RNA-encoded peptidoglycan hydrolases and their uses for treating bacterial infections - Patents.com

Abstract

The present invention provides agents and methods for treating bacterial infections using RNA: RNA encoding a peptidoglycan hydrolase, e.g., an endolysin, is formulated and administered such that the peptidoglycan hydrolase protein, e.g., an endolysin protein, can be produced and secreted by cells of a subject to combat bacterial infection.

Description

[Technical Field]

[0001] The present invention provides agents and methods for treating bacterial infections using RNA. RNA encoding a peptidoglycan hydrolase, e.g., an endolysin, is formulated and administered such that the peptidoglycan hydrolase protein, e.g., an endolysin protein, can be produced and secreted by cells of a subject to combat the bacterial infection. [Background technology]

[0002] Despite the widespread use of small-molecule antibiotics, humans still suffer from more than 2 billion bacterial infections each year. While antibiotics are suitable for a range of these infections, there are several situations in which antibiotics do not provide reasonable cure rates. Resistance rates are rising rapidly, and novel therapies against antibiotic-resistant pathogens are urgently needed. Additionally, antibiotics are often ineffective against bacterial biofilms, the preferred habitat for many bacterial pathogens. Finally, our microbiomes are colonized by beneficial bacteria, and in many situations, antibiotics do more harm than good (e.g., the gut microbiome, where antibiotic therapy may be the treatment of choice for C. difficile). From the patient's perspective, novel therapies are most urgently needed when patients suffer from chronic infections in which antibiotics provide short-term relief but not sustained cure.

[0003] For all these reasons, new antibacterial agents are urgently needed.

[0004] Bacteriophage-encoded peptidoglycan hydrolases, also known as endolysins or "enzybiotics," are promising alternatives to antibiotics (Fischetti, 2018; Schmelcher et al., 2012). These enzymes are produced late in the lytic cycle of phage-infected bacteria and cleave the peptidoglycan (PG) of the bacterial cell wall, thus lysing the cell and releasing progeny phages. These enzymes were initially described for Gram-positive bacteria, which lack a Gram-negative outer membrane that can limit access to the cell wall peptidoglycan. Endolysins have several advantages over antibiotics, notably a very narrow host range, usually restricted to a single genus or even a single species (Fischetti, 2010), and a low tendency to generate resistance within the host (Schuch et al., 2014). Bacteriophages that invade Gram-positive bacteria encode a wide variety of highly diverse endolysins. In general, endolysins have a modular structure consisting of one or more enzymatically active domains (EADs), typically located at the C-terminus of the protein, connected by a flexible interdomain linker to at least one cell wall-binding domain (CBD), both of which may contribute to specificity for a given genus or species of bacteria (Oliveira et al., 2013).

[0005] In addition to phage-endolysins, other bactericidal proteins, including autolysins and bacteriocins, are known, and their activities can be combined with those of endolysins (Linden and Nelson, 2018). Autolysins are structurally very similar to endolysins and are used by bacteria to remodel their cell walls by partially lysing them during growth in size or cell division. Bacteriocins are typically produced by bacteria to kill other bacteria. Bacteriocins may have architectures homologous to endolysins, such as the bacteriocin lysostaphin, a metallopeptidase produced by Staphylococcus simulans to specifically kill its close relative, Staphylococcus aureus. Alternatively, they may be unrelated antibacterial peptides, such as nisin, that perforate the inner membrane of Gram-negative bacteria.

[0006] To date, endolysin resistance in bacteria has not been reported, and these enzymes have demonstrated activity against bacteria in suspension and in biofilms, a bacterial population notorious for conferring antibiotic resistance and causing a range of hospital-acquired and recurrent infections. Additionally, engineered lysin has been shown to have 10-fold higher bactericidal activity compared to native lysin (Landlinger C. et al. (2021) Pathogens 10(1):54).

[0007] There is a need to provide agents and treatments for treating bacterial infections. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2017 / 060314 Brochure [Patent Document 2] International Publication No. 2007 / 036366 Pamphlet [Patent Document 3] International Application No. PCT / EP2019 / 056502 Brochure [Patent Document 4] International Publication No. 2016 / 005324 A1 Brochure [Patent Document 5] US Patent Application Publication No. 2005 / 0287153 [Patent Document 6] US Patent Application Publication No. 2007 / 0003549 [Patent Document 7] US Patent Application Publication No. 20070048282 [Patent Document 8] U.S. Patent No. 5,876,969 [Patent Document 9] International Publication No. 2011 / 124718 Brochure [Patent Document 10] International Publication No. 2013 / 075066 Brochure [Patent Document 11] International Publication No. 2011 / 0514789 Brochure [Patent Document 12] US Patent Application Publication No. 2005 / 0287153 [Patent Document 13] US Patent Application Publication No. 2007 / 0003549 [Patent Document 14] US Patent Application Publication No. 2007 / 0178082 [Patent Document 15] US Patent Application Publication No. 2007 / 0269422 [Patent Document 16] US Patent Application Publication No. 2010 / 0113339 [Patent Document 17] International Publication No. 2009 / 083804 Brochure [Patent Document 18] International Publication No. 2009 / 133208 Brochure [Patent Document 19] U.S. Patent No. 7,176,278 [Patent Document 20] U.S. Patent No. 8,158,579 [Patent Document 21] US Patent Application Publication No. 2007 / 0178082 [Patent Document 22] US Patent Application Publication No. 2014 / 0220017 [Patent Document 23] US Patent Application Publication No. 2017 / 0145062 [Patent Document 24] US Patent Application Publication No. 2012 / 0094909 [Patent Document 25] International Publication No. 2017 / 182524 Brochure [Patent Document 26] International Publication No. 2017 / 162461 Brochure [Patent Document 27] International Publication No. 2017 / 075531 Brochure [Patent Document 28] International Publication No. 2018 / 081480 Brochure [Patent Document 29] International Publication No. 2017 / 060314 Brochure [Non-patent literature]

[0009] [Non-Patent Document 1] Landlinger C. et al. (2021) Pathogens 10(1):54 [Non-patent document 2] Smith and Waterman, 1981, Ads App. Math., vol. 2, p. 482 [Non-patent document 3] Neddleman and Wunsch, 1970, J. Mol. Biol., 48, 443 [Non-patent document 4] Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 88, 2444 [Non-Patent Document 5] Molecular Cloning: A Laboratory Manual, 4th edition, edited by MR Green and J. Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012

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[0010] Herein, we describe an approach for treating bacterial infections in which RNA, such as nucleoside-modified RNA (modRNA) or self-amplifying RNA (saRNA), is used to express an endolysin in a patient's cells. The RNA encoding the endolysin is formulated and administered such that the endolysin protein is produced and secreted by the patient's cells to combat the bacterial infection. [Means for solving the problem]

[0011] In one aspect, the invention provides an RNA encoding an amino acid sequence comprising a peptidoglycan hydrolase.

[0012] In some embodiments, the peptidoglycan hydrolase degrades peptidoglycan in the bacterial cell wall.

[0013] In some embodiments, the peptidoglycan hydrolase is derived from a bacterium.

[0014] In some embodiments, the peptidoglycan hydrolase is derived from a bacteriophage.

[0015] In some embodiments, the peptidoglycan hydrolase is or is derived from an endolysin.

[0016] In some embodiments, the endolysin comprises one or more enzymatically active domains (EADs) connected to one or more cell wall binding domains (CBDs) by flexible inter-domain linkers.

[0017] In some embodiments, the peptidoglycan hydrolase has an altered glycosylation pattern to improve blood pharmacokinetics.

[0018] In some embodiments, the peptidoglycan hydrolase is modified to reduce glycosylation.

[0019] In some embodiments, the peptidoglycan hydrolase is modified by removing glycosylation sites.

[0020] In some embodiments, the peptidoglycan hydrolase is modified to reduce immunogenicity.

[0021] In some embodiments, the peptidoglycan hydrolase is modified by removing T-cell epitopes.

[0022] In some embodiments, the peptidoglycan hydrolase is or is derived from endolysin Lys26A (derived from P. aeruginosa phage JD010).

[0023] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1.

[0024] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2.

[0025] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, or a fragment of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3.

[0026] In some embodiments, the peptidoglycan hydrolase is a secreted peptidoglycan hydrolase.

[0027] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises a signal peptide.

[0028] In some embodiments, the peptidoglycan hydrolase is fused to a signal peptide.

[0029] In some embodiments, the signal peptide is cleaved during secretion or export.

[0030] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10, or a fragment of the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10.

[0031] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4.

[0032] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0033] In some embodiments, the amino acid sequence comprising a peptidoglycan hydrolase comprises an extended pharmacokinetic (PK) polypeptide.

[0034] In some embodiments, the peptidoglycan hydrolase is fused to a pharmacokinetic-modifying group.

[0035] In some embodiments, the pharmacokinetic-modifying group comprises a moiety that is heterologous to the peptidoglycan hydrolase.

[0036] In some embodiments, the pharmacokinetic-modifying group comprises a moiety selected from the group consisting of albumin, an immunoglobulin fragment, transferrin, Fn3, a functional variant thereof, or a functional fragment of albumin, an immunoglobulin fragment, transferrin, Fn3, or a functional variant thereof.

[0037] In some embodiments, the pharmacokinetic-modifying group comprises human albumin, a functional variant thereof, or a functional fragment of human albumin or a functional variant thereof.

[0038] In some embodiments, the pharmacokinetic-modifying group is fused to the N-terminus of the peptidoglycan hydrolase.

[0039] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises, from N-terminus to C-terminus, N-pharmacokinetic modifier-GS-linker-peptidoglycan hydrolase-C.

[0040] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0041] In some embodiments, the RNA comprises modified nucleosides in place of uridine.

[0042] In some embodiments, the RNA includes a modified nucleoside in place of each uridine.

[0043] In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0044] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0045] In some embodiments, the RNA is suitable for expression of a peptidoglycan hydrolase in a eukaryotic cell, e.g., a mammalian cell, such as a human cell. In some embodiments, the RNA comprises sequence elements that allow for expression of the peptidoglycan hydrolase in a eukaryotic cell, e.g., a mammalian cell, such as a human cell. In some embodiments, such sequence elements comprise a cap, a 5' UTR, a 3' UTR, and a polyA.

[0046] In some embodiments, the RNA comprises a cap0 5' cap.

[0047] In some embodiments, the RNA is 5' capped m2 7,2'O G(5')ppSp(5')G (especially its D1 diastereomer).

[0048] In some embodiments, the RNA comprises a cap1 5' cap.

[0049] In some embodiments, the RNA comprises a 5' cap m27, 3'-O Gppp(m1 2'-O )Contains ApG.

[0050] In some embodiments, the RNA comprises a 5'UTR.

[0051] In some embodiments, the 5'UTR comprises the nucleotide sequence of SEQ ID NO:11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:11.

[0052] In some embodiments, the RNA comprises a 3'UTR.

[0053] In some embodiments, the 3'UTR comprises the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0054] In some embodiments, the RNA comprises a polyA sequence.

[0055] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0056] In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0057] In some embodiments, the RNA is mRNA.

[0058] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6.

[0059] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0060] In some embodiments, the RNA is self-amplifying RNA.

[0061] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, or a fragment of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0062] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0063] In some embodiments, the RNA is formulated as a liquid, a solid, or a combination thereof.

[0064] In some embodiments, the RNA is formulated for injection.

[0065] In some embodiments, the RNA is formulated for intravenous administration.

[0066] In some embodiments, the RNA is formulated as a lipid particle.

[0067] In some embodiments, the RNA-lipid particle is a lipid nanoparticle (LNP).

[0068] In some embodiments, the RNA is present in a composition or medical preparation.

[0069] In some embodiments, the composition is a pharmaceutical composition.

[0070] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0071] In some embodiments, the medical preparation is a kit.

[0072] In some embodiments, the medical preparation further comprises instructions for using the RNA to treat or prevent a bacterial infection.

[0073] In some embodiments, the RNA is for administration to a human.

[0074] In some embodiments, the RNA is for pharmaceutical use.

[0075] In some embodiments, pharmaceutical uses include therapeutic or prophylactic treatment of a disease or disorder.

[0076] In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing a bacterial infection.

[0077] In one aspect, the invention provides a composition or medical preparation comprising an RNA encoding an amino acid sequence comprising a peptidoglycan hydrolase.

[0078] In some embodiments, the peptidoglycan hydrolase degrades peptidoglycan in the bacterial cell wall.

[0079] In some embodiments, the peptidoglycan hydrolase is derived from a bacterium.

[0080] In some embodiments, the peptidoglycan hydrolase is derived from a bacteriophage.

[0081] In some embodiments, the peptidoglycan hydrolase is or is derived from an endolysin.

[0082] In some embodiments, the endolysin comprises one or more enzymatically active domains (EADs) connected to one or more cell wall binding domains (CBDs) by flexible inter-domain linkers.

[0083] In some embodiments, the peptidoglycan hydrolase has an altered glycosylation pattern to improve blood pharmacokinetics.

[0084] In some embodiments, the peptidoglycan hydrolase is modified to reduce glycosylation.

[0085] In some embodiments, the peptidoglycan hydrolase is modified by removing glycosylation sites.

[0086] In some embodiments, the peptidoglycan hydrolase is modified to reduce immunogenicity.

[0087] In some embodiments, the peptidoglycan hydrolase is modified by removing T-cell epitopes.

[0088] In some embodiments, the peptidoglycan hydrolase is or is derived from endolysin Lys26A (derived from P. aeruginosa phage JD010).

[0089] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1.

[0090] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2.

[0091] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, or a fragment of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3.

[0092] In some embodiments, the peptidoglycan hydrolase is a secreted peptidoglycan hydrolase.

[0093] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises a signal peptide.

[0094] In some embodiments, the peptidoglycan hydrolase is fused to a signal peptide.

[0095] In some embodiments, the signal peptide is cleaved during secretion or export.

[0096] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10, or a fragment of the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10.

[0097] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4.

[0098] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0099] In some embodiments, the amino acid sequence comprising a peptidoglycan hydrolase comprises an extended pharmacokinetic (PK) polypeptide.

[0100] In some embodiments, the peptidoglycan hydrolase is fused to a pharmacokinetic-modifying group.

[0101] In some embodiments, the pharmacokinetic-modifying group comprises a moiety that is heterologous to the peptidoglycan hydrolase.

[0102] In some embodiments, the pharmacokinetic-modifying group comprises a moiety selected from the group consisting of albumin, an immunoglobulin fragment, transferrin, Fn3, a functional variant thereof, or a functional fragment of albumin, an immunoglobulin fragment, transferrin, Fn3, or a functional variant thereof.

[0103] In some embodiments, the pharmacokinetic-modifying group comprises human albumin, a functional variant thereof, or a functional fragment of human albumin or a functional variant thereof.

[0104] In some embodiments, the pharmacokinetic-modifying group is fused to the N-terminus of the peptidoglycan hydrolase.

[0105] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises, from N-terminus to C-terminus, N-pharmacokinetic modifier-GS-linker-peptidoglycan hydrolase-C.

[0106] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0107] In some embodiments, the RNA comprises modified nucleosides in place of uridine.

[0108] In some embodiments, the RNA includes a modified nucleoside in place of each uridine.

[0109] In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0110] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0111] In some embodiments, the RNA is suitable for expression of a peptidoglycan hydrolase in a eukaryotic cell, e.g., a mammalian cell, such as a human cell. In some embodiments, the RNA comprises sequence elements that allow for expression of the peptidoglycan hydrolase in a eukaryotic cell, e.g., a mammalian cell, such as a human cell. In some embodiments, such sequence elements comprise a cap, a 5' UTR, a 3' UTR, and a polyA.

[0112] In some embodiments, the RNA comprises a cap0 5' cap.

[0113] In some embodiments, the RNA is 5' capped m2 7,2'O G(5')ppSp(5')G (especially its D1 diastereomer).

[0114] In some embodiments, the RNA comprises a cap1 5' cap.

[0115] In some embodiments, the RNA is 5' capped m2 7,3'-O Gppp(m1 2'-O )Contains ApG.

[0116] In some embodiments, the RNA comprises a 5'UTR.

[0117] In some embodiments, the 5'UTR comprises the nucleotide sequence of SEQ ID NO:11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:11.

[0118] In some embodiments, the RNA comprises a 3'UTR.

[0119] In some embodiments, the 3'UTR comprises the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0120] In some embodiments, the RNA comprises a polyA sequence.

[0121] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0122] In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0123] In some embodiments, the RNA is mRNA.

[0124] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6.

[0125] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0126] In some embodiments, the RNA is self-amplifying RNA.

[0127] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, or a fragment of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0128] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0129] In some embodiments, the RNA is formulated as a liquid, a solid, or a combination thereof.

[0130] In some embodiments, the RNA is formulated for injection.

[0131] In some embodiments, the RNA is formulated for intravenous administration.

[0132] In some embodiments, the RNA is or will be formulated as a lipid particle.

[0133] In some embodiments, the RNA-lipid particle is a lipid nanoparticle (LNP).

[0134] In some embodiments, the composition is a pharmaceutical composition.

[0135] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0136] In some embodiments, the medical preparation is a kit.

[0137] In some embodiments, the kit further comprises instructions for using the RNA to treat or prevent a bacterial infection.

[0138] In some embodiments, the composition or medical preparation is for administration to a human.

[0139] In some embodiments, the composition or medical preparation is for pharmaceutical use.

[0140] In some embodiments, pharmaceutical uses include therapeutic or prophylactic treatment of a disease or disorder.

[0141] In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing a bacterial infection.

[0142] In one aspect, the invention provides a cell comprising an RNA as described herein. In one aspect, the invention provides a cell transfected with an RNA as described herein.

[0143] In some embodiments, the cells are human cells.

[0144] In one aspect, the invention provides a population of cells, wherein at least one cell is a cell described herein. In one aspect, the invention provides a population of cells, wherein the population of cells comprises a plurality of cells described herein.

[0145] In some embodiments, the cell or population of cells described herein is present in a subject.

[0146] In one aspect, the invention provides a method for producing a peptidoglycan hydrolase in vivo, the method comprising transfecting a cell or population of cells with an RNA as described herein.

[0147] In some embodiments, the cell or population of cells described herein is present in a subject.

[0148] In some embodiments, the subject has been administered an RNA or composition described herein.

[0149] In one aspect, the invention provides a method for treating a bacterial infection in a subject, the method comprising administering to the subject RNA encoding an amino acid sequence comprising a peptidoglycan hydrolase.

[0150] In some embodiments, the peptidoglycan hydrolase degrades peptidoglycan in the bacterial cell wall.

