Encapsulated RNA polynucleotides and methods of use

Abstract

The present disclosure relates to recombinant RNA molecules encoding oncolytic viral genomes. The present disclosure further relates to the encapsulation of recombinant RNA molecules and the use of recombinant RNA molecules and / or particles for the treatment and prevention of cancer. In one aspect, the present disclosure provides lipid nanoparticles (LNPs) comprising synthetic RNA viral genomes encoding oncolytic Coxsackievirus viruses, wherein the Coxsackievirus is a CVA21 strain selected from EF and KY strains.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 134,376, filed January 6, 2021, U.S. Provisional Patent Application No. 63 / 147,959, filed February 10, 2021, U.S. Provisional Patent Application No. 63 / 181,899, filed April 29, 2021, U.S. Provisional Patent Application No. 63 / 181,917, filed April 29, 2021, and U.S. Provisional Patent Application No. 63 / 181,663, filed April 29, 2021, the contents of each of which are incorporated by reference herein in their entirety.

[0002] Incorporating electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety. A computer-readable copy of the Sequence Listing (Filename: ONCR_023_03WO_SeqList_ST25.txt, Created: January 6, 2022, File Size: Approximately 524 KB).

[0003] Field The present disclosure relates generally to the fields of immunology, inflammation, and cancer therapy. More specifically, the present disclosure relates to oncolytic virus strains, the design of recombinant DNA molecules for viral genome expression, and particle-encapsulated viral genomes. The present disclosure further relates to the treatment and prevention of proliferative disorders such as cancer. [Background technology]

[0004] background Oncolytic viruses are replication-competent viruses with a lytic life cycle that can infect and lyse tumor cells. Direct tumor cell lysis not only results in cell death, but also generates innate and adaptive immune responses against tumor antigens that are taken up and presented by local antigen-presenting cells. Thus, oncolytic viruses combat tumor cell growth through both direct cell lysis and the promotion of antigen-specific adaptive responses that can sustain anti-tumor responses after viral clearance.

[0005] However, the clinical use of replication-competent viruses presents several challenges. Generally, viral exposure activates innate immune pathways, triggering a broad, nonspecific inflammatory response. If a patient has not previously been exposed to a virus, this initial innate immune response can lead to the development of an adaptive antiviral response and the production of neutralizing antibodies. If a patient has previously been exposed to a virus, pre-existing neutralizing antiviral antibodies can prevent the desired lytic effect. In both cases, the presence of neutralizing antibodies not only prevents viral lysis of target cells but also renders re-administration of viral therapeutics ineffective. These factors limit the use of viral therapeutics in the treatment of metastatic cancer because the effectiveness of repeated systemic administrations required for such cancer treatment is hindered by naturally occurring antiviral responses. Even in the absence of such obstacles, subsequent viral replication in non-diseased cells can result in substantial non-disease collateral damage to surrounding cells and tissues. Furthermore, different oncolytic virus strains typically exhibit significantly different tropisms and efficacies in killing cancer cells.

[0006] There remains a long-felt unmet need in the art for compositions and methods relating to the therapeutic use of replication-competent viruses, the engineering of viruses and corresponding expression templates, and the selection of appropriate viral strains for different therapeutic indications. The present disclosure provides such compositions and methods. Summary of the Invention

[0007] In one aspect, the present disclosure provides lipid nanoparticles (LNPs) comprising a synthetic RNA viral genome encoding an oncolytic Coxsackievirus, wherein the Coxsackievirus is a CVA21 strain selected from the EF and KY strains. In some embodiments, the Coxsackievirus is a CVA21-KY strain, and the CVA21-KY strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:5. In some embodiments, the Coxsackievirus is a CVA21-EF strain, and the CVA21-EF strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:9. In some embodiments, the Coxsackievirus comprises a 5'UTR (IRES) sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:6 or 10. In some embodiments, the Coxsackievirus comprises a P1 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7 or 11. In some embodiments, the Coxsackievirus comprises a 3D sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:8 or 12. In some embodiments, the synthetic RNA viral genome does not comprise a polynucleotide sequence having more than 95%, more than 90%, more than 85%, or more than 80% sequence identity to SEQ ID NO:1.

[0008] In one aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising a synthetic RNA viral genome encoding an oncolytic Coxsackievirus, wherein the Coxsackievirus is the CVA21 Kuykendall strain.

[0009] In one aspect, the disclosure provides a lipid nanoparticle (LNP) comprising a synthetic RNA viral genome encoding an oncolytic Seneca Valley Virus (SVV), wherein the synthetic RNA viral genome comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 68. In some embodiments, the synthetic RNA viral genome comprises a 5'UTR (IRES) sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to nucleic acids 1-670 of SEQ ID NO: 68. In some embodiments, the synthetic RNA viral genome encodes an SVV VP2 protein comprising a S177A mutation.

