, greater than 40 years of age, greater than 50 years of age, greater than 60 years of age, greater than 70 years of age, or older. In some aspects, the subject is greater than 50 years of age.
  • the subject is or is not at least, at most, exactly, between (inclusive or exclusive) any two of, or about 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. In some aspects, the subject may be or be about 50 years of age or older.
  • the subject is at least, at most, exactly, or between any two of 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. In some aspects the subject may be 50 years of age or older.
  • Synthetic genes of interest encoding fHbp-A05 and fHbp-B01 were codon optimized using the Genewiz codon optimization tool (Codon Optimization (genewiz.com)) and ordered as synthetic FragmentGENEs from Genewiz for human host expression. Fragments were directionally cloned into Pmel linearized BMD576 (see Example 8) using homologous regions via Gibson assembly.
  • the Gibson enzyme mix used in the cloning reaction removes the Pmel digested DNA termini, fusing the start and stop codons of the synthetic gene respectively, to the Kozak sequence of the 5’IITR and first nucleotide of the 3’ UTR sequence.
  • BMD576 includes a T7 promoter for in vitro transcription (IVT), and a 3’ 80 nt polyA adenylation site with a terminal, unique, BspQ1 restriction enzyme site for linearization of the DNA plasmid as IVT template.
  • DNA plasmids were linearized with BspQI prior to in vitro transcription.
  • the linearized templates were purified by treatment with Proteinase K (200 pg/mL)/SDS (0.5%) at 50°C for 30 minutes and phenol/chloroform extraction (equivalent volume of 25:24:1 premixed IAA reagent) followed by ethanol precipitation. 5 pg of BspQI linearized plasmid DNA was used as template in each 100 pL in vitro transcription reaction.
  • RNA was transcribed by T7 RNA polymerase (New England Biolabs), and the CleanCap® AG kit (TriLink) was used to simultaneously cap the newly transcribed modRNA molecules with m7G(5'’)ppp(5'’)(2'’OMeA)pG.
  • TriLink the CleanCap® AG kit
  • each reaction was treated with 0.3 U/pL DNasel for 30 minutes at 37°C, and modRNA containing N1 -methylpseudo UTP was precipitated by the addition of 2.5M LiCI solution and incubation for 30 minutes at -20°C.
  • RNA samples were collected by spinning LiCI incubated tubes at 15,000 rpm for 20 minutes at 4°C, washed twice with 70% ethanol and resuspended in nuclease free water. Size and integrity of the transcribed RNAs were confirmed using an Advanced Analytical Fragment Analyzer and RNA markers (Agilent). Between 330-650 pg of purified modRNA was generated in each reaction.
  • RNA 25 pL volumes of 4-fold serial dilutions of RNA (from 30 ng/well) were combined with lipofectamine (MessengerMax, Invitrogen) for 5 minutes at room temperature in a 96-well deep-well (2.2 mL) plate (Fisher Scientific). Expi293 suspension cells (ThermoFisher) were diluted in 0.45 mL of Opti-MEM growth media to a final concentration of 1 x10 6 /well with shaking for 24 hours at 37°C, 8% CO2 and 80% humidity overnight.
  • RNA 25 pL volumes of 4-fold serial dilutions of RNA (from 300 ng/well) were combined with lipofectamine (MessengerMax, Invitrogen) for 5 minutes at room temperature in a 96-well deep-well (2.2 mL) plate.
  • Expi293 suspension cells (ThermoFisher) were diluted in 0.45 mL of Opti-MEM growth media to a final concentration of 1 x10 6 /well with shaking for 24 hours at 37°C, 8% CO2, and 80% humidity overnight.
  • fHbp secreted into culture media supernatants was quantified by Octet biolayer interferometry 24 hour after the transfection of Expi293 cells with modRNA.
  • Antihuman FcProG biosensors were used to first bind the monoclonal mouse mAb MN86- 994-1 1 -1 followed by binding reactions with clarified transfection supernatants.
  • the biosensors were first hydrated with conditioned Expi293 media (200 pL/well) for 10 minutes at room temperature, followed by capture with saturating concentration of mAb (final 1 pg/mL, 200 pL/well) for 10 minutes at room temperature on an orbital shaker.
  • fHbp concentrations were determined by interpolating values from a parallel titration of purified recombinant fHbp protein standard (MnB Bulk DS Subfamily B batch G09710B306) starting at 20 pg/ml with 2-fold dilutions using linear regression analysis.