[0151] In some embodiments, the peptidoglycan hydrolase is derived from a bacterium.

[0152] In some embodiments, the peptidoglycan hydrolase is derived from a bacteriophage.

[0153] In some embodiments, the peptidoglycan hydrolase is or is derived from an endolysin.

[0154] In some embodiments, the endolysin comprises one or more enzymatically active domains (EADs) connected to one or more cell wall binding domains (CBDs) by flexible inter-domain linkers.

[0155] In some embodiments, the peptidoglycan hydrolase has an altered glycosylation pattern to improve blood pharmacokinetics.

[0156] In some embodiments, the peptidoglycan hydrolase is modified to reduce glycosylation.

[0157] In some embodiments, the peptidoglycan hydrolase is modified by removing glycosylation sites.

[0158] In some embodiments, the peptidoglycan hydrolase is modified to reduce immunogenicity.

[0159] In some embodiments, the peptidoglycan hydrolase is modified by removing T-cell epitopes.

[0160] In some embodiments, the peptidoglycan hydrolase is or is derived from endolysin Lys26A (derived from P. aeruginosa phage JD010).

[0161] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1.

[0162] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2.

[0163] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, or a fragment of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3.

[0164] In some embodiments, the peptidoglycan hydrolase is a secreted peptidoglycan hydrolase.

[0165] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises a signal peptide.

[0166] In some embodiments, the peptidoglycan hydrolase is fused to a signal peptide.

[0167] In some embodiments, the signal peptide is cleaved during secretion or export.

[0168] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10, or a fragment of the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10.

[0169] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4.

[0170] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0171] In some embodiments, the amino acid sequence comprising a peptidoglycan hydrolase comprises an extended pharmacokinetic (PK) polypeptide.

[0172] In some embodiments, the peptidoglycan hydrolase is fused to a pharmacokinetic-modifying group.

[0173] In some embodiments, the pharmacokinetic-modifying group comprises a moiety that is heterologous to the peptidoglycan hydrolase.

[0174] In some embodiments, the pharmacokinetic-modifying group comprises a moiety selected from the group consisting of albumin, an immunoglobulin fragment, transferrin, Fn3, a functional variant thereof, or a functional fragment of albumin, an immunoglobulin fragment, transferrin, Fn3, or a functional variant thereof.

[0175] In some embodiments, the pharmacokinetic-modifying group comprises human albumin, a functional variant thereof, or a functional fragment of human albumin or a functional variant thereof.

[0176] In some embodiments, the pharmacokinetic-modifying group is fused to the N-terminus of the peptidoglycan hydrolase.

[0177] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase comprises, from N-terminus to C-terminus, N-pharmacokinetic modifier-GS-linker-peptidoglycan hydrolase-C.

[0178] In some embodiments, the amino acid sequence comprising the peptidoglycan hydrolase is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and the codon-optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0179] In some embodiments, the RNA comprises modified nucleosides in place of uridine.

[0180] In some embodiments, the RNA includes a modified nucleoside in place of each uridine.

[0181] In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0182] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0183] In some embodiments, the RNA comprises a cap0 5' cap.

[0184] In some embodiments, the RNA is 5' capped m2 7,2'O G(5')ppSp(5')G (especially its D1 diastereomer).

[0185] In some embodiments, the RNA comprises a cap1 5' cap.

[0186] In some embodiments, the RNA is 5' capped m2 7,3'-O Gppp(m1 2'-O )Contains ApG.

[0187] In some embodiments, the RNA comprises a 5'UTR.

[0188] In some embodiments, the 5'UTR comprises the nucleotide sequence of SEQ ID NO:11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:11.

[0189] In some embodiments, the RNA comprises a 3'UTR.

[0190] In some embodiments, the 3'UTR comprises the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0191] In some embodiments, the RNA comprises a polyA sequence.

[0192] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0193] In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:13.

[0194] In some embodiments, the RNA is mRNA.

[0195] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6.

[0196] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0197] In some embodiments, the RNA is self-amplifying RNA.

[0198] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, or a fragment of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0199] In some embodiments, the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0200] In some embodiments, the RNA is formulated as a liquid, a solid, or a combination thereof.

[0201] In some embodiments, the RNA is administered by injection.

[0202] In some embodiments, the RNA is administered by intravenous administration.

[0203] In some embodiments, the RNA is formulated as a lipid particle.

[0204] In some embodiments, the RNA-lipid particle is a lipid nanoparticle (LNP).

[0205] In some embodiments, the RNA is formulated as a pharmaceutical composition.

[0206] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0207] In some embodiments, the subject is a human.

[0208] In some embodiments, the infection is a chronic infection.

[0209] In some embodiments, the bacteria causing the infection is a multidrug-resistant bacteria.

[0210] In some embodiments, the bacteria causing the infection are gram-negative bacteria, gram-positive bacteria, or both.

[0211] In some embodiments, the bacteria is Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Porphyromonas gingivalis, Helicobacter pylori, Chlamydia trachomatis, Enterobacteriaceae, E. coli, Klebsiella pneumoniae, Salmonella, Shigella, Borrelia, Campylobacter jejuni, Neisseria gonorrhoeae, Chlamydia trachomatis, trachomatis, Vibrio cholerae, and Fusobacterium nucleatum.

[0212] In some embodiments, the bacteria is resistant to a carbapenem.

[0213] In some embodiments, the bacteria is selected from Staphylococci, Streptococci, Staphylococcus aureus, Streptococcus pneumoniae, Clostridioides difficile, Cutibacterium acnes, Mycobacterium tuberculosis, Gardnerella, Enterococcus faecalis, Enterococcus faecium, Lactobacillus iners, Bacillus subtilis, and Bacillus anthracis.

[0214] In some embodiments, the bacteria is resistant to methicillin and vancomycin or is non-susceptible to penicillin.

[0215] In some embodiments, the bacteria is methicillin-resistant Staphylococcus aureus (MRSA).

[0216] In some embodiments, the invention provides an agent described herein, e.g., an RNA, composition, or medical preparation described herein, for use in the methods described herein, e.g., for treating or preventing a bacterial infection or a disease associated with or caused by a bacterial infection.

[0217] In some embodiments, the RNA described herein is a single-stranded RNA that can be translated into a corresponding protein upon entry into a cell, e.g., a recipient cell. In addition to a wild-type or codon-optimized sequence encoding an amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having peptidoglycan hydrolase activity, e.g., an endolysin, the RNA may contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized to maximize the effectiveness of the RNA in terms of stability and translation efficiency. In some embodiments, the RNA contains all of these elements. In some embodiments, the RNA contains beta-S-ARCA(D1)(m2 7,2'-O GppSpG) or m2 7,3'-O Gppp(m1 2'-O ) ApG may be used as a specific capping structure at the 5' end of an RNA drug substance. The 5'-UTR sequence may be the 5'-UTR sequence of human alpha-globin mRNA, optionally with an optimized "Kozak sequence" to increase translation efficiency. The 3'-UTR sequence may be a combination of two sequence elements (FI elements) derived from an "amino-terminal split enhancer" (AES) mRNA (termed F) and a mitochondrially encoded 12S ribosomal RNA (termed I) located between the coding sequence and the poly(A) tail to ensure higher maximum protein levels and extended mRNA duration. These were identified through an ex vivo selection process for sequences that confer RNA stability and increase total protein expression (see WO 2017 / 060314, which is incorporated herein by reference). Alternatively, the 3'-UTR may be two repeat 3'-UTRs of human beta-globin mRNA. Additionally, a 110-nucleotide long poly(A) tail consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence (of random nucleotides), and another 70 adenosine residues may be used. This poly(A) tail sequence was designed to enhance RNA stability and translation efficiency.

[0218] The encoded amino acid sequence may include amino acid sequences other than the amino acid sequence of a peptide or polypeptide with peptidoglycan hydrolase activity. Such other amino acid sequences may support the function or activity of the peptide or polypeptide with peptidoglycan hydrolase activity. In some embodiments, such other amino acid sequences include amino acid sequences that enhance protein stability or persistence in the body.

[0219] Furthermore, in the RNA, a secretory signal peptide may be fused to the peptidoglycan hydrolase coding region. A sequence encoding a short linker peptide consisting mainly of the amino acids glycine (G) and serine (S), which is commonly used in fusion proteins, may be used as the GS / linker.

[0220] RNA can be formulated into lipid particles to produce serum-stable formulations for intravenous (iv) administration.RNA can also be present in lipid nanoparticles (LNP).RNA-nanoparticles can target the liver, thereby resulting in efficient expression of encoded proteins.In one embodiment, the RNA described herein is N1-methylpseudouridine-modified dsRNA purified RNA, which is formulated as lipid nanoparticles for intravenous administration. [Brief explanation of the drawings]

[0221] [Figure 1]Bactericidal activity of 11 ricins expressed and secreted in P. pastoris. Supernatants from P. pastoris cultures expressing different ricin constructs were mixed with ricin buffer at the indicated dilutions and incubated with S. aureus cells. Bacterial growth was monitored by measuring the culture OD over the course of 24 hours. A.) Growth curves of S. aureus in the presence of supernatants from different ricin-expressing P. pastoris strains L0502*: Supernatants from L0502 were diluted with supernatants from wild-type P. pastoris (wt S / N) that do not express recombinant ricin. B.) Supernatants from different clones derived from ricin L0499 were incubated with S. aureus at the indicated dilutions, and bacterial growth was monitored over time. [Figure 2] Detection of RNA-encoded LysPA26 in HEK293T cells by anti-FLAG BV241-conjugated antibody staining and flow cytometry. HEK293T cells analyzed by flow cytometry were incubated with no RNA (untransfected control), 9_LysPA26, and 10_LysPA26. Viability indicates cell survival after transfection, transfection indicates the percentage of cells showing protein expression, and expression indicates the mean fluorescence percentage of positive cells. Bar height indicates the average of technical triplicates. [Figure 3] Western blot of denatured proteins detected in the supernatant (culture medium) and cell lysates of HEK cells transfected with 9_LysPA26 and 10_LysPA26 at concentrations of 350 ng and 1 μg. The positive control used was HVV aptamer-FLAG (not shown due to overexposure). The expected product size was approximately 17 kDa. [Figure 4]Schematic diagram of the basic structure of RNA, with a 5'-cap, 5'- and 3'-untranslated regions, and a poly(A) tail. (A) Nucleoside-modified mRNA. (B) Self-amplifying mRNA further encodes a viral replicase and a subgenomic promoter. Individual elements are not drawn to scale relative to their respective sequence lengths. mRNA = messenger ribonucleic acid, SGP = subgenomic promoter, sec = secretory signal peptide, UTR = untranslated region, PolyA = polyadenosine. [Figure 5] Expression of endolysin in HEK cells. HEK293T cells were transfected with 0.5 μg and 1 μg of modRNA and saRNA of constructs 9_LysPA26 and 10_LysPA26. Extracellular proteins and proteins in cell lysates were detected. The left panel shows the modRNA supernatant and cell lysate. The construct with a secretion signal (10_LysPA26) is clearly detectable in the supernatant at both concentrations, whereas the construct without a secretion signal (9_LysPA26) is undetectable. The same results were observed with saRNA. [Figure 6]Expression of DNA- and RNA-encoded lysostaphin in cells. A, B: Concentration- and time-dependent expression of L0483 from mRNA (pST4-T7-AGA-dEarI-hAg-Kozak-SS(IGHV1-2)-His-3C-hL0483-FI-A30LA70) and DNA (pcDNA3.1(+)-SS(IGHV1-2)-His-3C-hL0483) in HEK293T / 17 cells. (A) 0.9 × 10 HEK293T / 17 cells were transfected with 3, 5, or 7.5 μg of mRNA. 2.5 μg of DNA was transfected in parallel. Supernatants were collected after 24 h (lane 1), 48 h (lane 2), or 72 h (lane 3) of expression. (B) Quantification of the chemiluminescent signal in panel A using Image Lab 6. C, D: Expression of L0483 mRNA-LNP (1.5 μg) in HEK293T / 17 and CHO-K1 cells. Expression was compared to mRNA transfection performed with Ribojuice (construct PL084_pST4-T7-AGA-dEarI-hAg-Kozak-SS(IGkappa)-His-3C-hL0483(PM)-FLAG-FI-A30LA70). (C) Expression levels varied significantly between cell lines. Transfection with ribojuice resulted in lower expression. (D) Approximately 15-fold higher expression was observed with mRNA-LNP compared to ribojuice transfection of the same mRNA. [Figure 7] Expression of DNA- and RNA-encoded ricin in HEK293T / 17 cells. Expression of ricin variants by mRNA or DNA transfection. HEK293T / 17 cells were transfected with 5 μg of mRNA or 2.5 μg of DNA. Supernatants were analyzed by Western blot to show expression from DNA as well as mRNA. [Figure 8]Bactericidal activity (A–D) and enzymatic activity (E) of ricin expressed from mRNA in HEK293T / 17 cells. (A–D) Growth inhibition assay in duplicate. (A) Concentrated supernatant from HEK293T / 17 transfection with 2.5 μg of L0483 DNA in duplicate. (B) Growth control in duplicate. (C) Concentrated supernatant from HEK293T / 17 transfection with L0483 mRNA-LNP in duplicate. (D) Concentrated supernatant from HEK293T / 17 transfection with luciferase mRNA-LNP in duplicate. (E) Enzymatic activity of ricin expressed from mRNA in HEK293T / 17 cells. [Figure 9] PK parameters of recombinant and mRNA-delivered lysostaphin - measurement of lysine levels in mouse plasma. (A) Three mice were treated with 535 μg of recombinant lysostaphin, and plasma lysine levels were measured for 24 hours. (B) Three mice were treated with 107 μg of recombinant lysostaphin, and plasma lysine levels were measured for 24 hours. (C) Five mice were treated with 30 μg of L0483 mRNA-LNP, and plasma lysine levels were measured for 72 hours. [Figure 10] In vivo efficacy of lysostaphin in S. aureus mouse bacteremia. Reduction of bacterial burden in various organs is shown after administration of two different doses of recombinant lysostaphin or SOC (vancomycin). DETAILED DESCRIPTION OF THE INVENTION

[0222] Array Description The following table provides a list of certain sequences referenced herein.

[0223] [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

[0224] Detailed Description of the Invention Although the present disclosure is further described in more detail below, it should be understood that the present disclosure is not limited to the particular methodology, protocols, and reagents described herein, which may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0225] The elements of the present disclosure will be described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining any number of the explicitly described embodiments and / or preferred elements of the present disclosure. Furthermore, unless the context dictates otherwise, all permutations and combinations of all described elements should be considered to be disclosed by the description of this application.

[0226] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the art.

[0227] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated feature, element, member, integer, or step, or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step, or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the scope of a claim or the disclosure to the specified features, elements, members, integers, or steps, and to those that do not materially affect the basic and novel characteristics of the claim or the disclosure. The term "consisting of" limits the scope of a claim or the disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0228] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0229] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0230] The use of any and all examples or exemplary language (e.g., "e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the disclosure as otherwise claimed. Language in the specification should be construed to indicate any non-claimed element essential to the practice of the disclosure.

[0231] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes both cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0232] As used herein, "and / or" should be construed as meaning that each of the two specified features or components is specifically disclosed without regard to the presence or absence of the other. For example, "X and / or Y" should be construed as meaning that (i) X, (ii) Y, and (iii) each of X and Y are specifically disclosed as if individually set forth herein.

[0233] In the context of the present disclosure, the term "about" refers to a range of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the stated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example, ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±2% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±1% from the stated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the stated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the stated numerical value. As one skilled in the art would understand, the specific such deviation of the numerical value for a given technical effect will depend on the nature of the technical effect, for example, a natural or biological technical effect may generally have a greater such deviation than a man-made or engineered technical effect.

[0234] The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually set forth herein.

[0235]

[0001] Throughout the text of this specification, several documents are cited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate the present disclosure by virtue of prior invention.

[0236] definition The following definitions apply to all aspects of this disclosure: The following terms have the following meanings unless otherwise indicated: Any undefined terms have their art-recognized meanings.

[0237] Terms such as "reduce" or "inhibit," as used herein, refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0238] Terms like "enhance" as used herein refer to the ability to cause an overall increase in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0239] "Physiological pH," as used herein, refers to a pH of about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0240] As used in this disclosure, "% wt / vol" refers to mass / volume percent, a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0241] As used in this disclosure, "wt. %" refers to percent by weight, which is a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total mass of the total composition in grams (g).

[0242] As used in this disclosure, "mol %" is defined as the ratio of moles of one component to the total moles of all components multiplied by 100.

[0243] As used in this disclosure, "mol % total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like substances.

[0244] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is determined by the following formula:

number

[0245] According to the present disclosure, the term "ionic strength" refers, in some embodiments, to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, the presence of chelating agents makes their concentration or effective concentration (presence of free ions) low enough in some embodiments to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or substantially absent.

[0246] "Osmolality" refers to the concentration of a particular solute expressed as osmoles of solute per kilogram of solvent.

[0247] The term "lyophilize" or "freeze-drying" refers to freeze-drying a substance by freezing the substance and then reducing the ambient pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or below 1 Pa or lower) to directly sublimate the freezing medium of the substance from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0248] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with a fluid being atomized (atomized) in a vessel (spray dryer) and evaporating the solvent from the droplets formed to produce a dry powder.

[0249] The term "reconstitute" relates to the addition of a solvent, such as water, to a dry product to return it to a liquid state, such as its original liquid state.

[0250] The term "recombinant" in the context of this disclosure means "produced by genetic engineering." In some embodiments, a "recombinant entity" in the context of this disclosure does not occur in nature.

[0251] The term "naturally occurring," as used herein, refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes not only known objects, but also objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0252] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures may include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0253] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given based on EDTA disodium salt.

[0254] The term "cryoprotectant" refers to a substance added to a formulation to protect the active ingredient during the freezing step.

[0255] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0256] According to the present disclosure, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Although both "peptide" and "polypeptide" are protein molecules, the terms "protein" and "polypeptide" are generally used synonymously herein.

[0257] The term "portion" refers to a fraction. When referring to a particular structure such as an amino acid sequence or a protein, the term "portion" can refer to a contiguous or discontinuous fraction of said structure.

[0258] The terms "portion" and "fragment" are used interchangeably herein and refer to a contiguous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a contiguous element of said structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% (0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.

[0259] The term "fragment", when referring to an amino acid sequence (peptide or polypeptide), refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3'-terminal open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5'-terminal open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of the amino acid sequence. Fragments of an amino acid sequence include, for example, a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30, or up to 55 consecutive amino acids of the amino acid sequence.