[0010] In some embodiments, delivery of the LNP to a cell results in the production of viral particles by the cell, which are infectious and lytic. In some embodiments, the synthetic RNA viral genome further comprises a heterologous polynucleotide encoding an exogenous payload protein. In some embodiments, the LNP further comprises a second recombinant RNA molecule encoding an exogenous payload protein.

[0011] In some embodiments, the exogenous payload protein comprises or consists of an MLKL 4HB domain, a Gasdermin DN-terminal fragment, a Gasdermin EN-terminal fragment, an HMGB1 Box B domain, SMAC / Diablo, melittin, L-amino acid oxidase (LAAO), a disintegrin, TRAIL (TNFSF10), a nitroreductase, a reovirus FAST protein, leptin / FOSL2, α-1,3-galactosyltransferase, or adenosine deaminase 2 (ADA2). In some embodiments, the nitroreductase is NfsB or NfsA. In some embodiments, the reovirus FAST protein is ARV p14, BRV p15, or a p14-p15 hybrid. In some embodiments, the exogenous payload protein is a fluorescent protein, an enzyme protein, a cytokine, a chemokine, an antigen-binding molecule capable of binding to a cell surface receptor, or a ligand for a cell surface receptor. In some embodiments, the cytokine is selected from GM-CSF, IFNγ, IL-2, IL-7, IL-12, IL-18, IL-21, and IL-36γ. In some embodiments, the ligand for the cell surface receptor is Flt3 ligand or TNFSF14. In some embodiments, the chemokine is selected from CXCL10, CCL4, CCL21, and CCL5. In some embodiments, the antigen binding molecule is capable of binding to and inhibiting an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor is PD-1. In some embodiments, the antigen binding molecule is capable of binding to a tumor antigen. In some embodiments, the antigen binding molecule is a bispecific T cell engager molecule (BiTE) or a bispecific light T cell engager molecule (LiTE). In some embodiments, the tumor antigen is a viral antigen selected from HBV-core (hepatitis B core antigen), HBV-pol, HbS-Ag, HPV E6, HPV E7, Merkel cell polyoma large T antigen, and Epstein-Barr virus antigens EBNA2 or BZLF1. In some embodiments, the tumor antigen is DLL3 or EpCAM.

[0012] In some embodiments, the synthetic RNA viral genome and / or recombinant RNA molecule comprises a microRNA (miRNA) target sequence (miR-TS) cassette, wherein the miR-TS cassette comprises one or more miRNA target sequences. In some embodiments, the one or more miRNAs are selected from miR-124, miR-1, miR-143, miR-128, miR-219, miR-219a, miR-122, miR-204, miR-217, miR-137, miR-142, and miR-126. In some embodiments, the miR-TS cassette comprises a. one or more copies of a miR-124 target sequence, one or more copies of a miR-1 target sequence, and one or more copies of a miR-143 target sequence; b. one or more copies of a miR-128 target sequence, one or more copies of a miR-219a target sequence, and one or more copies of a miR-122 target sequence; c. one or more copies of a miR-128 target sequence, one or more copies of a miR-204 target sequence, and one or more copies of a miR-219 target sequence, or d. Contains one or more copies of a miR-217 target sequence, one or more copies of a miR-137 target sequence, and one or more copies of a miR-126 target sequence.

[0013] In some embodiments, the LNP comprises a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid. In some embodiments, the cationic lipid is a compound of formula (I): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: A is -N(CHR N1 )(CH2R N2 ), or a 4- to 7-membered heterocyclyl ring containing at least one N, wherein the 4- to 7-membered heterocyclyl ring is 3 and optionally substituted with Each X is independently -O-, -N(R 1 )-, or -N(R2 )- and R 1 is an optionally substituted C1-C 31 selected from the group consisting of aliphatic and steroidal; R 2 is an optionally substituted C1-C 31 selected from the group consisting of aliphatic and steroidal; R 3 is an optionally substituted C1-C6 aliphatic; R N1 and R N2 are each independently hydrogen, hydroxy-C1-C6 alkyl, C2-C6 alkenyl, or C3-C7 cycloalkyl; L 1 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 alkenylene chains, L 2 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 alkenylene chains, L 3 is a bond, an optionally substituted C1-C6 alkylene chain, or a divalent optionally substituted C3-C7 cycloalkylene, However, when A is -N(CH3)(CH3) and X is O, L 3 is not a C1-C6 alkylene chain.

[0014] In some embodiments, the number of carbon atoms between the S of the thiolate and the nearest N in A is 2 to 4. In some embodiments, the cationic lipid is a compound of formula (Ia): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: m is 0, 1, 2, 3, 4, 5, or 6.