  • samples of normalized cell pellets and supernatants were analyzed by SDS-PAGE followed by Western blot with detection by primary antibody mAB MN86-994-1 1 -1 (mouse) and secondary anti-mouse AP conjugate.
  • mRNA constructs were designed using the BMD576 backbone encoding secreted and anchored versions of both fHbp proteins.
  • Nonnative glycosylation that occurs on proteins produced in mammalian cells was predicted using the NetNGIyc N-glycoslyation prediction server, https:/ services.healthtech.dtu.dk/services/NetNGIyc-1 .0/ (Gupta, R. and S. Brunak, Pac Symp Biocomput, 2002: p. 310-22).
  • Asparagine 46 in fHbp-A05 and asparagine 47 in fHbp-B01 were predicted to be glycosylated during mammalian expression. These positions were mutated to serine in the expression constructs. The N-terminal cysteine present in both native sequences was removed to avoid potential mispairing disulfide bond formation.
  • fHbp is posttranslationally lipidated at the N-terminus, which mediates its association with the bacterial cell membrane (da Silva, R.A.G., et al., Br J Pharmacol, 2017. 174(14): p. 2247-2260). This type of lipidation does not occur in mammalian cells, thus in order to anchor fHbp to the mammalian cell surface, an alternative strategy of anchoring the protein via the N-terminus is required.
  • One option initially considered was to use a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • GPI anchors are attached to the C-terminus of proteins, they could not be applied to fHbp as it would be presented in the opposite orientation than in nature.
  • Various anchoring approaches were considered, such as lipidation and type I or II single pass transmembrane domains (Spiess, M., FEBS Lett, 1995. 369(1 ): p. 76-9). Lipidation primarily localizes proteins to the intracellular side of the plasma membrane, so it was not an option.
  • a type I transmembrane domain fusion protein would orient the fusion partner protein with the C-terminus facing into the cytoplasm; however a type II transmembrane domain or signal anchor would confer the appropriate orientation to fHbp with the C-terminus facing the extracellular milieu.
  • a type II signal anchor is a highly hydrophobic sequence that directs proteins to the plasma membrane. Notably, unlike a signal peptide, the type II signal anchor is not cleaved (Lemire, I., etal., Biochemical Journal, 1997. 322(1 ): p. 335-342). The C- terminus and functional domains of proteins with a type II signal anchor are located on the extracellular side of the cell membrane and are anchored to the cell via a transmembrane helix. The N-termini remain on the cytoplasmic side of the membrane.
  • NA Influenza neuraminidase
  • a viral protein that cleaves sialic acid residues on viral proteins that are added during protein production in the host cell ER, which is an essential process for virion release and budding (Gong, J., W. Xu, and J. Zhang, Curr Med Chem, 2007. 14(1 ): p. 1 13-22; McAuley, J.L., et al., Front Microbiol, 2019. 10: p. 39).
  • NA is a type II signal anchor protein which exists as a tetramer of four identical monomers, each composed of four distinct structural domains (the catalytic head, stalk, transmembrane region and the cytoplasmic tail) ( Figure 3).
  • Tetramerization is mediated primarily by residues in the N-terminal transmembrane domain, though cysteine residues in the stalk region may also be important for subunit interactions, by assisting with tetramer stabilization via promotion of intermolecular disulfide bond formation between monomers (Gong, J., W. Xu, and J. Zhang, Curr Med Chem, 2007. 14(1 ): p. 113-22; McAuley, J.L., et al., Front Microbiol, 2019. 10: p. 39).
  • the HINI The HINI
  • WSN/33 NA sequence was selected for evaluation in the initial fHbp mammalian expression experiments.
  • EXAMPLE 7 Cloning and Expression of Membrane-Anchored fHbp-A05 and fHbp-B01 from modRNA Transcripts
  • Table 1 below provides the amino acid and RNA sequences of several fHbp constructs.
  • Endotoxin levels for all DP modRNA LNPs measured with the LAL test cartridge system were 1 EU/mL (Endosafe). Testing by PCR for adventitious agents was done by Charles River Research Animal Diagnostic Services with their standard infectious disease panel. All samples were negative. (Signals 00803405-0002, 0003) Encapsulation efficiency was determined by RiboGreen assay, LNP size and polydispersity index (PDI) by dynamic light scattering (DLS) (Malvern).