[0260] "Variant," as used herein with respect to an amino acid sequence (peptide or polypeptide), means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified version of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences, such as 1 to about 10 or 1 to about 5 amino acid differences, compared to the parent.

[0261] "Wild-type" or "WT" or "native," as used herein, refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified.

[0262] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide or polypeptide) may include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0263] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting products. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal truncated variants and / or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous peptides or polypeptides and / or the replacement of an amino acid with another amino acid with similar properties are preferred. In some embodiments, the amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., the substitution of a similarly charged or uncharged amino acid. Conservative amino acid changes involve the substitution of one member of an amino acid family for the side chain. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: - glycine, alanine; - valine, isoleucine, leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - serine, threonine; - lysine, arginine; and - Phenylalanine, tyrosine.

[0264] In some embodiments, the degree of similarity, such as identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is measured over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is determined over, for example, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is determined over the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, such as sequence identity, can be performed using a best sequence alignment using tools known in the art, such as Align using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, and Gap Extend 0.5.

[0265] "Sequence similarity" indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0266] The terms "% identical" and "% identity" or similar terms are intended to refer, inter alia, to the percentage of nucleotides or amino acids that are identical between the sequences to be compared after optimal alignment. The percentage is purely statistical; the differences between the two sequences may, but need not, be randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison may be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math., 2, 482; with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol., 48, 443; with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 88, 2444; or with the aid of computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, Inc., 575 Science Drive, Madison, Wisconsin). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include (i) an expectation threshold set to 10, (ii) a word size set to 28, (iii) a maximum number of matches within the query range set to 0, (iv) match / mismatch scores set to 1, -2, (v) gap costs set to linear, and (vi) use of a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include (i) an expectation threshold set to 10, (ii) a word size set to 3, (iii) a maximum number of matches within the query range set to 0, (iv) a matrix set to BLOSUM62, (v) gap costs set to presence: 11, extension: 1, and (vi) a conditional composition score matrix adjustment.

[0267] The percentage identity is obtained by determining the number of identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0268] In some embodiments, the degree of similarity or identity is determined over a region of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is determined over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, over consecutive nucleotides. In some embodiments, the degree of similarity or identity is determined over the entire length of the reference amino acid sequence.

[0269] Homologous amino acid sequences, according to the present disclosure, exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and for example at least 95%, at least 98%, or at least 99% identity of the amino acid residues.

[0270] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described in detail, for example, in Molecular Cloning: A Laboratory Manual, 4th Edition, edited by M.R. Green and J. Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, solid-phase synthesis and similar methods.

[0271] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. In the context of peptidoglycan hydrolases, one particular function is the activity of degrading peptidoglycan exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant," as used herein, particularly refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered compared to the amino acid sequence of the parent molecule or sequence, and that is still capable of performing one or more of the functions of the parent molecule or sequence, e.g., degrading peptidoglycan. In some embodiments, alterations to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however in other embodiments the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0272] An amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, one of skill in the art will understand that peptidoglycan hydrolases suitable for use herein may be altered to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0273] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition in a manufacturing process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0274] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient, or the cells may be present in vitro, e.g., outside the patient's body. Thus, according to the present invention, cells for transfection with nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or body of a patient. According to the present disclosure, transfection may be transient or stable. In some transfection applications, transient expression of the transfected genetic material is sufficient. Introducing RNA into cells can result in transient expression of the encoded protein. Because nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, the exogenous nucleic acid will be diluted or degraded during mitosis. In cells that allow episomal amplification of nucleic acids, the dilution rate is significantly reduced. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must be performed. Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system for transfection. Generally, the nucleic acid encoding the antigen is transiently introduced into the cell. When RNA is introduced into the cell, the encoded protein can be transiently expressed.

[0275] The present disclosure includes peptide or polypeptide analogs. According to the present disclosure, a peptide or polypeptide analog is a modified form of the peptide or polypeptide from which it is derived, and retains at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide retains at least one pharmacological activity of the peptide or polypeptide from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecules associated with the peptide or polypeptide, such as carbohydrates, lipids, and / or peptides or polypeptides. In some embodiments, a peptide or polypeptide "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the peptides and polypeptides.

[0276] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining together of two or more elements, components, or domains.

[0277] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0278] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0279] According to various embodiments of the present disclosure, nucleic acids, such as RNA, encoding a peptide or polypeptide are taken up or introduced, i.e., transfected or transduced, into cells, which may be present in vitro or present in a subject, resulting in expression of the peptide or polypeptide. The cells may, for example, produce the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or in the nucleus), secrete the encoded peptide or polypeptide, and / or express it on their surface. In some embodiments, the cells secrete the encoded peptide or polypeptide.

[0280] In accordance with the present disclosure, terms such as "expressing a nucleic acid" and "encoding a nucleic acid" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide or polypeptide, the nucleic acid is expressed when present in an appropriate environment, e.g., a cell, such that said peptide or polypeptide can be produced.

[0281] The term "expression", as used herein, includes transcription and / or translation of a particular nucleotide sequence.

[0282] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code of a DNA sequence is transcribed into RNA (in particular mRNA), which can then be translated into peptides or proteins.

[0283] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of amino acid sequences to make peptides or polypeptides.

[0284] The medical preparations described herein, particularly kits, may include instructional materials or instructions for use. As used herein, "instructional materials" or "instructions" includes publications, recordings, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional materials for kits of the present invention may, for example, be affixed to a container containing a composition of the present invention or may be shipped together with a container containing the composition. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the recipient use the instructional materials and the composition in conjunction.

[0285] A prodrug of a particular compound described herein is a compound that, when administered to an individual, undergoes chemical conversion under physiological conditions to provide the particular compound. Additionally, prodrugs can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the particular compound when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Exemplary prodrugs are in vivo hydrolyzable esters (using an alcohol or carboxy group contained in the particular compound) or amides (using an amino or carboxy group contained in the particular compound). Specifically, any amino group contained in the particular compound that has at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0286] In this specification, the structural formula of a compound may represent a particular isomer of said compound, but it should be understood that the present invention includes all isomers, such as structurally occurring geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, and tautomers, as well as mixtures of isomers, and is not limited to the description of the formula.

[0287] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains a pair of equal amounts of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even in pure form, spontaneously and reversibly interconvert by the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is keto-enol tautomer isomer. "Conformers" are stereoisomers that are formally interconvertible by simple rotation about a single bond, and include in particular those resulting in different three-dimensional configurations of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat cyclohexane.

[0288] The term "average diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in the so-called Z-average, which has the dimension of length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. Vol. 57, 1972, pp. 4814-4820, ISO 13321). Herein, the terms "average diameter," "diameter," or "size" of particles are used synonymously with this Z-average value.

[0289] In some embodiments, the "polydispersity index", as mentioned in the definition of "average diameter", can be calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.

[0290] The "radius of gyration" of the particle around the axis of rotation (herein referred to as R g) is the radial distance from the axis of rotation at which the moment of inertia about a given axis would be the same as the actual mass distribution if the total mass of the particle were assumed to be concentrated. Mathematically, R g is the root mean square distance of a particle component from either the center of mass or a given axis. For example, for a polymer consisting of n mass elements of mass mi (i = 1, 2, 3, . . . , n) located at a certain distance si from the center of mass, R g is the si over all mass elements 2 is the square root of the mass average of

[0291]

number

[0292] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, the small scattering vector

number

number

[0293] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius relates to the mobility of a particle, taking into account not only size but also solvent effects. For example, a smaller charged particle exhibiting stronger hydration may have a larger hydrodynamic radius than a larger charged particle exhibiting weaker hydration. This is because the smaller particle drags more water molecules with it as it moves through the solution. Because the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is calculated using the Stokes-Einstein equation:

number

[0294] The expression "light scattering," as used herein, refers to the physical process in which light is forced to deviate from its straight-line trajectory along one or more paths due to local inhomogeneities in the medium through which it passes.

[0295] The term "UV" means ultraviolet and refers to the band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, i.e., shorter than visible light but longer than X-rays.

[0296] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" as used herein means that scattered light at different, discrete angles can be detected, e.g., measured by a single detector moving over a range that includes a selected specific angle, or by an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser light source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the radius of gyration (R g ) and thus the size of the particles can be determined. Preferably, the Zimm plot is calculated using the following formula:

number

number

number

[0297] The terms "dynamic light scattering" or "DLS," as used herein, refer to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is shone onto a sample through a polarizer. The scattered light then passes through a second polarizer where it is detected, and the resulting image is projected onto a screen. When particles in solution are struck by light, they diffract the light in all directions. The diffracted light from the particles can either interfere constructively (bright areas) or destructively (dark areas). This process is repeated at short intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity of each spot over time.

[0298] The terms "static light scattering" or "SLS," as used herein, refer to a technique for determining the size and size distribution profile of particles, particularly related to their radius of gyration, and / or their molar mass. High-intensity monochromatic light, usually a laser, is projected into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. An accurate measurement of both the molar mass and the radial size of all macromolecules requires angular dependence. Therefore, simultaneous measurements at several angles relative to the direction of incident light are known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS) and are generally considered the standard implementation of static light scattering.

[0299] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a mixture of DNA and RNA. The nucleic acid may exist as a single-stranded or double-stranded, linear, or covalently closed circular molecule. The nucleic acid may be isolated. As used herein, the term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in the case of DNA, e.g., by polymerase chain reaction (PCR), or in the case of RNA, by in vitro transcription (e.g., using RNA polymerase); (ii) produced recombinantly by cloning; (iii) purified by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0300] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without a phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0301] The five standard nucleosides that typically make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated by the single-letter codes U, A, T, C, and G, respectively. However, thymidine contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine, and is therefore more commonly designated "dT" (the "d" stands for "deoxy"). This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. The remaining three nucleosides are designated A, C, and G in RNA but dA, dC, and dG in DNA.

[0302] In some embodiments, the modified purine (A or G) or pyrimidine (C, T, or U) base moiety is modified with one or more alkyl groups, e.g., one or more C1-4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, e.g., N 7 -C1~4 alkyl-guanine, N 6 -C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(1)-C1-4 alkyl-uracil, preferably N7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0303] As used herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or most deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking the hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the ends of DNA. As used herein, it is also contemplated that the nucleotides of DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For purposes of the present disclosure, such modified DNA is considered to be an analog of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogs).

[0304] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0305] The term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, the RNA contains mostly or entirely ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to internal RNA nucleotides or the addition of non-nucleotide material to the ends of the RNA. It is also contemplated herein that the nucleotides of the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of the present disclosure, such altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0306] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activator RNA (such as small activator RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0307] The term "in vitro transcription" or "IVT," as used herein, means that transcription (i.e., production of RNA) is performed in a cell-free manner. That is, IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0308] RNA According to the present disclosure, the term "RNA" includes "mRNA." According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced by using a DNA template. Generally, mRNA encodes a peptide or polypeptide.

[0309] Although mRNA is single stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0310] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0311] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide or polypeptide.

[0312] In some embodiments, preferably the mRNA encoding the peptide or polypeptide has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0313] As is established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 4th Edition, edited by M.R. Green and J. Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2012. Furthermore, various in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (TranscriptAid™ T7 kit, MEGAscript™ T7 kit, MAXIscript™, etc.), New England BioLabs Inc. (HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit, etc.), Promega (RiboMAX™, HeLaScribe™, Riboprobe™ system, etc.), Jena Bioscience (SP6 or T7 transcription kit, etc.), and Epicentre (AmpliScribe™, etc.). When providing modified mRNA, modified naturally occurring nucleotides, non-naturally occurring nucleotides, and / or corresponding modified nucleotides, such as modified non-naturally occurring nucleotides, may be incorporated during synthesis (preferably in vitro transcription), or modifications may be made to and / or added to the mRNA after transcription.

[0314] In some embodiments, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 promoter or an SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0315] In some embodiments of the present disclosure, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In certain embodiments, the replicon or self-replicating RNA is derived from or includes elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are a typical example of a positive-strand RNA virus. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5'-cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication, as well as protein modification) and structural proteins (which form the virus particle). Typically, there are two open reading frames (ORFs) in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by a first ORF that begins near the 5' end of the genome, while the structural proteins of alphaviruses are encoded together by a second ORF that is found downstream of the first ORF and extends to near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., 87, 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly like messenger RNA to translate the open reading frame encoding the nonstructural polyprotein (nsP1234).

[0316] Alphavirus-derived vectors have been proposed for delivering exogenous genetic information to target cells or organisms. A simple approach involves replacing the open reading frame encoding an alphavirus structural protein with an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase and the other nucleic acid molecule is capable of being replicated in trans by the replicase (hence the term trans-replication system). Trans-replication requires that both of these nucleic acid molecules be present in a given host cell. The nucleic acid molecule capable of being replicated in trans by the replicase must contain certain alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0317] In some embodiments of the present disclosure, the RNA comprises one or more modifications, for example, to increase its stability, increase translation efficiency, decrease immunogenicity, and / or decrease cytotoxicity. For example, to increase expression of the RNA, the RNA may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in International Publication No. WO 2007 / 036366 and International Application No. PCT / EP2019 / 056502, and include: 5'-cap structures; extension or truncation of naturally occurring poly(A) tails; alteration of the 5'- and / or 3'-untranslated regions (UTRs), such as the introduction of UTRs not associated with the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA).

[0318] In some embodiments, the RNA comprises a 5'-cap structure. In some embodiments, the RNA does not have an uncapped 5'-triphosphate. In some embodiments, the RNA may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found at the 5'-end of an RNA molecule, generally comprising guanosine 5'-triphosphate (Gppp) connected via its triphosphate moiety to the 5'-end of the next nucleotide in the RNA (i.e., the guanosine is connected to the rest of the RNA by a 5'-to-5' triphosphate linkage). Guanosine is an N 7 The cap structure m 7 Gppp). The term "5'-cap analog" includes 5'-caps that are based on a traditional 5'-cap but are modified at either the 2'- or 3'-position of the m7 guanosine structure to prevent incorporation of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also referred to as anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those with one or more substitutions at the bridging and non-bridging oxygens of the phosphate bridge, such as phosphorothioate-modified 5'-cap analogs at the β-phosphate (e.g., m27,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA)), as described in International Application No. PCT / EP2019 / 056502. Providing RNA with a 5'-cap structure as described herein can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5'-cap compound, with the 5'-cap structure being co-transcriptionally incorporated into the generated RNA strand, or the RNA can be generated, for example, by in vitro transcription, and the 5'-cap structure can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0319] In some embodiments, the RNA is m2 7,2'O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3 'O G(5')ppp(5')G, and m2 7,3'-O Gppp(m1 2'-O ) ApG.

[0320] In some embodiments, the RNA comprises cap0, cap1, or cap2, preferably cap1 or cap2. According to the present disclosure, the term "cap0" refers to the structure "m7GpppN", where N is any nucleoside with an OH group at the 2' position. According to the present disclosure, the term "cap1" refers to the structure "m 7 According to the present disclosure, the term "cap2" refers to the structure "m7GpppNmNm", where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0321] The 5'-cap analog beta-S-ARCA (β-S-ARCA) has the following structure: [ka]

[0322] The "D1" diastereomer of beta-S-ARCA, or "beta-S-ARCA(D1)," is the diastereomer of beta-S-ARCA that elutes first from an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)). The HPLC is preferably analytical HPLC. In some embodiments, a 5 μm, 4.6×250 mm Supelcosil LC-18-T RP column, capable of applying a flow rate of 1.3 ml / min, is preferably used for the separation. In some embodiments, a gradient of methanol in ammonium acetate is used, e.g., a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH 5.9, within 15 minutes. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with 280 nm excitation and 337 nm detection.

[0323] 5'-cap analog m2, a component of cap1 7,3'-O Gppp(m1 2'-O )ApG(m2 7,3'O G(5')ppp(5')m 2'-O ApG) has the following structure: [ka]

[0324] An exemplary cap0 mRNA containing β-S-ARCA and mRNA has the following structure: [ka]

[0325] An exemplary cap0 mRNA comprising m27,3'OG(5')ppp(5')G and mRNA has the following structure: [ka]

[0326] An exemplary cap1 mRNA comprising m27,3'-OGppp(m12'-O)ApG and mRNA has the following structure: [ka]

[0327] As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA molecule. Poly-A tails or poly-A sequences are known to those skilled in the art and may follow the 3'-UTR of the RNAs described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. Uninterrupted poly-A tails are typical in nature. The RNAs disclosed herein may have a poly-A tail attached to the free 3' end of the RNA after transcription by a template-independent RNA polymerase, or may have a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0328] Poly(A) tails of approximately 120 A nucleotides have been shown to have beneficial effects on RNA levels in transfected eukaryotic cells, as well as on the levels of proteins translated from open reading frames located upstream (5') of the poly(A) tail (Holtkamp et al., 2006, Blood, 108:4009-4017).

[0329] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, although the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consisting of" means that all nucleotides in the poly-A tail, i.e., 100% of the number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0330] In some embodiments, a poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is called a poly(A) cassette.

[0331] In some embodiments, a poly(A) cassette present on the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 can be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an even distribution of the four nucleotides (dA, dC, dG, and dT), e.g., 5-50 nucleotides in length, have been shown to promote uninterrupted propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level and to be associated with beneficial properties at the RNA level, including support for RNA stability and translation efficiency. Thus, in some embodiments, the poly-A tail included in the RNA molecules described herein consists essentially of A nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such random sequences may be 5-50, 10-30, or 10-20 nucleotides in length.

[0332] In some embodiments, the 3' end of the poly-A tail is not flanked by nucleotides other than A nucleotides, i.e., the poly-A tail is not blocked at its 3' end by nucleotides other than A, and is not followed at its 3' end by nucleotides other than A.

[0333] In some embodiments, the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail set forth in SEQ ID NO: 13. In some embodiments, the polyA tail comprises at least 100 nucleotides, in some embodiments, the polyA tail comprises about 150 nucleotides, in some embodiments, the polyA tail comprises about 120 nucleotides.

[0334] In some embodiments, the RNA used in the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region of a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region of an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) may be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" generally does not include a polyA sequence. Thus, the 3'-UTR is upstream of the polyA sequence (if present), e.g., directly adjacent to the polyA sequence. Incorporation of a 3'-UTR into the 3' untranslated region of an RNA (preferably mRNA) molecule can result in enhanced translation efficiency. Synergistic effects may be achieved by incorporating two or more such 3'-UTRs (the 3'-UTRs are preferably arranged in a head-to-tail orientation; see, e.g., Holtkamp et al., Blood 108:4009-4017 (2006)). The 3'-UTR may be autologous or heterologous to the RNA (e.g., mRNA) being introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as alpha2-globin, alpha1-globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin gene or mRNA. For example, an RNA (e.g., an mRNA) may be modified by replacing or inserting one or more, e.g., two, copies of a 3'-UTR from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0335] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 11. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 12.