[0015] In some embodiments, A is an optionally substituted 5-6 membered heterocyclyl ring. In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0016] In some embodiments, the cationic lipid is selected from DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA (MC3), COATSOME® SS-LC (formerly SS-18 / 4PE-13), COATSOME® SS-EC (formerly SS-33 / 4PE-15), COATSOME® SS-OC, COATSOME® SS-OP, di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L-319), or N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP).

[0017] In some embodiments, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0018] In some embodiments, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the helper lipid is 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0019] In some embodiments, the structural lipid is cholesterol.

[0020] In some embodiments, the PEG lipid is a compound of formula (A"): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1” is a bond, -[(CH2) 0-3 -C(O)O] 1-3 -, -(CH2) 0-3 -C(O)O-(CH2) 1-3 -OC(O)- or -C(O)N(H)-; R P1” is C5-C 25 Alkyl or C5-C 25 is alkenyl, R P2” is hydrogen or -CH3, The LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%.

[0021] In some embodiments, L P1” is a bond, —CHC(O)O—, —CHCHC(O)O—, —CHC(O)OCHC(O)O—, —CHC(O)OCHCHOC(O)—, or —C(O)N(H)—.

[0022] In some embodiments, L P1” is a bond. In some embodiments, R P2” is hydrogen. In some embodiments, the PEG lipid is a compound of formula (B): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, R B1 is C5-C 25 Alkyl or C5-C25 is alkenyl, The LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%.

[0023] In some embodiments, the PEG lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), 1,2-dipalmitoyl-rac-glycerol methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG), and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-amine).

[0024] In some embodiments, the PEG lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-PEG5K), 1,2-dipalmitoyl-rac-glycerol methoxypolyethylene glycol-2000 (DPG-PEG2K), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-5000 (DSG-PEG5K), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DSG-PEG2K), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-5000 (DMG-PEG5K), and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DMG-PEG2K).

[0025] In some embodiments, the cationic lipid comprises COATSOME® SS-OC, the helper lipid comprises DSPC, the structural lipid comprises cholesterol (Chol), and the PEG lipid comprises DPG-PEG2000.

[0026] In some embodiments, the cationic lipid comprises COATSOME® SS-OC, the helper lipid comprises DSPC, the structural lipid comprises cholesterol (Chol), and the PEG lipid is a compound of formula (A″): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1” is a bond, R P1” is C5-C 25 Alkyl or C5-C 25 is alkenyl, R P2” is hydrogen, The LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%.

[0027] In some embodiments, the PEG lipid is selected from the group consisting of BRIJ™ S100, BRIJ™ S20, BRIJ™ O20, and BRIJ™ C20. In some embodiments, the PEG lipid is BRIJ™ S100.

[0028] In some embodiments, the ratio of SS-OC:DSPC:Chol:PEG lipids (as a percentage of the total lipid content) is A:B:C:D, where A+B+C+D=100%; e. A=40%~60%, B=10%~25%, C=20%~30%, and D=0.01%~3%, f. A=45%~50%, B=20%~25%, C=25%~30%, and D=0.01%~1%, or g. A = approximately 49%, B = approximately 22%, C = approximately 28%, and D = approximately 0.5%.

[0029] In some embodiments, the ratio of SS-OC:DSPC:Chol:PEG lipids (as a percentage of the total lipid content) is A:B:C:D, where A+B+C+D=100%; h. A=40%~60%, B=10%~30%, C=20%~45%, and D=0%~3%, i. A=40%~60%, B=10%~30%, C=25%~45%, and D=0.01%~3%, j. A=45%~55%, B=10%~20%, C=30%~40%, and D=1%~2%, k. A=45%~50%, B=10%~15%, C=35%~40%, and D=1%~2%, or l. A = approximately 49%, B = approximately 11%, C = approximately 38%, and D = approximately 1.5%.

[0030] In some embodiments, the ratio of SS-OC:DSPC:Chol:PEG lipids (as a percentage of the total lipid content) is about A:B:C:D, where A+B+C+D=100%; m. A=45%~65%, B=5%~20%, C=20%~45%, and D=0%~3%, n. A=50%~60%, B=5%~15%, C=30%~45%, and D=0.01%~3%, o. A=55%~60%, B=5%~15%, C=30%~40%, and D=1%~2%; p. A=55%~60%, B=5%~10%, C=30%~35%, and D=1%~2%, or q. A = approximately 58%, B = approximately 7%, C = approximately 33%, and D = approximately 1.5%.