  • mice 1 and 2 were terminally bled at week 6 and cages 3 and 4 will be terminally bled at week 10.
  • the serum bactericidal assay quantifies functional N. meningitidis antibody titers as the reciprocal of the highest test serum dilution resulting in at least a 50 percent reduction in the bacteria.
  • Performing the hSBA requires 3 days: one day for initiating the culture of target bacteria, a second day to perform the assay and a third day to enumerate surviving target bacteria and determine assay results.
  • bacteria stock is streaked on agar plates for overnight growth of isolated bacterial colonies.
  • bacterial colonies are resuspended in liquid medium for mixing with human serum samples and a source of complement, which is a group of proteins that aid in the destruction of bacteria.
  • the mixture is then incubated to allow the antibodies in the serum to bind to the bacteria and activate the complement system, leading to bacterial lysis. At the end of the incubation, a portion of this mixture is transferred to filter plates for growth of remaining viable bacterium. Colonies generated by surviving bacteria are enumerated on the third day to determine the effectiveness of the antibodies. This is calculated by quantifying if there was at least a 50 percent reduction in the number of viable bacteria, when compared to the number of viable bacteria in control wells containing all assay components except test serum.
  • RNA molecule comprising at least one open reading frame encoding a Nesiseria meningitidis polypeptide.
  • RNA molecule of paragraph 1 wherein the Neisseria meningitidis polypeptide is a full-length, truncated, fragment, or variant thereof.
  • RNA molecule of paragraph 1 wherein the Neisseria meningitidis polypeptide comprises at least one mutation.
  • RNA molecule of paragraph 3 wherein the at least one mutation occurs at a predicted glycosylation site.
  • RNA molecule of paragraph 4 wherein the predicted glycosylation site is an asparagine residue in the corresponding polypeptide sequence.
  • RNA molecule of paragraph 1 wherein the polypeptide is human factor H binding protein (fHbp).
  • RNA molecule of paragraph 4 wherein the fHbp polypeptide is from serogroup B.
  • RNA molecule of paragraph 5 wherein the fHbp polypeptide is from the subfamily A strain.
  • RNA molecule of paragraph 6 wherein the polypeptide is fHbp-A05.
  • RNA molecule of paragraph 5 wherein the fHbp polypeptide is from the subfamily B strain.
  • RNA molecule of paragraph 8 wherein the polypeptide is fHbp-B01 .
  • RNA molecule of paragraph 4 wherein the open reading frame further encodes an N-terminal secretion signal polypeptide.
  • RNA molecule of paragraph 10 wherein the N-terminal secretion signal is fused in-frame at the N-terminal position of the fHbp.
  • RNA molecule of paragraph 11 wherein the secretion signal is a murine IgK secretion signal.
  • RNA molecule of paragraph 11 further comprising a linker sequence between the N-terminal secretion signal and the fHbp.
  • the open reading frame further encodes an influenza neuraminidase (NA) structural domain polypeptide, or fragment thereof.
  • NA influenza neuraminidase
  • RNA molecule of paragraph 14 wherein the NA structural domain, or fragment thereof, is fused in-frame at the N-terminal position of the fHbp.
  • RNA molecule of paragraph 15 further comprising a linker sequence between the NA structural domain, or fragment thereof, and the fHbp.
  • RNA molecule of paragraph 14 wherein the NA structural domain, or fragment thereof, comprises the cytoplasmic tail, the transmembrane domain, and the first 7 amino acids of the stalk region.
  • RNA molecule of any one of paragraphs 1 to 20, wherein the open reading frame comprises a nucleic acid sequence of Table 1 , including but not limited to any of SEQ ID NOs: 4, 6, 8, and 10.
  • RNA molecule of any one of paragraphs 1 to 20, wherein the open reading frame comprises a nucleic acid sequence having at least 90%, 95, 96%, 97%, 98% or 99% identity to the sequence of any of SEQ ID NOs: 4, 6, 8, and 10.
  • RNA molecule of paragraph 1 comprising in the 5’ to 3’ direction: a. an open reading frame encoding an influenza neuraminidase (NA) structural domain polypeptide, or fragment thereof; and b. an open reading frame encoding a fHbp polypeptide.