[0336] In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:11.

[0337] In some embodiments, the RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

[0338] The RNA may have modified ribonucleotides to increase stability, and / or reduce immunogenicity, and / or reduce cytotoxicity. For example, in some embodiments, uridines in the RNA described herein are replaced (partially or completely, preferably completely) with modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0339] In some embodiments, the modified uridine replacing the uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0340] In some embodiments, the modified nucleoside that replaces (partially or completely, preferably completely) uridine in the RNA is 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), lysine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methyl- 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-uridine Methyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine The modified uridine may be any one or more of idouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (cmnm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (mcm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0341] RNA (preferably, mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "psi-modified," although the term "m1psi-modified" means that the RNA (preferably, mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably, mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such psi-modified or m1psi-modified or m5U-modified RNA typically exhibits reduced immunogenicity compared to the unmodified form and is therefore preferred for applications where induction of an immune response needs to be avoided or minimized. In some embodiments, the RNA (preferably, mRNA) contains N(1)-methylpseudouridine, completely replacing uridine.

[0342] The codons of the RNA used in the present disclosure may be further optimized, for example, to increase the GC content of the RNA and / or to replace codons that are rare in a cell (or subject) in which the peptide or polypeptide of interest is to be expressed with codons that are synonymously frequent in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA used in the present disclosure is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence. This includes embodiments in which one or more sequence regions of the coding sequence have been codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0343] The term "codon optimization" refers to changing the codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without changing the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, the coding region may be codon-optimized for optimal expression in a subject to be treated using the RNA described herein. Codon optimization is based on the finding that translation efficiency is also determined by differences in the frequency of tRNA occurrence in cells. Therefore, the sequence of the RNA may be modified so that codons available for frequently occurring tRNAs are inserted in place of "rare codons."

[0344] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of an RNA described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA, and the amino acid sequence encoded by the RNA is preferably unaltered 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 the efficient translation of that RNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Taking into account the fact that several codons encode the same amino acid (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing such codons with other codons that encode the same amino acid but do not contain A and / or U nucleotides or have a lower content of A and / or U nucleotides.

[0345] In various embodiments, the G / C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild-type RNA.

[0346] Combinations of the above-mentioned modifications, i.e., incorporation of a 5'-cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), substitution of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine, and / or pseudouridine (ψ) or N(1)-methylpseudouridine (mψ) or 5-methyluridine (m5U) for uridine), and codon optimization, have a synergistic effect on improving RNA (preferably mRNA) stability and translation efficiency. Thus, in some embodiments, the RNA used in the present disclosure includes a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications, namely: (i) incorporation of a 5' cap structure; (ii) incorporation of a polyA sequence, unmasking of a polyA sequence; (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) substitution of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine, and / or pseudouridine (ψ) or N(1)-methylpseudouridine (mψ) or 5-methyluridine (m5U) for uridine); and (v) codon optimization.

[0347] Some aspects of the present disclosure include targeted delivery of the RNA disclosed herein to certain cells or tissues.

[0348] Lipid-based RNA delivery systems have an inherent preference for the liver. Liver accumulation is caused by discontinuities in the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ is the liver and the target tissue is liver tissue. Delivery to such target tissue is preferred, particularly when it is desired that the RNA or the encoded peptide or polypeptide be present in this organ or tissue, and / or when it is desired to express the encoded peptide or polypeptide in large amounts, and / or when systemic presence of the encoded peptide or polypeptide, particularly in significant amounts, is desired or required.

[0349] In some embodiments, after administration of the RNA described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytoplasm of the target cell. In some embodiments, the RNA is RNA that encodes a peptide or polypeptide, and the RNA is translated by the target cell to produce the peptide or polypeptide. In some embodiments, the target cell is a liver cell. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a lymph node cell. In some embodiments, the target cell is a lung cell. In some embodiments, the target cell is a skin cell. Thus, the RNA particles described herein can be used to deliver RNA to such target cells.

[0350] Peptidoglycan hydrolases / endolysins and RNAs encoding peptidoglycan hydrolases / endolysins "Encoding" refers to the inherent property of a particular sequence of nucleotides of a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for synthesizing other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be said to encode the protein or other product of that gene or cDNA.

[0351] In some embodiments, RNA, such as mRNA, used in the present disclosure comprises a nucleic acid sequence encoding a peptidoglycan hydrolase, such as an endolysin.

[0352] In some embodiments, the RNA, such as mRNA, used in the present disclosure comprises a nucleic acid sequence encoding a peptidoglycan hydrolase, such as an endolysin, and is capable of expressing said peptidoglycan hydrolase, such as an endolysin, particularly when introduced into a cell or a subject, preferably a human cell or subject. Thus, in some embodiments, the RNA, such as mRNA, used in the present disclosure comprises a coding region (open reading frame (ORF)) encoding a peptidoglycan hydrolase, such as an endolysin. In this regard, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with a start codon and ending with a stop codon. In some embodiments, the RNA, such as mRNA, used in the present disclosure comprises nucleic acid sequences encoding more than one peptidoglycan hydrolase, such as an endolysin, for example, two, three, four, or more peptidoglycan hydrolase, such as an endolysin. In some embodiments, the one or more peptidoglycan hydrolase, such as an endolysin, encoded by the RNA may comprise or consist of a naturally occurring sequence, may comprise or consist of a variant of a naturally occurring sequence, or may comprise or consist of a non-naturally occurring sequence, e.g., a recombinant sequence. In some embodiments, the peptides or polypeptides encoded by the RNAs described herein may consist of or include one or more peptidoglycan hydrolases such as endolysins, and may include additional sequences such as secretion signals, extended PK groups, tags, and any other sequences. In some embodiments, the additional sequences are fused to the one or more peptidoglycan hydrolases such as endolysins, and in some embodiments, separated by a linker. In such embodiments, the one or more peptidoglycan hydrolases such as endolysins can be considered pharmaceutically active peptides or polypeptides, even if the additional sequences support the function or effect of the one or more peptidoglycan hydrolases such as endolysins.

[0353] According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" refers to a peptide or polypeptide that can be used to treat an individual in whom expression of the peptide or polypeptide is believed to be beneficial, e.g., in ameliorating the symptoms of a disease. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to ameliorate, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or pathology. A pharmaceutically active peptide or polypeptide may also have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutically active peptide or polypeptide" includes whole peptides or polypeptides and can also refer to pharmaceutically active fragments thereof. The term can also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0354] The peptidoglycan hydrolases, such as endolysins, described herein can be prepared as fusion or chimeric polypeptides comprising a peptidoglycan hydrolase moiety, such as an endolysin moiety, and a heterologous polypeptide (i.e., a polypeptide that is not a peptidoglycan hydrolase, such as an endolysin). The peptidoglycan hydrolase, such as an endolysin, may be fused to an extended PK group that increases its circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of peptides or polypeptides, such as peptidoglycan hydrolases, such as endolysins, or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0355] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses the properties of a compound, including, by way of example, absorption, distribution, metabolism, and excretion by a subject. As used herein, an "extended PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Patent Application Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, July 2016; 16(7):903-15, which is incorporated herein by reference in its entirety. As used herein, an "extended PK" peptidoglycan hydrolase, such as an "extended PK" endolysin, refers to a peptidoglycan hydrolase moiety, such as an endolysin moiety, combined with an extended PK group. In some embodiments, an extended PK peptidoglycan hydrolase, such as an extended PK endolysin, is a fusion protein in which a peptidoglycan hydrolase moiety, such as an endolysin moiety, is linked or fused to an extended PK group.

[0356] In certain embodiments, the serum half-life of an extended PK peptidoglycan hydrolase, such as an extended PK endolysin, is increased compared to the peptidoglycan hydrolase, such as an endolysin, alone (i.e., the peptidoglycan hydrolase, such as an endolysin, that is not fused to an extended PK group). In certain embodiments, the serum half-life of an extended PK peptidoglycan hydrolase, such as an extended PK endolysin, is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the peptidoglycan hydrolase, such as an endolysin, alone. In certain embodiments, the serum half-life of an extended PK peptidoglycan hydrolase, such as an extended PK endolysin, is at least 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 10x, 12x, 13x, 15x, 17x, 20x, 22x, 25x, 27x, 30x, 35x, 40x, or 50x greater than the serum half-life of the peptidoglycan hydrolase, such as an endolysin, alone. In certain embodiments, the serum half-life of the extended PK peptidoglycan hydrolase, such as an extended PK endolysin, is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0357] As used herein, "half-life" refers to the time it takes for the serum or plasma concentration of a compound, such as a peptide or polypeptide, to be reduced by 50% in vivo, e.g., by degradation and / or clearance or sequestration by natural mechanisms. Extended PK peptidoglycan hydrolases, such as extended PK endolysins, suitable for use in the present invention are stabilized in vivo, e.g., by fusion with serum albumin (e.g., HSA or MSA), which resists degradation and / or clearance or sequestration. Half-life can be determined by any method known per se, such as pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art. Suitable techniques may generally include, for example, administering a suitable dose of an amino acid sequence or compound to a subject; periodically collecting blood or other samples from the subject; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating, from a plot of the data thus obtained, the time until the level or concentration of the amino acid sequence or compound is reduced by 50% compared to the initial level at the time of administration. Further information is provided in standard handbooks such as, for example, Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd ed., Marcel Dekker (1982).

[0358] In certain embodiments, the extended PK group includes serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0359] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a protein such as a therapeutic protein, particularly a peptidoglycan hydrolase such as an endolysin. Albumin fusion proteins can be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding albumin. Once part of the albumin fusion protein, the therapeutic protein and albumin may each be referred to as a "portion," "region," or "moiety" of the albumin fusion protein (e.g., a "therapeutic protein portion" or an "albumin protein portion"). In highly preferred embodiments, the albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to, the mature form of a therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In some embodiments, the albumin fusion protein is processed by host cells, such as liver cells, in the target organ of the administered RNA and secreted into the circulatory system. Processing of the nascent albumin fusion protein in the secretory pathway of the host cell used to express the RNA includes, but is not limited to, signal peptide cleavage, disulfide bond formation, correct folding, carbohydrate addition and processing (e.g., N-linked and O-linked glycosylation), specific proteolytic cleavage, and / or assembly into multimeric proteins. The albumin fusion protein is preferably encoded by the RNA in an unprocessed form, particularly with the signal peptide at its N-terminus, and, after secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, the "processed form of the albumin fusion protein" refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as the "mature albumin fusion protein."

[0360] In preferred embodiments, albumin fusion proteins comprising a therapeutic protein have increased plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the time from in vivo administration of a therapeutic protein and its transport into the bloodstream until the therapeutic protein is degraded and excreted from the bloodstream into organs such as the kidneys or liver, resulting in its eventual elimination from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

[0361] As used herein, "albumin" refers generically to an albumin protein or amino acid sequence, or an albumin fragment or variant having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from another vertebrate, or a variant of such a molecule. Albumin may be derived from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of an albumin fusion protein may be derived from a different animal than the therapeutic protein portion.

[0362] In certain embodiments, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0363] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and include human serum albumin (and fragments and variants thereof), as well as albumin (and fragments and variants thereof) from other species.

[0364] As used herein, an albumin fragment sufficient to prolong the therapeutic activity or plasma stability of a therapeutic protein refers to an albumin fragment of sufficient length or structure to stabilize or prolong the therapeutic activity or plasma stability of that protein, such that the plasma stability of the therapeutic protein portion of an albumin fusion protein is extended or prolonged compared to the plasma stability of the unfused state.

[0365] The albumin portion of the albumin fusion protein may comprise the full-length albumin sequence or may comprise one or more fragments thereof that are capable of stabilizing or extending therapeutic activity or plasma stability. Such fragments may be 10 amino acids or longer, may comprise about 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may comprise part or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains can be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0366] Generally speaking, an albumin fragment or variant will be at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0367] According to the present disclosure, the albumin may be a naturally occurring albumin or a fragment or variant thereof. The albumin may be human albumin or may be derived from any vertebrate, particularly any mammal.

[0368] Preferably, the albumin fusion protein comprises albumin as the N-terminal moiety and a therapeutic protein as the C-terminal moiety. Alternatively, albumin fusion proteins comprising albumin as the C-terminal moiety and a therapeutic protein as the N-terminal moiety can also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N- and C-termini of albumin. In a preferred embodiment, the therapeutic proteins fused to the N- and C-termini are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused to the N- and C-termini are different therapeutic proteins. In some embodiments, both different therapeutic proteins are peptidoglycan hydrolases, such as endolysins.

[0369] In some embodiments, the therapeutic protein is conjugated to albumin via a peptide linker. The linker peptide between the fusion moieties provides greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety for, for example, binding to its cognate receptor. The linker peptide may be composed of amino acids and may be flexible or more rigid. The linker sequence may be protease or chemically cleavable.

[0370] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of the two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain, wherein the Fc domain does not include an Fv domain. In certain embodiments, an Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain, as used herein, generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1 domain, hinge domain, CH2 domain, and / or CH3 domain, as well as fragments of such peptides comprising, for example, only the hinge domain, CH2 domain, and CH3 domain.The Fc domain may be derived from any species and / or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. Fc domains encompass native Fc molecules and Fc variant molecules. As provided herein, one of skill in the art will understand that any Fc domain may be engineered to differ in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain exhibits reduced effector function (e.g., FcγR binding).

[0371] The Fc domains of the polypeptides described herein may be derived from different immunoglobulin molecules. For example, the Fc domain of the polypeptide may comprise a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0372] In certain embodiments, the extended PK group includes an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are included in the term "Fc domain" for purposes of this disclosure). The Fc domain does not include the variable region that binds to the antigen. Fc domains suitable for use in the present disclosure can be obtained from a number of different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain may also be derived from the immunoglobulin of another mammalian species, including, for example, a rodent species (e.g., mouse, rat, rabbit, guinea pig) or a non-human primate species (e.g., chimpanzee, macaque).

[0373] Furthermore, the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0374] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly available deposits. Constant region domains, including Fc domain sequences, can be selected to lack particular effector functions and / or to have specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from such sequences using art-recognized techniques.

[0375] In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in U.S. Patent Application Publication Nos. 2005 / 0287153, 2007 / 0003549, 2007 / 0178082, 2007 / 0269422, 2010 / 0113339, WO 2009 / 083804, and WO 2009 / 133208, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent Nos. 7,176,278 and 8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin-binding protein, such as those disclosed in U.S. Patent Application Publication Nos. 2007 / 0178082, 2014 / 0220017, and 2017 / 0145062, which are incorporated herein by reference in their entireties. In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as the protein disclosed in U.S. Patent Application Publication No. 2012 / 0094909, which is incorporated herein by reference in its entirety. Methods for fabricating fibronectin-based scaffold domain proteins are also disclosed in U.S. Patent Application Publication No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3(HSA), an Fn3 protein that binds to human serum albumin.

[0376] In certain aspects, one or more peptide linkers can be used in extended PK peptidoglycan hydrolases, such as extended PK endolysins, suitable for use in accordance with the present disclosure. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains of a linear amino acid sequence of a polypeptide chain (e.g., an extended PK portion and a peptidoglycan hydrolase portion, such as an endolysin portion). For example, a peptide linker can be used to connect a peptidoglycan hydrolase portion, such as an endolysin portion, to an HSA domain.

[0377] Linkers suitable for fusing extended PK groups to peptidoglycan hydrolases, such as endolysins, are well known in the art. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine-polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0378] In some embodiments, the RNA encoding a peptidoglycan hydrolase, such as an endolysin, is a single-stranded 5'-capped RNA that is translated into the respective protein upon entry into the cells of a subject to which the RNA is administered, e.g., liver cells, or cells of a tissue affected by a bacterial infection. Preferably, the RNA contains structural elements (5' cap, 5' UTR, 3' UTR, poly(A) sequence) optimized to maximize the effectiveness of the RNA in terms of stability and translation efficiency.

[0379] In some embodiments, beta-S-ARCA (D1) is used as the specific capping structure for the 5' end of the RNA. In some embodiments, m27,3'-OGppp(m12'-O)ApG is used as the specific capping structure for the 5' end of the RNA. In some embodiments, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the poly(A) sequence is 110 nucleotides in length and consists of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence was designed to enhance RNA stability and translation efficiency in dendritic cells. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

[0380] In some embodiments, the RNAs described herein have the structure: Cap-5'UTR-peptidoglycan hydrolase-3'UTR-Poly A. In some embodiments, the RNAs described herein have the structure: Cap-5'UTR-SP-peptidoglycan hydrolase-3'UTR-Poly A, where SP comprises the signal peptide coding sequence.

[0381] In some embodiments, RNA encoding a peptidoglycan hydrolase, such as an endolysin, is expressed in cells of a subject to provide a peptidoglycan hydrolase, such as an endolysin. In some embodiments, the peptidoglycan hydrolase, such as an endolysin, is secreted. In some embodiments, RNA encoding a peptidoglycan hydrolase, such as an endolysin, is transiently expressed in cells of a subject. In some embodiments, RNA encoding a peptidoglycan hydrolase, such as an endolysin, is administered systemically. In some embodiments, systemic administration of RNA encoding a peptidoglycan hydrolase, such as an endolysin, results in expression of RNA encoding a peptidoglycan hydrolase, such as an endolysin, in the liver. In some embodiments, systemic administration of RNA encoding a peptidoglycan hydrolase, such as an endolysin, results in expression of RNA encoding a peptidoglycan hydrolase, such as an endolysin, in cells of a tissue affected by a bacterial infection, e.g., lung cells.

[0382] The term "peptidoglycan hydrolase," as used herein, refers to any polypeptide capable of hydrolyzing bacterial peptidoglycan, such as that of Gram-negative bacteria. The term is not limited to a specific enzymatic cleavage mechanism. Three major classes of bacterial peptidoglycan hydrolases are glycosidases, which cleave the glycan backbone; amidases, which cleave side chain peptides; and peptidases (endopeptidases and carboxypeptidases), which cleave within peptide side chains. The term encompasses naturally occurring peptidoglycan hydrolases, such as those of eukaryotic, prokaryotic, or viral (e.g., bacteriophage) origin. The term encompasses, for example, lysozyme, endolysin, tail spike depolymerase, virion-associated peptidoglycan hydrolase (VAPGH), bacteriocins (e.g., lysostaphin), and autolysin. A "peptidoglycan hydrolase" may also be a synthetic or engineered polypeptide (e.g., a recombinant polypeptide) capable of hydrolyzing bacterial peptidoglycan. For example, enzymatically active shuffled endolysins in which two or more endolysin domains have been swapped / exchanged qualify as "peptidoglycan hydrolases," as do truncated endolysins in which only the enzymatically active domains remain. Peptidoglycan hydrolase activity can be measured by assays well known in the art, for example, antibacterial assays, such as those described by Briers et al. (J. Biochem. Biophys Methods; 2007; 70:531-533) or Donovan et al. (J. FEMS Microbiol Lett. 2006 December; 265(1)).

[0383] Endolysins include bacteriophage-encoded enzymes that act by hydrolyzing the host cell wall, subsequently allowing the release of bacteriophage progeny. The lytic activity of endolysins is classified into different types: (i) acetylmuramidases, (ii) transglycosylases, (iii) glucosaminidases, (iv) amidases, and (v) endopeptidases.

[0384] Endolysins generally have molecular weights ranging from 15 to 40 kDa. The majority of endolysins have a modular structure. Modular endolysins are often characterized by the presence of one or two (multi-domain) N-terminal enzymatically active domains (EADs) connected to a C-terminal cell wall-binding domain (CBD) by a short, flexible linker region. The enzymatically active domains (EADs) of modular endolysins function to cleave various specific peptidoglycan bonds in the murein layer of the host bacterium, while the cell wall-binding domains (CBDs) recognize and bind to various epitopes in the cell wall, thereby correctly anchoring the catalytic activity of the EADs.

[0385] Generally, endolysins that act on Gram-negative bacteria have a simple globular structure with an EAD without a CBD. Recent studies have also demonstrated the existence of Gram-negative phage endolysins with a globular structure containing one or two CBDs at the N-terminus but with the EAD module at the C-terminus.

[0386] EADs can be divided into three classes of six different enzymatic activities based on their mode of action and individual enzyme specificity. (i) Glycosidases generally cleave the β-1,4 glycosidic bonds that link the alternating polymeric structures of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in the peptidoglycan layer. (ii) Amidase catalyzes the cleavage of the amide bond between MurNAc and L-alanine, the first amino acid in the peptide stem. (iii) The endopeptidase cleaves the bond between two amino acids of the stem peptide. The bond cleavage can occur either within the interpeptide bridge or within the stem peptide-interpeptide bridge.

[0387] As used herein, the term "endolysin" includes peptidoglycan hydrolases encoded by bacteriophages (or bacterial viruses) and includes any peptidoglycan hydrolases suitable for hydrolyzing bacterial cell walls. Such peptidoglycan hydrolases may be naturally occurring enzymes of bacteriophages, modified enzymes of bacteriophages, and non-naturally occurring enzymes, e.g., recombinant enzymes, such as enzymes with non-naturally occurring arrangements of domains that may be derived from naturally occurring enzymes. In some embodiments, endolysins are enzymes that have been genetically engineered to alter their host range and efficiency. In some embodiments, endolysins include endolysins comprising naturally occurring domains and naturally occurring endolysins, e.g., endolysins comprising an N-terminal naturally occurring catalytic domain and a C-terminal naturally occurring cell wall binding domain, chimeric lysins characterized by shuffling of the natural domains to create desired properties, artilysins characterized by the addition of an LPS disrupting agent to disrupt Gram-negative outer membranes, and truncated lysines in which the C-terminal cell wall binding domain has been removed, e.g., to increase activity.

[0388] In some embodiments, the endolysin is derived from a bacteriophage (bacterial virus). In some embodiments, the endolysin may be a β-(1,4)-glycosylase (lysozyme), transglycosylase, amidase, or endopeptidase. In some embodiments, an "endolysin" comprises at least one "enzymatically active domain" (EAD) having at least one of the following activities: endopeptidase, N-acetyl-muramoyl-L-alanine-amidase (amidase), N-acetyl-muramidase, N-acetyl-glucosaminidase (lysozyme), or transglycosylase. In some embodiments, the endolysin may also comprise an enzymatically inactive region that binds to the cell wall of the host bacterium, a so-called CBD (cell wall binding domain). The endolysin may comprise one or more CBDs, for example, two or more CBDs. However, the term "endolysin" as used herein also includes enzymes that have at least one EAD but no CBD. Generally, the cell wall-binding domain can bind to various components on the surface of bacteria. Preferably, the cell wall-binding domain is a peptidoglycan-binding domain and binds to the peptidoglycan structure of bacteria. The different domains of an endolysin may be connected by a domain linker.

[0389] In Gram-positive bacteria, the plasma membrane is surrounded by a peptidoglycan layer. The main purpose of the cell wall of Gram-positive bacteria is to maintain the shape of the bacterium and resist pressure within the bacterial cell. Peptidoglycan, or murein, is a polymer composed of sugars and amino acids. The sugar components are composed of N-acetylglucosamine and N-acetylmuramic acid residues linked in a β-(1,4) configuration. Peptide chains of 3–5 amino acids are attached to the N-acetylmuramic acid. Peptide chains can crosslink with other peptide chains to form a 3D mesh-like layer. Peptide chains may contain both D- and L-amino acid residues, and their composition can vary depending on the type of bacteria.

[0390] In contrast to Gram-positive bacteria, Gram-negative bacteria have an outer membrane with a characteristic asymmetric bilayer: an inner monolayer containing phospholipids (mainly phosphatidylethanolamine) and an outer monolayer composed primarily of lipopolysaccharide (LPS).

[0391] The term "cell wall," as used herein, refers to any component that forms the outer cell enclosure of a bacterium and thus ensures the integrity of the bacterium. In particular, the term "cell wall," as used herein, refers to the peptidoglycan, the outer membrane of Gram-negative bacteria, the bacterial cell membrane, which has lipopolysaccharide, but also to additional layers deposited on the peptidoglycan, for example, as a capsule, outer protein layer, or slime.

[0392] The term "EAD" as used herein refers to the enzymatically active domain of an endolysin. The EAD is responsible for the hydrolysis of bacterial peptidoglycan. The EAD exhibits at least one enzymatic activity of an endolysin. The EAD may also be composed of more than one enzymatically active module. The term "EAD" is used herein synonymously with the term "catalytic domain."

[0393] Most Gram-negative bacteria and many Gram-positive bacteria form bacterial biofilms. A biofilm is defined as a collection of microorganisms attached to an aggregate or surface. The attached bacteria are often surrounded and protected by extracellular polymeric substances produced by Gram-negative and Gram-positive bacteria. Biofilms make bacteria more resistant to antibacterial agents such as antibiotics, disinfectants, and cell wall-degrading enzymes. Furthermore, biofilm treatment is currently not feasible because the extracellular polymeric substances protect the bacteria from degradation by antibacterial agents, disinfectants, or biofilm-degrading agents.

[0394] In some embodiments, the endolysin is or is derived from a Gram-negative phage endolysin. In some embodiments, the endolysin is or is derived from a Pseudomonas phage endolysin. In some embodiments, the endolysin is or is derived from a Pseudomonas aeruginosa phage endolysin. In some embodiments, the endolysin is or is derived from endolysin LysPA26 having the following sequence:

[0395] [ka]

[0396] For the purposes of this disclosure, the above sequence is considered to be the wild-type LysPA26 amino acid sequence. The position numbers of the LysPA26 protein indicated herein are relative to the corresponding positions in the amino acid sequence according to SEQ ID NO: 1 and LysPA26 protein variants.

[0397] In some embodiments, the encoded amino acid sequence comprises, consists essentially of, or consists of an endolysin, such as LysPA26.

[0398] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 6-138 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 6-138 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 6-138 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 6-138 of SEQ ID NO:1. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 6-138 of SEQ ID NO:1.

[0399] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 2 to 145 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 2 to 145 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 2 to 145 of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 2 to 145 of SEQ ID NO:1. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 2 to 145 of SEQ ID NO:1.

[0400] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a functional fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:1.

[0401] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:2.

[0402] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, and / or (ii) the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a functional fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:2 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:1.

[0403] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3, or a fragment of the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 3.

[0404] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, or a fragment of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, and / or (ii) the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a functional fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:3, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:1.

[0405] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:6.

[0406] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, and / or (ii) the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a functional fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:6, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:1.

[0407] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8, or a fragment of the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 8.

[0408] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, or a fragment of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, and / or (ii) the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a functional fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:8, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:1.

[0409] According to certain embodiments, the signal peptide (or signal sequence) is fused, either directly or via a linker, to a peptidoglycan hydrolase, such as an endolysin. Thus, in some embodiments, the signal peptide is fused to the above-described amino acid sequence derived from LysPA26 endolysin, or a functional fragment thereof contained in the above-described encoded amino acid sequence.

[0410] In some embodiments, the open reading frames of the RNA described herein encode polypeptides that include a signal sequence that is functional, for example, in mammalian cells.

[0411] In some embodiments, the signal sequence employed is "endogenous" in nature, in that it is associated with (eg, linked to) the encoded polypeptide.

[0412] In some embodiments, the signal sequence used is heterologous to the encoded polypeptide, e.g., is not naturally part of a polypeptide (e.g., protein) whose sequence is included in the encoded polypeptide.

[0413] In some embodiments, a signal peptide is a sequence typically characterized by a length of about 15 to 30 amino acids.

[0414] In many embodiments, but not limited to, the signal peptide is located at the N-terminus of the encoded polypeptide described herein. In some embodiments, signal peptides are preferably encoded by the RNAs of the present disclosure and enable the transport of the polypeptides to which they are associated to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosomal-lysosomal compartment.

[0415] In some embodiments, the RNA sequence encodes a peptidoglycan hydrolase, e.g., an endolysin, which may include or be otherwise linked to a signal sequence (e.g., a secretory sequence) such as those listed in Table 2, or a sequence having 1, 2, 3, 4, or 5 amino acid differences thereto. In some embodiments, a signal sequence such as MRVMAPRTLILLLSGALALTETWAGS, or a sequence having 1, 2, 3, 4, or up to 5 amino acid differences thereto, is used. In some embodiments, a sequence such as MRVMAPRTLILLLSGALALTETWAGS, or a sequence having 1, 2, 3, 4, or up to 5 amino acid differences thereto, is used.

[0416] In some embodiments, the signal sequence is selected from those included in Table 2 below.

[0417] [Table 2]

[0418] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a functional fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:4.

[0419] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:5.

[0420] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, and / or (ii) the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a functional fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:5, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:4.

[0421] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:7.

[0422] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, and / or (ii) the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a functional fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:7, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:4.

[0423] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO:9.

[0424] In some embodiments, the RNA encodes an amino acid sequence comprising (i) the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, and / or (ii) the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a functional fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO:9, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:4.

[0425] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 14, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14, or a functional fragment of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 14.

[0426] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 15 or 16, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16, or a fragment of the nucleotide sequence of SEQ ID NO: 15 or 16 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 15 or 16.

[0427] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 15 or 16, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15 or 16. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 14, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14, or a functional fragment of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 15 or 16, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 14.

[0428] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 17, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17, or a functional fragment of the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 17.

[0429] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 18 or 19, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 18 or 19, or a fragment of the nucleotide sequence of SEQ ID NO: 18 or 19 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 18 or 19. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 18 or 19.

[0430] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 18 or 19, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 18 or 19, or a nucleotide sequence of SEQ ID NO: 18 or 19 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 18 or 19. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 17, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17, or a functional fragment of the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 18 or 19, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 17.

[0431] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:20, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:20, or a functional fragment of the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:20.

[0432] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 21 or 22, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21 or 22, or a fragment of the nucleotide sequence of SEQ ID NO: 21 or 22 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21 or 22. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 21 or 22.

[0433] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO:21 or 22, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:21 or 22, or a nucleotide sequence of SEQ ID NO:21 or 22 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:21 or 22. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 20, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 20, or a functional fragment of the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 21 or 22 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 20.

[0434] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:23, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:23, or a functional fragment of the amino acid sequence of SEQ ID NO:23 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:23.

[0435] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 24 or 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 24 or 25, or a fragment of the nucleotide sequence of SEQ ID NO: 24 or 25 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 24 or 25. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 24 or 25.

[0436] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO:24 or 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:24 or 25, or a nucleotide sequence of SEQ ID NO:24 or 25 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:24 or 25. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 23, or a functional fragment of the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 24 or 25 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 23.

[0437] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:26, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:26, or a functional fragment of the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:26.

[0438] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 27 or 28, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 27 or 28, or a fragment of the nucleotide sequence of SEQ ID NO: 27 or 28 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 27 or 28. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 27 or 28.

[0439] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO:27 or 28, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:27 or 28, or a nucleotide sequence of SEQ ID NO:27 or 28 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:27 or 28. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 26, or a functional fragment of the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 27 or 28, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 26.

[0440] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29, or a functional fragment of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 29.

[0441] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 30 or 31, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30 or 31, or a fragment of the nucleotide sequence of SEQ ID NO: 30 or 31 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30 or 31. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 30 or 31.

[0442] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 30 or 31, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30 or 31, or a nucleotide sequence of SEQ ID NO: 30 or 31 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30 or 31. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29, or a functional fragment of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 30 or 31, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29.

[0443] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 32, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 32, or a functional fragment of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 32.

[0444] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 33 or 34, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 33 or 34, or a fragment of the nucleotide sequence of SEQ ID NO: 33 or 34 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 33 or 34. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 33 or 34.

[0445] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 33 or 34, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 33 or 34, or a nucleotide sequence of SEQ ID NO: 33 or 34 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 33 or 34. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 32, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 32, or a functional fragment of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 33 or 34, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 32.

[0446] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 35, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 35, or a functional fragment of the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 35.

[0447] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 36 or 37, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 36 or 37, or a fragment of the nucleotide sequence of SEQ ID NO: 36 or 37 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 36 or 37. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 36 or 37.

[0448] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 36 or 37, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 36 or 37, or a nucleotide sequence of SEQ ID NO: 36 or 37 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 36 or 37. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 35, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 35, or a functional fragment of the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 36 or 37, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 35.

[0449] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 38, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 38, or a functional fragment of the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 38.

[0450] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 39 or 40, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 39 or 40, or a fragment of the nucleotide sequence of SEQ ID NO: 39 or 40 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 39 or 40. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 39 or 40.

[0451] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 39 or 40, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 39 or 40, or a nucleotide sequence of SEQ ID NO: 39 or 40 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 39 or 40. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 38, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 38, or a functional fragment of the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 39 or 40 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 38.

[0452] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:41, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:41, or a functional fragment of the amino acid sequence of SEQ ID NO:41 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO:41.

[0453] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 42 or 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 42 or 43, or a fragment of the nucleotide sequence of SEQ ID NO: 42 or 43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 42 or 43. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 42 or 43.

[0454] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 42 or 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 42 or 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 42 or 43. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 41, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 41, or a functional fragment of the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 42 or 43, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 41.

[0455] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 44, or a functional fragment of the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 44.

[0456] In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 45 or 46, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 45 or 46, or a fragment of the nucleotide sequence of SEQ ID NO: 45 or 46 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 45 or 46. In some embodiments, the RNA comprises the nucleotide sequence of SEQ ID NO: 45 or 46.

[0457] In some embodiments, the RNA comprises (i) the nucleotide sequence of SEQ ID NO: 45 or 46, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 45 or 46, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 45 or 46. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 44, or a functional fragment of the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the RNA (i) comprises the nucleotide sequence of SEQ ID NO: 45 or 46, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 44.

[0458] RNA Embodiments Described Herein Below, embodiments of RNA for use herein are described, and certain terms used in describing those elements have the following meanings. hAg-Kozak: 5'-UTR sequence of human alpha-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency. SP: signal peptide. Endolysin: A sequence encoding the corresponding endolysin or fragment. FI elements: The 3'-UTR is a combination of two sequence elements derived from the "amino-terminal split enhancer" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified through an ex vivo selection process for sequences that confer RNA stability and increase total protein expression. A30L70: a 110 nucleotide long poly(A) tail consisting of a stretch of 30 adenosine residues followed by a 10 nucleotide linker sequence and another 70 adenosine residues.

[0459] In some embodiments, the RNA described herein has the following structure: Cap-hAg-Kozak-Endolysin-FI-A30L70

[0460] In some embodiments, the RNA described herein has the following structure: Cap-hAg-Kozak-SP-Endolysin-FI-A30L70

[0461] In some embodiments, the encoded amino acid sequences described herein have the following structure: SP-endolysin

[0462] In some embodiments, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, SP comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, FI comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, A30L70 comprises the nucleotide sequence of SEQ ID NO: 13.

[0463] In some embodiments, the RNA used in this disclosure is non-immunogenic.

[0464] The term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA"), as used herein, refers to RNA that, when administered to a mammal, does not induce a response by the immune system or that induces a response that is weaker than the response that would be induced by the same RNA, i.e., standard RNA (stdRNA), except that the RNA has not been subjected to the modifications and processing that render the non-immunogenic RNA non-immunogenic. In certain embodiments, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated innate immune receptor activation, and / or by limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA) during, e.g., in vitro transcription, and / or by removing double-stranded RNA (dsRNA) after, e.g., in vitro transcription. In certain embodiments, the non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated innate immune receptor activation and / or by removing double-stranded RNA (dsRNA), e.g., after in vitro transcription.

[0465] To render non-immunogenic RNA (particularly mRNA) non-immunogenic by incorporating a modified nucleoside, any modified nucleoside can be used as long as it reduces or suppresses the immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated innate immune receptor activation. In some embodiments, the modified nucleoside comprises replacing one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (cmnm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (mcm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5In certain embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0466] In some embodiments, replacing one or more uridines with a nucleoside comprising a modified nucleobase comprises replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridines.

[0467] During the synthesis of RNA by in vitro transcription (IVT) using T7 RNA polymerase, abnormal activity of the enzyme produces significant amounts of aberrant products, including double-stranded RNA (dsRNA). dsRNA induces inflammatory cytokines and activates effector enzymes, leading to protein synthesis inhibition. The formation of dsRNA during in vitro transcription (IVT) RNA synthesis can be limited, for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP can be added one or more times during RNA synthesis. dsRNA can also be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using, for example, a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used, using Escherichia coli RNase III, which specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA by using cellulose material.In some embodiments, RNA preparation is contacted with cellulose material, and ssRNA is separated from cellulose material under the conditions that allow dsRNA to bind to cellulose material, and allow ssRNA not to bind to cellulose material.Suitable method for providing ssRNA is disclosed in, for example, WO2017 / 182524.