  • NA neuraminidase
  • fHbp fHbp polypeptide
  • RNA molecule of paragraph 1 comprising in the 5’ to 3’ direction: a. an open reading frame encoding an N-terminal secretion signal polypeptide; and b. an open reading frame encoding a fHbp polypeptide.
  • RNA molecule of paragraph 29 further comprising a linker sequence located between the open reading frame encoding the N-terminal secretion signal polypeptide and the open reading frame encoding the fHbp polypeptide.
  • RNA molecule of any one of paragraphs 1 to 31 further comprising a 5' untranslated region (5' UTR).
  • RNA molecule of paragraph 32, wherein the 5' UTR comprises a sequence of SEQ ID NO: 1 .
  • RNA molecule any one of paragraphs 1 to 31 , further comprising a 3' untranslated region (3' UTR).
  • composition of paragraph 34, wherein the 3' UTR comprises a sequence of SEQ ID NO: 2.
  • RNA molecule of any one of paragraphs 1 to 35 wherein the RNA molecule comprises a 5' cap moiety.
  • RNA molecule of paragraph 36 wherein the RNA molecule comprises a 5' cap moiety comprising (3'OMe) - m2 7 ’ 3 '°Gppp (mi 2 ’ °)ApG.
  • RNA molecule of any one of paragraphs 1 to 37 further comprising a 3' poly-A tail.
  • RNA molecule of any one of paragraphs 1 to 31 wherein the RNA molecule comprises a 5' UTR and 3' UTR.
  • RNA molecule of any one of paragraphs 1 to 31 wherein the RNA molecule comprises a 5' cap, 5' UTR, and 3' UTR.
  • RNA molecule of any one of paragraphs 1 to 31 wherein the RNA molecule comprises a 5' cap, 5' UTR, 3' UTR, and poly-A tail.
  • the RNA molecule of any of paragraphs 1 to 31 comprising a 5' UTR comprising the sequence of SEQ ID NO: 1 , an open reading frame comprising the sequence of any of SEQ ID NOs: 3, 5, 7, and 9 and a 3' UTR comprising the sequence of SEQ ID NO: 2.
  • RNA molecule of any one of paragraphs 1 to 43, wherein the open reading frame comprises a G/C content of at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, of or of about 50% to 75% or 55% to 70%, or of or of about 58%, 66%, or 62%.
  • RNA molecule of any of paragraphs 1 to 45 wherein the RNA molecule comprises stabilized RNA.
  • RNA molecule of paragraph 47 wherein the modified nucleotide is pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1 -methyl-1 -deazapseudouridine, 2-thio-1 -methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 -methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyl uridine.
  • the modified nucleotide is pseudouridine, N1
  • RNA molecule of paragraph 47 or 48, wherein the modified nucleotide is N1 -methylpseudouridine (4»).
  • RNA molecule of any one of paragraphs 1 to 50 wherein the RNA is mRNA or self-replicating RNA.
  • RNA molecule of paragraph 51 wherein the RNA is a mRNA.
  • 53 A composition comprising the RNA molecule of any one of paragraphs 1 to 52, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • composition of paragraph 53 wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, and at least a first and second structural lipid.
  • composition of paragraph 53 or 54, wherein the lipid nanoparticle comprises a cationic lipid comprises a cationic lipid.
  • composition of paragraph 55, wherein the cationic lipid is (4- hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl)bis(2-hexyldecanoate) (ALC-0315).
  • PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, glycol-lipids including PEG- c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), and PEG- 2000-DMG, PEGylated diacylglycerol (PEG-DAG) such
  • composition of any one of paragraphs 56 to 58, wherein the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).
  • composition of paragraph 60, wherein the neutral lipid is 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE),
  • composition of paragraph 60 or 61 , wherein the neutral lipid is 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • composition of any one of paragraphs 53 to 65 comprising an RNA molecule at a concentration of or of about 0.01 to 0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising a cationic lipid at a concentration of or of about 0.8 to 0.95 mg/mL, a PEGylated lipid at a concentration of or of about 0.05 to 0.15 mg/mL, a neutral lipid at a concentration of or of about 0.1 to 0.25 mg/mL and a steroid or steroid analog at a concentration of or of about 0.3 to 0.45 mg/mL.