[0468] As the term is used herein, "removing" or "removal" refers to the property of separating a population of a first substance, such as non-immunogenic RNA, from the vicinity of a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, a population of a first substance characterized by being devoid of the population of the second substance will have a measurably lower content of the second substance compared to an unseparated mixture of the first and second substances.

[0469] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, for example, dsRNA (particularly mRNA) is removed from non-immunogenic RNA so that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, non-immunogenic RNA (particularly mRNA) does not contain or is essentially free of dsRNA. In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside-modified RNA. In some embodiments, non-immunogenic RNA (particularly mRNA) compositions comprise single-stranded nucleoside-modified RNA (particularly mRNA) and are substantially free of double-stranded RNA (dsRNA). In some embodiments, non-immunogenic RNA (particularly mRNA) compositions comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single-stranded nucleoside-modified RNA compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0470] The amount of dsRNA can be determined by various methods.For example, sample can be contacted with dsRNA specific antibody, and the amount of antibody that binds to RNA can be considered as the measure of the amount of dsRNA in sample.The sample that contains known amount of dsRNA can be used as reference.

[0471] For example, RNA can be spotted on membrane, for example nylon blotting membrane.Membrane can be blocked with TBS-T buffer solution (20mM TRIS pH 7.4, 137mM NaCl, 0.1% (volume / volume) TWEEN-20) containing 5% (weight / volume) skim milk powder.For detecting dsRNA, membrane can be incubated with dsRNA-specific antibody, for example, dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary).For example, after washing with TBS-T, membrane can be incubated with secondary antibody, for example, HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, catalog number 715-035-150), and the signal provided by secondary antibody can be detected.

[0472] In some embodiments, non-immunogenic RNA (especially mRNA) is translated more efficiently in cells than standard RNA with the same sequence. In some embodiments, translation is enhanced 2-fold relative to the unmodified counterpart. In some embodiments, translation is enhanced 3-fold. In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, the fold is 10-1000 fold. In some embodiments, the fold is 10-100 fold. In some embodiments, the fold is 10-200 fold. In some embodiments, the fold is 10-300 fold. In some embodiments, the fold is 10-500 fold. In some embodiments, the fold is 20-1000 fold. In some embodiments, the fold is 30-1000 fold. In some embodiments, the fold is 50-1000 fold. In some embodiments, the fold is 100-1000 fold. In some embodiments, the fold is 200-1000 fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0473] In some embodiments, non-immunogenic RNA (especially mRNA) exhibits significantly lower natural immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits two-fold less natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced by three-fold. In some embodiments, the natural immunogenicity is reduced by four-fold. In some embodiments, the natural immunogenicity is reduced by five-fold. In some embodiments, the natural immunogenicity is reduced by six-fold. In some embodiments, the natural immunogenicity is reduced by seven-fold. In some embodiments, the natural immunogenicity is reduced by eight-fold. In some embodiments, the natural immunogenicity is reduced by nine-fold. In some embodiments, the natural immunogenicity is reduced by ten-fold. In some embodiments, the natural immunogenicity is reduced by fifteen-fold. In some embodiments, the natural immunogenicity is reduced by twenty-fold. In some embodiments, the natural immunogenicity is reduced by fifty-fold. In some embodiments, the natural immunogenicity is reduced 100-fold. In some embodiments, the natural immunogenicity is reduced 200-fold. In some embodiments, the natural immunogenicity is reduced 500-fold. In some embodiments, the natural immunogenicity is reduced 1000-fold. In some embodiments, the natural immunogenicity is reduced 2000-fold.

[0474] The term "exhibiting significantly lower natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction in the extent to which an effective amount of non-immunogenic RNA (especially mRNA) can be administered without eliciting a detectable natural immune response. In some embodiments, this term refers to a reduction in the extent to which non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0475] "Immunogenicity" is the ability of a foreign substance, such as RNA, to evoke an immune response in humans or other animals. The innate immune system is a relatively nonspecific, immediate component of the immune system. It is one of the two major components of the vertebrate immune system, along with the adaptive immune system.

[0476] In some embodiments, the RNA described herein is single-stranded RNA. In some embodiments, the RNA described herein is linear RNA. In some embodiments, the single-stranded RNA is coding RNA in that its nucleotide sequence comprises an open reading frame. In some embodiments, the single-stranded RNA has a nucleotide sequence that encodes a polypeptide or multiple polypeptides of the present disclosure.

[0477] In many embodiments, the related RNA is mRNA.

[0478] In some embodiments, the RNA comprises unmodified uridine residues, and RNA comprising only unmodified uridine residues may be referred to as "uRNA." In some embodiments, the RNA comprises one or more modified uridine residues, and in some embodiments, such RNA (e.g., RNA comprising entirely modified uridine residues) is referred to as "modRNA." In some embodiments, the RNA may be self-amplifying RNA (saRNA). In some embodiments, the RNA may be trans-amplifying RNA (see, e.g., WO 2017 / 162461).

[0479] In some embodiments, the RNA (e.g., single-stranded RNA) described herein has a length of at least 500 ribonucleotides (e.g., at least 600 ribonucleotides, at least 700 ribonucleotides, at least 800 ribonucleotides, at least 900 ribonucleotides, at least 1000 ribonucleotides, at least 1250 ribonucleotides, at least 1500 ribonucleotides, at least 1750 ribonucleotides, at least 2000 ribonucleotides, at least 2500 ribonucleotides, at least 3000 ribonucleotides, at least 3500 ribonucleotides, at least 4000 ribonucleotides, at least 4500 ribonucleotides, at least 5000 ribonucleotides, or longer). In some embodiments, the RNA described herein is a single-stranded RNA having a length of between about 800 ribonucleotides and 5000 ribonucleotides.

[0480] In some embodiments, the associated RNA comprises a polypeptide-encoding portion or portions. In certain embodiments, such one or more portions encode at least one or more polypeptides that are or include a peptidoglycan hydrolase, e.g., an endolysin. In certain embodiments, the encoded peptidoglycan hydrolase, e.g., an endolysin, may be a variant of a wild-type polypeptide.

[0481] In some embodiments, the single-stranded RNA (e.g., mRNA) may include a secretory signal coding region (e.g., a secretory signal coding region that enables one or more encoded target entities (including peptidoglycan hydrolases, e.g., endolysins) to be secreted upon translation by a cell). In some embodiments, such a secretory signal coding region may be or include a non-human secretory signal. In some embodiments, such a secretory signal coding region may be or include a human secretory signal.

[0482] In some embodiments, a single-stranded RNA (e.g., an mRNA) may contain at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, a 3' untranslated region (UTR), a 5' UTR, a cap structure, a polyadenine (polyA) tail, and any combination thereof.

[0483] At least four formats useful for RNA pharmaceutical compositions can be used herein: unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), self-amplifying mRNA (saRNA), and trans-amplifying RNA.

[0484] Characteristics of the modified uridine (e.g., pseudouridine) platform can include reduced adjuvant effects, a blunted ability to activate immune innate immune sensors, and thereby increased polypeptide (e.g., protein) expression.

[0485] Characteristics of self-amplifying platforms can include, for example, longer duration of polypeptide (e.g., protein) expression, better tolerance and safety, and a greater likelihood of efficacy even at very low RNA doses.

[0486] In some embodiments, a self-amplifying platform (e.g., RNA) comprises two nucleic acid molecules, one nucleic acid molecule encoding a replicase (e.g., a viral replicase) and another nucleic acid molecule capable of replication in trans (e.g., a replicon) by the replicase (a trans-replication system). In some embodiments, a self-amplifying platform (e.g., RNA) comprises multiple nucleic acid molecules, the nucleic acids encoding multiple replicases and / or replicons.

[0487] In some embodiments, the trans-replication system comprises the presence of both nucleic acid molecules in a single host cell.

[0488] In some such embodiments, the nucleic acid encoding the replicase (e.g., a viral replicase) is not capable of autonomous replication in the target cell and / or target organism, hi some such embodiments, the nucleic acid encoding the replicase (e.g., a viral replicase) lacks at least one conserved sequence element important for (+)-strand-templated (-)-strand synthesis and / or (-)-strand-templated (+)-strand synthesis.

[0489] In some embodiments, the self-amplifying RNA comprises a 3' untranslated region (UTR), a 5' UTR, a cap structure, a polyadenine (polyA) tail, and any combination thereof.

[0490] In some embodiments, the self-amplifying platform does not require viral particle propagation (e.g., does not involve unwanted viral particle formation). In some embodiments, the self-amplifying platform is not capable of forming viral particles.

[0491] particle The RNA, particularly mRNA, described herein may be present in particles comprising (i) RNA and (ii) at least one cationic or cationic ionizable compound, such as a polymer or lipid, that forms a complex with the RNA. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged RNA are involved in particle formation, which results in complex formation and spontaneous formation of RNA particles.

[0492] Various types of RNA-containing particles suitable for delivering particulate RNA have been previously described (see, for example, Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA can physically protect the RNA from degradation and, depending on the specific chemical properties, aid in cellular uptake and endosomal escape.

[0493] In the context of the present disclosure, the term "particle" refers to a structured entity formed by molecules or molecular complexes, particularly particle-forming compounds. In some embodiments, a particle comprises an envelope (e.g., one or more layers or lamellae) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the term "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) that do not necessarily have to be amphiphilic. Thus, a particle may have a monolayer or multilayer structure, and the substances that make up one or more layers or lamellae may comprise one or more types of amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids), optionally in combination with additional substances (e.g., additional lipids) that do not necessarily have to be amphiphilic. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized miniature structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0494] "RNA particles" can be used to deliver RNA to a desired target site (e.g., cells, tissues, organs, etc.). The RNA particles may be formed from lipids containing at least one cationic or cationic ionizable lipid or lipid-like substance. Without intending to be bound by any theory, the cationic or cationic ionizable lipid or lipid-like substance forms aggregates with the RNA, which aggregates result in colloidally stable particles.

[0495] The RNA particles described herein include lipid nanoparticle (LNP)-based formulations and lipoplex (LPX)-based formulations.

[0496] Generally, lipoplexes (LPX) can be obtained by mixing two aqueous phases: one containing RNA and one containing a lipid dispersion. In some embodiments, the lipid phase comprises liposomes.

[0497] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles, in which the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes in nanoparticle formation is that the lipids in the required mixture are capable of forming a lamellar (bilayer) phase in the aqueous environment to which they are applied.

[0498] In some embodiments, liposomes comprise a unilamellar or multilamellar phospholipid bilayer surrounding an aqueous core (also referred to herein as an aqueous lumen). Liposomes can be prepared from substances with polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids used to formulate liposomes designed for RNA delivery are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0499] In some embodiments, the lipoplexes are multilamellar liposome-based formulations formed upon electrostatic interaction between cationic liposomes and RNA. In some embodiments, the formed lipoplexes have a distinct internal arrangement of molecules resulting from the conversion of the liposomal structure to small RNA lipoplexes. In some embodiments, such formulations are characterized by poor RNA encapsulation and incomplete RNA capture.

[0500] In some embodiments, the LPX particles comprise amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids, and RNA (particularly mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made of one or more amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids) and negatively charged RNA (particularly mRNA) result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic or cationic ionizable amphiphilic lipids, such as DOTMA and / or DODMA, and additional lipids, such as DOPE. In some embodiments, the RNA (particularly mRNA) lipoplex particles are nanoparticles.

[0501] Generally, lipid nanoparticles (LNPs) can be obtained by directly mixing RNA in an aqueous phase and lipids in a phase containing an organic solvent such as ethanol, in which case lipids or lipid mixtures that do not form a lamellar (bilayer) phase in water can be used for particle formation.

[0502] In some embodiments, LNPs comprise or consist of cationic / ionizable lipids and helper lipids such as phospholipids, cholesterol, and / or polyethylene glycol (PEG) lipids. In some embodiments, in the RNA LNPs described herein, the RNA is associated with ionizable lipids that occupy the central core of the LNP. In some embodiments, the PEG lipids, along with the phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and charged and uncharged forms of ionizable lipids may be dispersed throughout the LNP.

[0503] In some embodiments, RNA (e.g., mRNA) may be non-covalently associated with a particle described herein. In embodiments, the RNA (especially mRNA) may be attached to the outer surface of the particle (surface RNA (especially surface mRNA)) and / or may be contained within the particle (encapsulated RNA (especially encapsulated mRNA)).

[0504] In some embodiments, the particles described herein (e.g., LNP and LPX) have a diameter in the range of about 10 to about 2000 nm, e.g., at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most 1900 nm (e.g., at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), for example, about 20 to about 1500 nm, for example, about 30 to 1200 nm, about 40 to 1100 nm , about 50 to 1000 nm, about 60 to 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, about 50 to 500 nm, or about 100 to 500 nm, for example, 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, or 70 to 150 nm.

[0505] In some embodiments, the particles described herein (e.g., LNP and LPX) may have a particle size of about 50 nM to about 1000 nM, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm , about 50nm to about 250nm, about 50nm to about 200nm, about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 700nm, about 100nm to about 600nm m, about 100nm to about 500nm, about 100nm to about 450nm, about 100nm to about 400nm, about 100nm to about 350nm, about 100nm to about 300nm, about 100nm to about 25 0nm, approximately 100nm ~ approximately 200nm, approximately 150nm ~ approximately 1000nm, approximately 150nm ~ approximately 800nm, approximately 150nm ~ approximately 700nm, approximately 150nm ~ approximately 600nm, approximately 150nm ~ Approximately 500nm, approximately 150nm to approximately 450nm, approximately 150nm to approximately 400nm, approximately 150nm to approximately 350nm, approximately 150nm to approximately 300nm, approximately 150nm to approximately 250nm, approximately 150nm The average diameter ranges from about 200 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm.

[0506] In some embodiments, the particle described herein is nanoparticle.The term " nanoparticle " refers to the nano-sized particle that comprises nucleic acid (particularly mRNA) described herein and at least one cationic or cationic ionizable lipid, and the three external dimensions of the particle are all nano-sized, that is, at least about 1 nm and less than about 1000 nm.Preferably, the size of the particle is its diameter.

[0507] The RNA particles (particularly mRNA particles) described herein may exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the RNA particles may exhibit a polydispersity index ranging from about 0.01 to about 0.4, or from about 0.1 to about 0.3.

[0508] The N / P ratio indicates the ratio of nitrogen groups in the lipid to the number of phosphate groups in the RNA. This correlates with the charge ratio, since nitrogen atoms are usually positively charged (depending on the pH) and phosphate groups are negatively charged. The N / P ratio is pH dependent, since there is a charge balance. Lipid formulations are often formed with N / P ratios greater than 4 and up to 12, as positively charged nanoparticles are thought to favor transfection. In this case, the RNA is thought to bind fully to the nanoparticles.

[0509] The RNA particles (particularly mRNA particles) described herein can be prepared using a wide range of methods that may include obtaining a colloid from at least one cationic or cationic ionizable lipid and mixing the colloid with RNA to obtain the RNA particles.

[0510] The term "colloid," as used herein, refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are fine, with particle sizes ranging from 1 to 1000 nanometers. This mixture may be referred to as a colloid or colloidal suspension. The term "colloid" may refer only to the particles in the mixture, rather than the entire suspension.

[0511] For the preparation of colloids comprising at least one cationic or cationic-ionizable lipid, methods conventionally used and appropriately adapted for the preparation of liposomal vesicles are applicable herein. The most commonly used methods for preparing liposomal vesicles share the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

[0512] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film on the bottom of a flask. The resulting lipid film is hydrated using an appropriate aqueous medium to produce a liposome dispersion. An additional miniaturization step may also be included.

[0513] Reverse phase evaporation is an alternative to film hydration for preparing liposomal vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of the mixture is required to homogenize the system. Removal of the organic phase under reduced pressure results in a milky gel, which then transforms into a liposomal suspension.

[0514] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution with a needle. This disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation such as liposome formation. Generally, the RNA (especially mRNA) lipoplex particles described herein can be obtained by adding RNA (especially mRNA) to a colloidal liposome dispersion. In one embodiment, such a colloidal liposome dispersion is formed using the ethanol injection technique as follows: An ethanol solution containing a lipid, such as a cationic or cationic ionizable lipid such as DOTMA, and additional lipids is injected into an aqueous solution with stirring. In some embodiments, the RNA (especially mRNA) lipoplex particles described herein can be obtained without using an extrusion process.

[0515] The term "extrude" or "extrusion" refers to the creation of particles having a defined cross-sectional profile. In particular, the term refers to the reduction in particle size by forcing the particles through a filter with defined pores.

[0516] Other methods that have the characteristic of not containing organic solvents can also be used to prepare colloids according to the present disclosure.

[0517] In some embodiments, LNPs comprise four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid. In some embodiments, LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with RNA in an aqueous buffer. While the RNA particles described herein may comprise a polymer-conjugated lipid such as a PEG-lipid, RNA particles that do not comprise a polymer-conjugated lipid such as a PEG-lipid are also provided herein.

[0518] In some embodiments, LNPs comprising RNA and at least one cationic or cationic-ionizable lipid described herein are prepared by (a) preparing an RNA solution comprising water and a buffer system, (b) preparing an ethanol solution comprising a cationic or cationic-ionizable lipid and, if present, one or more additional lipids, and (c) combining the RNA solution prepared in (a) with the ethanol solution prepared in (b), thereby preparing a formulation comprising the LNP. Step (c) may be followed by one or more steps selected from dilution and filtration, such as tangential flow filtration.

[0519] In some embodiments, LNPs comprising RNA and at least one cationic or cationic ionizable lipid described herein are prepared by (a') preparing a liposome or colloid preparation of the cationic or cationic ionizable lipid and, if present, one or more additional lipids in an aqueous phase, (b') preparing an RNA solution comprising water and a buffer system, and (c') mixing the liposome or colloid preparation prepared in (a') with the RNA solution prepared in (b'). Step (c') may be followed by one or more steps selected from dilution and filtration, such as tangential flow filtration.

[0520] The present disclosure describes particles comprising RNA (especially mRNA) and at least one cationic or cationic ionizable lipid associated with RNA to form RNA particles, and compositions comprising such particles. RNA particles may contain RNA complexed in various forms by non-covalent interactions with the particles. The particles described herein are not virus particles, and in particular are not infectious virus particles, i.e., the particles described herein cannot infect cells with the virus.

[0521] Suitable cationic or cationic ionizable lipids are lipids that form RNA particles and are included in the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with RNA to form an RNA particle. Such components include any component that may be part of an RNA particle.

[0522] In some embodiments, an RNA particle (particularly an mRNA particle) comprises more than one type of RNA molecule, and the molecular parameters of the RNA molecules may be similar or different from each other in terms of molar mass or basic structural elements such as molecular architecture, capping, coding regions, or other features.

[0523] In a particulate formulation, each RNA species can be formulated separately as an individual particulate formulation. In that case, each individual particulate formulation will contain one RNA species. The individual particulate formulations may exist as separate entities, for example, in separate containers. Such a formulation can be obtained by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing particles to form. Each particle will exclusively contain the specific RNA species provided when forming the particle (individual particulate formulation). In some embodiments, a composition, such as a pharmaceutical composition, contains more than one individual particulate formulation. The corresponding pharmaceutical composition is called a mixed particulate formulation. A mixed particulate formulation according to the present invention can be obtained by separately forming the individual particulate formulations, followed by a step of mixing the individual particulate formulations. The mixing step can result in a formulation containing a mixed population of RNA-containing particles. The individual particulate populations may be present together in a single container containing a mixed population of individual particulate formulations. Alternatively, all RNA species of a pharmaceutical composition can be formulated together as a composite particulate formulation. Such preparation can be obtained by providing the complex preparation (typically complex solution) of all RNA species together with particle forming agent, thereby allowing particle formation.In contrast to mixed particulate preparation, complex particulate preparation will typically comprise particles that contain more than one RNA species.In complex particulate composition, different RNA species typically exist together in a single particle.

[0524] polymer Polymers are widely used materials for nanoparticle-based delivery due to their high degree of chemical flexibility. Cationic polymers are typically used to electrostatically condense negatively charged RNA into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range of 5.5 to 7.5, which are thought to cause an ionic imbalance that leads to endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied in nucleic acid delivery and are suitable as cationic polymers herein. In addition, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) have gained widespread use in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers for the present invention.

[0525] "Polymer," as used herein, refers to its conventional meaning: a molecular structure comprising one or more repeating units (monomers) covalently joined. The repeating units may all be identical, or in some cases, more than one type of repeating unit may be present in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. Also, in some cases, additional moieties, such as targeting moieties, may be present in the polymer.

[0526] When more than one type of repeat unit is present in a polymer, the polymer is said to be a "copolymer." It should be understood that a polymer as used herein may also be a copolymer. The repeat units forming a copolymer may be arranged in any manner. For example, the repeat units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., including one or more regions each including a first repeat unit (e.g., a first block) and one or more regions each including a second repeat unit (e.g., a second block). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more different blocks.

[0527] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, meaning that the polymer can be chemically and / or biologically broken down in a physiological environment, such as within the body.

[0528] In certain embodiments, the polymer may be protamine or a polyalkyleneimine.

[0529] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (e.g., fish) specifically associated with DNA in place of somatic histones. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, water-soluble, heat-resistant, and yields primarily arginine upon hydrolysis. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0530] According to the present disclosure, the term "protamine", as used herein, is intended to include any protamine amino acid sequence, including fragments thereof, obtained or derived from natural or biological sources, and multimeric forms of said amino acid sequence or fragments thereof, as well as (synthetic) polypeptides that are artificial, specifically designed for a particular purpose, and cannot be isolated from natural or biological sources.

[0531] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-10 2 ~10 7 Da, preferably 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0532] According to the present disclosure, linear polyalkyleneimines, such as linear polyethyleneimine (PEI), are preferred.

[0533] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In one embodiment, cationic polymers contemplated for use herein include any cationic polymer that can associate with nucleic acids, for example, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is surrounded or encapsulated.

[0534] The particles described herein may also include polymers other than cationic polymers, i.e., non-cationic and / or anionic polymers, collectively referred to herein as non-cationic polymers.

[0535] lipids The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, the amphiphilic nature allows the molecules to self-assemble into organized structures and distinct phases. One such phase consists of lipid bilayers when they exist in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, such as carbohydrate groups, phosphate groups, carboxyl groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0536] As used herein, the term "hydrophobic" refers to any molecule, moiety, or group that is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least six carbon atoms. Hydrophobic groups can have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, so long as they are substantially immiscible or insoluble in aqueous solution.

[0537] The term "hydrocarbon" includes alkyl, alkenyl, or alkynyl as defined herein. It should be understood that one or more of the hydrogens in the alkyl, alkenyl, or alkynyl may be replaced with other atoms, such as halogen, oxygen, or sulfur. Unless otherwise stated, hydrocarbon groups can also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively non-polar.

[0538] The term "alkyl" refers to a saturated, straight- or branched-chain monovalent hydrocarbon moiety which may have 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. Examples of non-polar alkyl groups include, but are not limited to, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

[0539] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond and which may contain a total of 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms.

[0540] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond and a total of 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. An alkynyl group may optionally have one or more carbon-carbon double bonds.

[0541] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Additionally, the polar portion may have either a formal positive or a formal negative charge. Alternatively, the polar portion may have both a formal positive and negative charge and may be a zwitterion or an inner salt. For purposes of this disclosure, an amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0542] The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances structurally and / or functionally related to lipids but that cannot be considered lipids in the strict sense, particularly amphiphiles. For example, the term includes compounds that can form amphiphilic layers when present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, including surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules containing hydrophilic and hydrophobic moieties with different structural organizations that may or may not resemble the structure of lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSLs) and synthetic function-spacer-sterol constructs (FSSs), as well as artificial amphiphilic molecules. Lipids are generally cylindrical in shape. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Lipids have low solubility as monomers and tend to aggregate into water-insoluble planar bilayers. Conventional surfactant monomers are generally conical in shape. Hydrophilic head groups tend to occupy more molecular space than linear alkyl chains. In some embodiments, surfactants tend to aggregate into spherical or ellipsoidal micelles that are water-soluble. Lipids have the same basic structure as surfactants—polar hydrophilic head groups and nonpolar hydrophobic tails—but lipids differ from surfactants in the shape of the monomer, the type of aggregates they form in solution, and the concentration range required for aggregation. As used herein, the term "lipid" should be interpreted to cover both lipids and lipid-like substances, unless otherwise indicated herein or clearly contradicted by the context.

[0543] Lipids are generally classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used synonymously with fat, which is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as steroids, i.e., cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and their derivatives, and mixtures thereof.

[0544] Fatty acids or fatty acid residues are a diverse group of molecules made up of hydrocarbon chains terminated with carboxylic acid groups; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain is typically 4 to 24 carbon atoms long and may be saturated or unsaturated, and may be attached to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, the possibility of either cis or trans geometric isomers exists, thereby significantly affecting the molecular configuration. Cis double bonds cause the fatty acid chain to bend, and the effect of having more double bonds in the chain is even greater. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0545] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified with a different fatty acid. An additional subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol by glycosidic linkages.

[0546] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked by ester linkages to two fatty acid-derived "tails" and a phosphate ester linkage to one "head" group. Examples of glycerophospholipids, commonly called phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0547] Sphingolipids are a family of complex compounds that share a common structural feature: a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acylsphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The major sphingophospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine, and fungi contain phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

[0548] Sterol lipids, such as cholesterol and its derivatives or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0549] Saccharolipids are compounds in which fatty acids are directly linked to a sugar backbone, forming structures compatible with membrane bilayers. In saccharolipids, the glycerol backbone present in glycerolipids and glycerophospholipids is replaced with a monosaccharide. The best-known saccharolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with up to seven fatty acid acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, a hexacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0550] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes as well as by repetitive multimodular enzymes that share mechanistic features with fatty acid synthases. Polyketides comprise a large number of secondary metabolites and natural products derived from animal, plant, bacterial, fungal, and marine sources, and possess great structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0551] According to the present disclosure, lipids and lipid-like substances may be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0552] Cationic / cationic ionizable lipids The RNA particles described herein comprise at least one cationic or cationic ionizable lipid as particle-forming agent.The cationic or cationic ionizable lipid contemplated for use in the present invention includes any cationic or cationic ionizable lipid (including lipid-like substances) that can electrostatically bind to nucleic acid.In some embodiments, the cationic or cationic ionizable lipid contemplated for use herein can be associated with nucleic acid, for example, by forming a complex with nucleic acid or forming a vesicle in which nucleic acid is surrounded or enclosed.

[0553] As used herein, "cationic lipid" refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl chain, or more acyl chains, and the head group of the lipid typically carries a positive charge.

[0554] In some embodiments, cationic lipids only have a net positive charge at a certain pH, particularly acidic pH, but at a different, preferably higher pH, such as physiological pH, they preferably do not have a net positive charge, preferably have no charge, that is, are neutral.This ionizable behavior is believed to enhance efficacy by helping escape from endosomes and reducing toxicity, compared with particles that remain cationic at physiological pH.

[0555] As used herein, "cationic ionizable lipid" refers to a lipid or lipid-like substance that has a net positive charge or is neutral, that is, is not permanently cationic.Therefore, depending on the pH of the composition in which the cationic ionizable lipid is dissolved, the cationic ionizable lipid is positively charged or neutral.For the purposes of this disclosure, cationic ionizable lipid is covered by the term "cationic lipid" unless the context is inconsistent.

[0556] In some embodiments, the cationic or cationic-ionizable lipid comprises a head group that includes at least one nitrogen atom (N) that is positively charged or protonatable, e.g., under physiological conditions.

[0557] Examples of cationic or cationic ionizable lipids include N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium chloride (DMRIE). Dimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecanoate, ... Decylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoe thyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N -dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy) (c)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2 (Dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N), 12 -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

[0558] In some embodiments, the cationic or cationic ionizable lipid is DOTMA. In some embodiments, the cationic or cationic ionizable lipid is DODMA.

[0559] DOTMA is a cationic lipid with a quaternary amine head group. The structure of DOTMA can be represented as follows: [ka]

[0560] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows: [ka]

[0561] In some embodiments, the cationic or cationic-ionizable lipid may comprise from about 10 mol% to about 95 mol%, from about 20 mol% to about 95 mol%, from about 20 mol% to about 90 mol%, from about 30 mol% to about 90 mol%, from about 40 mol% to about 90 mol%, or from about 40 mol% to about 80 mol% of the total lipid present in the particle.

[0562] Additional fats The particles described herein may also contain lipids (including lipid-like substances) other than cationic or cationic ionizable lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationic ionizable lipids or lipid-like substances). Anionic and neutral lipids or lipid-like substances are collectively referred to herein as non-cationic lipids. In addition to cationic or cationic ionizable lipids, optimizing the formulation of RNA particles by adding other hydrophobic moieties, such as cholesterol and lipids, can enhance particle stability and RNA delivery efficacy.

[0563] One or more additional lipids may or may not affect the overall charge of RNA particles.In some embodiments, one or more additional lipids are non-cationic lipids or lipid-like substances.Non-cationic lipids may, for example, comprise one or more anionic lipids and / or neutral lipids.As used herein, " anionic lipids " refers to any lipid that is negatively charged at selected pH.As used herein, " neutral lipids " refers to any of the many lipid species that exist in either uncharged or neutral zwitterionic form at selected pH.

[0564] In some embodiments, the RNA particles described herein (particularly particles comprising mRNA) comprise a cationic or cationic ionizable lipid and one or more additional lipids.

[0565] Without wishing to be bound by theory, the amount of cationic or cationic ionizable lipid relative to the amount of one or more additional lipids can affect important RNA particle characteristics such as RNA charge, particle size, stability, tissue selectivity, and biological activity. Thus, in some embodiments, the molar ratio of cationic or cationic ionizable lipid to one or more additional lipids is from about 10:0 to about 1:9, from about 4:1 to about 1:2, from about 4:1 to about 1:1, from about 3:1 to about 1:1, or from about 3:1 to about 2:1.

[0566] In some embodiments, the one or more additional lipids included in the RNA particles described herein (particularly particles comprising mRNA) include one or more of the following: neutral lipids, steroids, and combinations thereof.

[0567] In some embodiments, one or more additional lipids comprise a neutral lipid that is a phospholipid.In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin.Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin.Such phospholipids include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), Ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 lysoPC), and phosphatidylcholine. phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DPPE), E), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and additional phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM.In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM, hi some embodiments, the neutral lipid is DOPE.

[0568] In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and derivatives thereof, and mixtures thereof.

[0569] Thus, in some embodiments, the RNA particles described herein (particularly particles comprising mRNA) comprise (1) a cationic or cationic ionizable lipid and a phospholipid such as DOPE, or (2) a cationic or cationic ionizable lipid and a phospholipid such as DOPE and cholesterol.

[0570] In some embodiments, the RNA particles described herein (particularly particles comprising mRNA) comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE, and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE, and cholesterol.

[0571] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows: [ka]

[0572] The structure of cholesterol can be represented as follows: [ka]

[0573] In some embodiments, the particles described herein do not include a polymer-conjugated lipid, such as a pegylated lipid. The term "pegylated lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0574] In some embodiments, the additional lipid (e.g., one or more phospholipids and / or cholesterol) may comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 2 mol% to about 80 mol%, about 5 mol% to about 80 mol%, about 5 mol% to about 60 mol%, about 5 mol% to about 50 mol%, about 7.5 mol% to about 50 mol%, or about 10 mol% to about 40 mol% of the total lipid present in the particle. In some embodiments, the additional lipid (e.g., one or more phospholipids and / or cholesterol) comprises about 10 mol%, about 15 mol%, or about 20 mol% of the total lipid present in the particle.

[0575] In some embodiments, the additional lipid comprises a mixture of (i) a phospholipid, such as DOPE, and (ii) cholesterol or a derivative thereof, in which the molar ratio of the phospholipid, such as DOPE, to cholesterol or a derivative thereof is about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.

[0576] Polymer-conjugated lipids In some embodiments, particle may comprise at least one polymer-conjugated lipid.Polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.In some embodiments, polymer-conjugated lipid is PEG-conjugated lipid, also referred to herein as PEGylated lipid or PEG lipid.

[0577] In some embodiments, the polymer-conjugated lipids are designed to sterically stabilize the lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer, hi some embodiments, the polymer-conjugated lipids can reduce association with serum proteins and / or resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0578] Polyethylene glycol (PEG) conjugated lipids A variety of PEG-conjugated lipids are known in the art, including, but not limited to, PEGylated diacylglycerols (PEG-DAGs) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAGs) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0579] In some embodiments, the particles may comprise one or more PEG-conjugated or PEGylated lipids as described in International Publication No. WO 2017 / 075531 and Japanese Publication No. 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0580] Lipoplex Particle Embodiments In some embodiments of the present disclosure, the RNA described herein may be present in an RNA lipoplex particle.

[0581] Lipoplexes (LPXs) are generally electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic RNA. The resulting lipoplexes have a distinct internal molecular arrangement resulting from the conversion of the liposomal structure into a small RNA-lipoplex. These formulations are generally characterized by poor RNA encapsulation and incomplete RNA capture.

[0582] In certain embodiments, the RNA lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0583] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In particular embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0584] The RNA lipoplex particles described herein, in some embodiments, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In embodiments, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0585] The RNA lipoplex particles and compositions comprising the RNA lipoplex particles described herein are useful for delivering RNA to target tissues following parenteral administration, particularly following intravenous administration.

[0586] Lipid Nanoparticle (LNP) Embodiments In some embodiments, the RNA described herein is present in the form of a lipid nanoparticle (LNP), which may comprise any lipid capable of forming a particle having one or more RNA molecules attached thereto or encapsulating one or more RNA molecules.

[0587] LNPs typically contain four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid such as a PEG-lipid. LNPs can be prepared by mixing lipids dissolved in ethanol with RNA in an aqueous buffer.

[0588] In some embodiments, in the RNA LNPs described herein, the RNA is bound to an ionizable lipid that occupies the central core of the LNP. The PEG lipid, together with the phospholipid, forms the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and charged and uncharged forms of the ionizable lipid may be dispersed throughout the LNP.

[0589] In some embodiments, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and PEGylated lipids.

[0590] In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and RNA encapsulated within or associated with the lipid nanoparticle.

[0591] In some embodiments, the LNP comprises 40 to 60 mol percent, 40 to 55 mol percent, 45 to 55 mol percent, or 45 to 50 mol percent cationic lipid.

[0592] In some embodiments, the neutral lipid is present at a concentration ranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent.

[0593] In some embodiments, the steroid is present at a concentration ranging from 30 to 50 mol percent, 30 to 45 mol percent, 35 to 45 mol percent, or 35 to 43 mol percent.

[0594] In some embodiments, the LNP comprises 1 to 10 mol percent, 1 to 5 mol percent, or 1 to 2.5 mol percent of polymer-conjugated lipid.

[0595] In some embodiments, the LNP comprises 45-55 mol percent cationic lipid, 5-15 mol percent neutral lipid, 30-45 mol percent steroid, 1-5 mol percent polymer-conjugated lipid, and RNA encapsulated within or associated with the lipid nanoparticle.

[0596] In some embodiments, the mole percent is determined based on the total moles of lipid present in the lipid nanoparticle. In some embodiments, the mole percent is determined based on the total moles of cationic lipid, neutral lipid, steroid, and polymer-conjugated lipid present in the lipid nanoparticle.

[0597] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0598] In some embodiments, the steroid is cholesterol.

[0599] In some embodiments, the polymer-conjugated lipid is a PEGylated lipid. In some embodiments, the PEGylated lipid has the following structure: [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: R 12 and R 13are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester linkages, and w has an average value in the range of 30 to 60. In some embodiments, R 12 and R 13 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R 12 and R 13 is each independently a linear saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0600] In some embodiments, the PEGylated lipid has the following structure: [ka] is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide having the formula:

[0601] In some embodiments, the PEGylated lipid has the following structure: [ka] wherein n is in the range of 30 to 60, for example, an average value of about 50; 2000 -C-DMA or includes it.

[0602] In some embodiments, the PEGylated lipid has the following structure: [ka] The polymer is or comprises DMG-PEG 2000 having the formula:

[0603] In some embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond, G 1 and G 2 are each independently unsubstituted C1-C 12 Alkylene or C1-C 12 is alkenylene, G 3 is C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1~C 12 is alkyl, R 1 and R 2 are each independently C6 to C 24 Alkyl or C6-C 24 is alkenyl, R 3 H, OR 5, CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR5C(=O)R 4 and R 4 is C1~C 12 is alkyl, R 5 is H or C1-C6 alkyl, x is 0, 1, or 2.

[0604] In some of the above-described embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] and During the ceremony, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is independently in each occurrence H, OH, or C1-C 24 is alkyl, n is an integer from 1 to 15.

[0605] In some of the above-described embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0606] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] and In the formula, y and z are each independently an integer ranging from 1 to 12.