  • LNP lipid nanoparticle
  • composition of any one of paragraphs 53 to 66 comprising an RNA molecule at a concentration of or of about 0.01 to 0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising (4-hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl)bis(2- hexyldecanoate) (ALC-0315) at a concentration of or of about 0.8 to 0.95 mg/mL, 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of or of about 0.05 to 0.15 mg/mL, 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) at a concentration of or of about 0.1 to 0.25 mg/mL and cholesterol at a concentration of or of about 0.3 to 0.45 mg/mL.
  • LNP lipid nanoparticle
  • ALC-0315 lipid nanoparticle
  • composition of any one of paragraphs 53 to 67 comprising an RNA molecule at a concentration of or of about 0.06 mg/mL formulated in a lipid nanoparticle (LNP). 69.
  • composition of any one of paragraphs 53 to 69 further comprising at least a buffer and a stabilizing agent, and optionally, a salt diluent.
  • Tris buffer comprises tromethamine and Tris hydrochloride (HCI).
  • composition of paragraph 72, wherein the tromethamine is at a concentration of or of about 0.1 to 0.3 mg/mL or of or of about 0.01 to 0.15 mg/mL.
  • composition of paragraph 72 or 73, wherein and the Tris HCI is at a concentration of or of about 1 .25 to 1 .40 mg/mL or of or of about 0.5 to 0.65 mg/mL.
  • composition of paragraph 75 comprising an RNA molecule at a concentration of or of about 0.01 to 0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising a cationic lipid at a concentration of or of about 0.8 to 0.95 mg/mL, a PEGylated lipid at a concentration of or of about 0.05 to 0.15 mg/mL, a neutral lipid at a concentration of or of about 0.1 to 0.25 mg/mL and a steroid or steroid analog at a concentration of or of about 0.3 to 0.45 mg/mL, and further comprising a Tris buffer comprising tromethamine at a concentration of or of about 0.1 to 0.3 mg/mL and Tris hydrochloride (HCI) at a concentration of or of about 1 .25 to 1 .40 mg/mL, and sucrose at a concentration of or of about 95 to 110 mg/mL, wherein the composition is a liquid composition.
  • LNP lipid nanoparticle
  • composition of paragraph 75 comprising an RNA molecule at a concentration of or of about 0.01 to 0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising a cationic lipid at a concentration of or of about 0.8 to 0.95 mg/mL, a PEGylated lipid at a concentration of or of about 0.05 to 0.15 mg/mL, a neutral lipid at a concentration of or of about 0.1 to 0.25 mg/mL and a steroid or steroid analog at a concentration of or of about 0.3 to 0.45 mg/mL, and further comprising a Tris buffer comprising tromethamine at a concentration of or of about 0.01 to 0.15 mg/mL and Tris hydrochloride (HCI) at a concentration of or of about 0.5 to 0.65 mg/mL, sucrose at a concentration of or of about 35 to 50 mg/mL.
  • LNP lipid nanoparticle
  • HAI Tris hydrochloride
  • composition of paragraph 77 further comprising or comprising about 5 to 15 mM Tris buffer, 200 to 400 mM sucrose at a pH of or of about 7.0 to 8.0, and optionally, 0.9% sodium chloride diluent to reconstitute.
  • a method of inducing an immune response against Neisseria meningitidis in a subject comprising administering to the subject an effective amount of the RNA molecule and/or composition of any one of paragraphs 1 to 80.
  • a method of preventing, treating and/or ameliorating an infection, disease or condition in a subject comprising administering to a subject an effective amount of the RNA molecule and/or composition of any one of paragraphs 1 to 80.
  • RNA molecule and/or composition of any one of paragraphs 1 to 80 in the manufacture of a medicament for use in inducing an immune response against Neisseria meningitids in a subject.
  • RNA molecule or composition of any one of paragraphs 1 to 80 in the manufacture of a medicament for use in preventing, treating, and/or ameliorating an infection, disease, or condition in a subject.
  • RNA molecule and/or composition is administered by intradermal or intramuscular injection.

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Abstract

La présente divulgation concerne des molécules d'ARN codant pour un polypeptide de Neisseria meningitidis. La présente divulgation concerne en outre des compositions comprenant les molécules d'ARN formulées dans une nanoparticule lipidique (ARN-LNP). La présente divulgation concerne en outre l'utilisation des molécules d'ARN, des ARN-LNP et des compositions pour le traitement et/ou la prévention d'une maladie méningococcique.
PCT/IB2024/062402 2023-12-13 2024-12-10 Molécules d'arn Pending WO2025126019A1 (fr)

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