[0607] In any of the above embodiments of formula (III), L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2Each of is —O(C═O)—. In some different embodiments of any of the above, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0608] In some different embodiments of formula (III), the lipid has either of the following structures (IIIE) or (IIIF): [ka]

[0609] In some of the above-described embodiments of formula (III), the lipid has any one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ). [ka]

[0610] In some of the above embodiments of Formula (III), n is an integer ranging from 2 to 12, such as from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0611] In some other of the above embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0612] In some of the above embodiments of formula (III), R 6 is H. In other embodiments of the above embodiments, R 6 is C1~C 24 In another embodiment, R 6 is OH.

[0613] In some embodiments of Formula (III), G 3 is unsubstituted. In other embodiments, G is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0614] In some other above-mentioned embodiments of formula (III), R 1 or R 2 Or both are C6-C 24 For example, in some embodiments, R 1 and R 2 are each independently of the following structure: [ka] and During the ceremony, R 7a and R 7b is independently in each occurrence H or C1-C 12 is alkyl, a is an integer from 2 to 12, R 7a , R 7b , and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0615] In some of the above embodiments of formula (III), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a is H in each occurrence. In other variations of the above embodiments, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0616] In different embodiments of formula (III), R 1 or R 2 or both have one of the following structures: [ka]

[0617] In some of the above embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0618] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below.

[0619] Representative compounds of formula (III). [Table 3A] [Table 3B] [Table 3C] [Table 3D] [Table 3E] [Table 3F] [Table 3G]

[0620] Further representative cationic lipids are as follows: [Table 4]

[0621] In some embodiments, the LNP comprises a cationic lipid shown in the table above, e.g., a cationic lipid of Formula (B) or Formula (D), particularly a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is DMG-PEG 2000.

[0622] A variety of lipids (including, for example, cationic lipids, neutral lipids, and polymer-conjugated lipids) are known in the art and can be used herein to form lipid nanoparticles, for example, lipid nanoparticles targeted to specific cell types (e.g., liver cells). In some embodiments, the neutral lipid can be or include a phospholipid or a derivative thereof (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol. In some embodiments, the polymer-conjugated lipid can be a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or a derivative thereof).

[0623] In some embodiments, the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159. ALC-0159: [ka]

[0624] In some embodiments, the RNA described herein is formulated in a composition comprising a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and RNA.

[0625] In some embodiments, the composition comprises a lipid of formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159. In some embodiments, the cationic lipid is a lipid of formula (III), the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid is ALC-0159.

[0626] In some embodiments, the composition comprises ALC-0315, RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159. In some embodiments, the cationic lipid is ALC-0315, the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid is ALC-0159.

[0627] ALC-0315 = ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate [ka]

[0628] ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide / 2-[2-(ω-methoxy(polyethylene glycol 2000)ethoxy]-N,N-ditetradecylacetamide [ka]

[0629] DSPC = 1,2-distearoyl-sn-glycero-3-phosphocholine [ka]

[0630] cholesterol: [ka]

[0631] In some embodiments, the composition comprises ALC-0366, RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159. In some embodiments, the cationic lipid is ALC-0366, the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid is ALC-0159.

[0632] ALC-0366 = ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) [ka]

[0633] In some embodiments, the composition comprises 3D-P-DMA, RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is PEG. 2000 In some embodiments, the cationic lipid is 3D-P-DMA, the neutral lipid is DSPC, the steroid is cholesterol, and the pegylated lipid is PEG. 2000 -C-DMA.

[0634] 3D-P-DMA = (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate [ka]

[0635] "PEG 2000 -C-DMA": 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2kDa)-C-DMA or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate(2000)) [ka] where n has a value in the range of 30 to 60, for example an average value of about 50.

[0636] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6.

[0637] Compositions containing RNA Compositions comprising one or more RNAs described herein, for example in the form of RNA particles, may include salts, buffers, or other ingredients, as further described below.

[0638] In some embodiments, the salt for use in the compositions described herein comprises sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmotic agent to pretreat the RNA before mixing with lipids. In some embodiments, the compositions described herein may comprise alternative organic or inorganic salts. Alternative salts include, but are not limited to, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassi...

Claims

1. A composition or medical preparation comprising an RNA encoding an amino acid sequence comprising a peptidoglycan hydrolase.

2. 2. The composition or medical preparation of claim 1, wherein the peptidoglycan hydrolase degrades peptidoglycan in bacterial cell walls.

3. 3. A composition or medical preparation according to claim 1 or 2, wherein the peptidoglycan hydrolase is derived from a bacteriophage.

4. A composition or medical preparation according to any one of claims 1 to 3, wherein the peptidoglycan hydrolase is or is derived from an endolysin.

5. A composition or medical preparation according to any one of claims 1 to 4, wherein the peptidoglycan hydrolase has been modified to reduce glycosylation.

6. A composition or medical preparation according to any one of claims 1 to 5, wherein the peptidoglycan hydrolase is modified by removing glycosylation sites.

7. A composition or medical preparation according to any one of claims 1 to 6, wherein the peptidoglycan hydrolase has been modified to reduce immunogenicity.

8. A composition or medical preparation according to any one of claims 1 to 7, wherein the peptidoglycan hydrolase is modified by removing T-cell epitopes.

9. 9. The composition or medical preparation of any one of claims 1 to 8, wherein the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:

1.

10. 10. The composition or medical preparation of any one of claims 1 to 9, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

2.

11. 10. The composition or medical preparation of any one of claims 1 to 9, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, or a fragment of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

3.

12. A composition or medical preparation according to any one of claims 1 to 11, wherein the peptidoglycan hydrolase is a secreted peptidoglycan hydrolase.

13. A composition or medical preparation according to any one of claims 1 to 12, wherein the amino acid sequence comprising the peptidoglycan hydrolase comprises a signal peptide.

14. A composition or medical preparation according to any one of claims 1 to 13, wherein the peptidoglycan hydrolase is fused to a signal peptide.

15. 15. A composition or medical preparation according to claim 13 or 14, wherein the signal peptide is cleaved during secretion or export.

16. 16. The composition or medical preparation of any one of claims 13 to 15, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a fragment of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:

10.

17. 17. The composition or medical preparation of any one of claims 1 to 16, wherein the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:

4.

18. 18. The composition or medical preparation of any one of claims 1 to 17, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

5.

19. 19. The composition or medical preparation of any one of claims 1 to 18, wherein the amino acid sequence comprising a peptidoglycan hydrolase comprises an extended pharmacokinetic (PK) polypeptide.

20. 20. The composition or medical preparation of any one of claims 1 to 19, wherein the peptidoglycan hydrolase is fused to a pharmacokinetic-modifying group.

21. 21. The composition or pharmaceutical preparation of claim 20, wherein the pharmacokinetic-modifying group comprises a moiety that is heterologous to the peptidoglycan hydrolase.

22. 22. The composition or medical preparation of claim 20 or 21, wherein the pharmacokinetic modifying group comprises a moiety selected from the group consisting of albumin, an immunoglobulin fragment, transferrin, Fn3, a functional variant thereof, or a functional fragment of albumin, an immunoglobulin fragment, transferrin, Fn3, or a functional variant thereof.

23. 23. The composition or medical preparation of any one of claims 20 to 22, wherein the pharmacokinetic modifying group comprises human albumin, a functional variant thereof, or a functional fragment of human albumin or a functional variant thereof.

24. 24. A composition or medical preparation according to any one of claims 1 to 23, wherein the amino acid sequence comprising the peptidoglycan hydrolase is encoded by a coding sequence which has been codon optimised and / or has an increased G / C content compared to a wild-type coding sequence, and wherein the codon optimisation and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

25. 25. The composition or medical preparation of any one of claims 1 to 24, wherein the RNA comprises modified nucleosides instead of uridine.

26. 26. The composition or medical preparation of any one of claims 1 to 25, wherein the RNA comprises a modified nucleoside in place of each uridine.

27. 27. The composition or medical preparation of claim 25 or 26, wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

28. 28. The composition or medical preparation of any one of claims 25 to 27, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

29. 29. The composition or medical preparation of any one of claims 1 to 28, wherein the RNA comprises a cap 05' cap.

30. The RNA is a 5' capped 2 7,2'O 30. A composition or medical preparation according to any one of claims 1 to 29, comprising G(5')ppSp(5')G, in particular its D1 diastereomer.

31. 29. The composition or medical preparation of any one of claims 1 to 28, wherein the RNA comprises a cap1 5' cap.

32. The RNA is a 5' capped 2 7,3'-O Gppp(m 1 2'-O 32. A composition or medical preparation according to any one of claims 1 to 28 and 31, comprising ApG.

33. 33. The composition or medical preparation of any one of claims 1 to 32, wherein the RNA comprises a 5'UTR.

34. 34. The composition or medical preparation of claim 33, wherein the 5'UTR comprises a nucleotide sequence of SEQ ID NO:11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

11.

35. 35. The composition or medical preparation of any one of claims 1 to 34, wherein the RNA comprises a 3'UTR.

36. 36. The composition or medical preparation of claim 35, wherein the 3'UTR comprises a nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

12.

37. The composition or medical preparation according to any one of claims 1 to 36, wherein said RNA comprises a polyA sequence.

38. 38. The composition or medical preparation of claim 37, wherein the polyA sequence comprises at least 100 nucleotides.

39. 39. A composition or medical preparation according to claim 37 or 38, wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

13.

40. The composition or medical preparation of any one of claims 1 to 39, wherein said RNA is mRNA.

41. 41. The composition or medical preparation of any one of claims 1 to 40, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

6.

42. 41. The composition or medical preparation of any one of claims 1 to 40, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

7.

43. 40. The composition or medical preparation of any one of claims 1 to 39, wherein the RNA is a self-amplifying RNA.

44. 44. The composition or medical preparation of any one of claims 1 to 39 and 43, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, or a fragment of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

8.

45. 44. The composition or medical preparation of any one of claims 1 to 39 and 43, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

9.

46. 46. ​​The composition or medical preparation of any one of claims 1 to 45, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

47. 47. The composition or medical preparation of any one of claims 1 to 46, wherein the RNA is formulated for injection.

48. 48. The composition or medical preparation of any one of claims 1 to 47, wherein the RNA is formulated for intravenous administration.

49. 49. The composition or medical preparation of any one of claims 1 to 48, wherein the RNA is or is to be formulated as a lipid particle.

50. 50. The composition or medical preparation of claim 49, wherein the RNA-lipid particle is a lipid nanoparticle (LNP).

51. A composition or medical preparation according to any one of claims 1 to 50, which is a pharmaceutical composition.

52. 52. The composition or medical preparation of claim 51, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and / or excipients.

53. The composition or medical preparation according to any one of claims 1 to 50, wherein the medical preparation is a kit.

54. 54. The composition or medical preparation of claim 53, further comprising instructions for use of said RNA to treat or prevent a bacterial infection.

55. A composition or medical preparation according to any one of claims 1 to 54, intended for administration to a human.

56. A composition or medical preparation according to any one of claims 1 to 55 for pharmaceutical use.

57. 57. The composition or medical preparation of claim 56, wherein the pharmaceutical use includes the therapeutic or prophylactic treatment of a disease or disorder.

58. 58. The composition or medical preparation of claim 57, wherein the therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing a bacterial infection.

59. A method for treating a bacterial infection in a subject, the method comprising administering to the subject RNA encoding an amino acid sequence comprising a peptidoglycan hydrolase.

60. 60. The method of claim 59, wherein the peptidoglycan hydrolase degrades peptidoglycan in a bacterial cell wall.

61. 61. The method of claim 59 or 60, wherein the peptidoglycan hydrolase is derived from a bacteriophage.

62. 62. The method of any one of claims 59 to 61, wherein the peptidoglycan hydrolase is or is derived from an endolysin.

63. 63. The method of any one of claims 59 to 62, wherein the peptidoglycan hydrolase has been modified to reduce glycosylation.

64. 64. The method of any one of claims 59 to 63, wherein the peptidoglycan hydrolase is modified by removing glycosylation sites.

65. 65. The method of any one of claims 59 to 64, wherein the peptidoglycan hydrolase has been modified to reduce immunogenicity.

66. 66. The method of any one of claims 59 to 65, wherein the peptidoglycan hydrolase is modified by removing T cell epitopes.

67. 67. The method of any one of claims 59 to 66, wherein the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1, or a fragment of the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:

1.

68. 68. The method of any one of claims 59 to 67, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:2, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2, or a fragment of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

2.

69. 68. The method of any one of claims 59 to 67, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:3, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3, or a fragment of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

3.

70. The method of any one of claims 59 to 69, wherein the peptidoglycan hydrolase is a secreted peptidoglycan hydrolase.

71. The method of any one of claims 59 to 70, wherein the amino acid sequence comprising the peptidoglycan hydrolase comprises a signal peptide.

72. The method of any one of claims 59 to 71, wherein the peptidoglycan hydrolase is fused to a signal peptide.

73. 73. The method of claim 71 or 72, wherein the signal peptide is cleaved during secretion or export.

74. 74. The method of any one of claims 71 to 73, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a fragment of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:

10.

75. 75. The method of any one of claims 59 to 74, wherein the amino acid sequence comprising the peptidoglycan hydrolase comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:

4.

76. 75. The method of any one of claims 59 to 74, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

5.

77. 77. The method of any one of claims 59 to 76, wherein the amino acid sequence comprising a peptidoglycan hydrolase comprises an extended pharmacokinetic (PK) polypeptide.

78. 78. The method of any one of claims 59 to 77, wherein the peptidoglycan hydrolase is fused to a pharmacokinetic-modifying group.

79. 79. The method of claim 78, wherein the pharmacokinetic-modifying group comprises a moiety that is heterologous to the peptidoglycan hydrolase.

80. 80. The method of claim 78 or 79, wherein the pharmacokinetic-modifying group comprises a moiety selected from the group consisting of albumin, an immunoglobulin fragment, transferrin, Fn3, a functional variant thereof, or a functional fragment of albumin, an immunoglobulin fragment, transferrin, Fn3, or a functional variant thereof.

81. 81. The method of any one of claims 78 to 80, wherein the pharmacokinetic modifying group comprises human albumin, a functional variant thereof, or a functional fragment of human albumin or a functional variant thereof.

82. 82. The method of any one of claims 59 to 81, wherein the amino acid sequence comprising the peptidoglycan hydrolase is encoded by a coding sequence that has been codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and wherein the codon-optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

83. 83. The method of any one of claims 59 to 82, wherein the RNA comprises modified nucleosides instead of uridine.

84. 84. The method of any one of claims 59 to 83, wherein the RNA comprises a modified nucleoside in place of each uridine.

85. 85. The method of claim 83 or 84, wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

86. 86. The method of any one of claims 83 to 85, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

87. 87. The method of any one of claims 59 to 86, wherein the RNA comprises a cap0 5' cap.

88. The RNA is a 5' capped 2 7,2'O 88. The method of any one of claims 59 to 87, comprising G(5')ppSp(5')G (particularly the D1 diastereomer thereof).

89. 87. The method of any one of claims 59 to 86, wherein the RNA comprises a cap1 5' cap.

90. The RNA is a 5' capped 2 7,3'-O Gppp(m 1 2'-O 90. The method of any one of claims 59 to 86 and 89, comprising ApG.

91. 91. The method of any one of claims 59 to 90, wherein the RNA comprises a 5'UTR.

92. 92. The method of claim 91, wherein the 5'UTR comprises a nucleotide sequence of SEQ ID NO:11 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

11.

93. 93. The method of any one of claims 59 to 92, wherein the RNA comprises a 3'UTR.

94. 94. The method of claim 93, wherein the 3'UTR comprises a nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

12.

95. The method of any one of claims 59 to 94, wherein the RNA comprises a polyA sequence.

96. 96. The method of claim 95, wherein the polyA sequence comprises at least 100 nucleotides.

97. 97. The method of claim 95 or 96, wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

13.

98. 98. The method of any one of claims 59 to 97, wherein the RNA is mRNA.

99. 99. The method of any one of claims 59 to 98, wherein the RNA encoding an amino acid sequence comprising a peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

6.

100. 99. The method of any one of claims 59 to 98, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

7.

101. 98. The method of any one of claims 59 to 97, wherein the RNA is a self-amplifying RNA.

102. 102. The method of any one of claims 59 to 97 and 101, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:8, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8, or a fragment of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

8.

103. 102. The method of any one of claims 59 to 97 and 101, wherein the RNA encoding the amino acid sequence comprising the peptidoglycan hydrolase comprises the nucleotide sequence of SEQ ID NO:9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9, or a fragment of the nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:

9.

104. 104. The method of any one of claims 59 to 103, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

105. The method of any one of claims 59 to 104, wherein the RNA is administered by injection.

106. The method of any one of claims 59 to 105, wherein the RNA is administered by intravenous administration.

107. 107. The method of any one of claims 59 to 106, wherein the RNA is formulated as a lipid particle.

108. The method of claim 107, wherein the RNA-lipid particle is a lipid nanoparticle (LNP).

109. The method of any one of claims 59 to 108, wherein the RNA is formulated as a pharmaceutical composition.

110. 110. The method of claim 109, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and / or excipients.

111. The method of any one of claims 59 to 110, wherein the subject is a human.

112. The method according to any one of claims 59 to 111, wherein the infection is a chronic infection.

113. The method according to any one of claims 59 to 112, wherein the bacteria causing the infection are multidrug resistant bacteria.

114. 114. The method of any one of claims 59 to 113, wherein the bacteria causing the infection are gram-negative bacteria, gram-positive bacteria, or both.

115. 115. The method of any one of claims 59 to 114, wherein the bacterium is selected from Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Porphyromonas gingivalis, Helicobacter pylori, Chlamydia trachomatis, Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Shigella spp., Borrelia spp., Campylobacter jejuni, Neisseria gonorrhoeae, Chlamydia trachomatis, Vibrio cholerae, and Fusobacterium nucleatum.

116. The method of any one of claims 59 to 115, wherein the bacteria is resistant to a carbapenem.

117. 115. The method of any one of claims 59 to 114, wherein the bacteria is selected from Staphylococcus, Streptococcus, Staphylococcus aureus, Streptococcus pneumoniae, Clostridioides difficile, Cutibacterium acnes, Mycobacterium tuberculosis, Gardnerella spp., Enterococcus faecalis, Enterococcus faecium, Lactobacillus inellus, Bacillus subtilis, and Bacillus anthracis.

118. 118. The method of any one of claims 59 to 117, wherein the bacteria is resistant to methicillin and vancomycin or is non-susceptible to penicillin.

119. The method of any one of claims 59 to 114, 117 and 118, wherein the bacterium is Methicillin-resistant Staphylococcus aureus (MRSA).

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