[0031] In one aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a. A synthetic RNA viral genome encoding Seneca Valley Virus (SVV), and b. A compound comprising a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is a compound of formula (A"): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1” is a bond, -[(CH2) 0-3 -C(O)O] 1-3 -, -(CH2) 0-3 -C(O)O-(CH2) 1-3 -OC(O)- or -C(O)N(H)-; R P1” is C5-C 25 Alkyl or C5-C 25 is alkenyl, R P2” is hydrogen or -CH3, the LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%; Lipid nanoparticles (LNPs) are provided.

[0032] In one aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a. A synthetic RNA viral genome encoding a Coxsackievirus, and b. A compound comprising a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is a compound of formula (A"): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1” is a bond, -[(CH2) 0-3 -C(O)O] 1-3 -, -(CH2) 0-3 -C(O)O-(CH2) 1-3 -OC(O)- or -C(O)N(H)-; R P1” is C5-C25 Alkyl or C5-C 25 is alkenyl, R P2” is hydrogen or -CH3, the LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%; Lipid nanoparticles (LNPs) are provided.

[0033] In some embodiments, R 1 is C 16 -C 18 Alkyl or C 16 -C 18 In some embodiments, L is alkenyl. P1” is a bond, —CHC(O)O—, —CHCHC(O)O—, —CHC(O)OCHC(O)O—, —CHC(O)OCHCHOC(O)—, or —C(O)N(H)—. In some embodiments, L P1” is a bond. In some embodiments, R P2” is hydrogen. In some embodiments, the PEG lipid is a compound of formula (A"-f1), formula (A"-f2), or formula (A"-f3): [ka] or a pharmaceutically acceptable salt thereof.

[0034] In one aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a. A synthetic RNA viral genome encoding Seneca Valley Virus (SVV), and b. A polymer comprising a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is Compounds of formula (B): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, R B1 is C5-C 25Alkyl or C5-C 25 is alkenyl, the LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%; Lipid nanoparticles (LNPs) are provided.

[0035] In one aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a. A synthetic RNA viral genome encoding a Coxsackievirus, and b. A polymer comprising a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is Compounds of formula (B): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, R B1 is C5-C 25 Alkyl or C5-C 25 is alkenyl, the LNPs have a molar ratio of PEG-lipid of about 0.001% to about 5%; Lipid nanoparticles (LNPs) are provided. In some embodiments, R 1 is C 15 -C 17 Alkyl or C 15 -C 17 It is alkenyl.

[0036] In some embodiments, the PEG lipid is a compound of formula (Ba) or formula (Bb): [ka] or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, n averages about 20, about 40, about 50, or about 100. In some embodiments, n averages about 100. In some embodiments, the PEG lipid comprises a PEG moiety having an average molecular weight of about 200 daltons to about 10,000 daltons, about 500 daltons to about 7,000 daltons, or about 800 daltons to about 6,000 daltons.

[0038] In some embodiments, the PEG lipid is HO-PEG-CH(CH) 16 CH3, HO-PEG20-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 14 CH3, HO-PEG20-C 18 H 35 , HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13 CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3, HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG40-C(O)-CH2(CH2) 15 CH3, and HO-PEG50-C(O)-CH2(CH2) 15 CH3.

[0039] In some embodiments, the LNPs induce a reduced immune response in vivo compared to a control LNP lacking the PEG-lipid of Formula (A") and / or the ionizable lipid of Formula (I), optionally wherein the PEG-lipid in the control LNP is PEG2K-DPG or PEG2K-DMG. In some embodiments, the immune response is accelerated blood clearance (ABC) of the LNPs and / or an anti-PEG IgM response.

[0040] In some embodiments, the cationic lipid is a compound of formula (I): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: A is -N(CHR N1 )(CH2R N2 ), or a 4- to 7-membered heterocyclyl ring containing at least one N, wherein the 4- to 7-membered heterocyclyl ring is 3 and optionally substituted with Each X is independently -O-, -N(R 1 )-, or -N(R 2 )- and R 1 is an optionally substituted C1-C 31 selected from the group consisting of aliphatic and steroidal; R 2 is an optionally substituted C1-C 31 selected from the group consisting of aliphatic and steroidal; R 3 is an optionally substituted C1-C6 aliphatic; R N1 and R N2 are each independently hydrogen, hydroxy-C1-C6 alkyl, C2-C6 alkenyl, or C3-C7 cycloalkyl; L 1 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 alkenylene chains, L 2 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 alkenylene chains, L 3 is a bond, an optionally substituted C1-C6 alkylene chain, or a divalent optionally substituted C3-C7 cycloalkylene, However, when A is -N(CH3)(CH3) and X is O, L 3 is not a C1-C6 alkylene chain.

[0041] In some embodiments, the number of carbon atoms between S and the nearest N in A of the thiolate is 2-4.

[0042] In some embodiments, the cationic lipid is a compound of formula (Ia): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: m is 0, 1, 2, 3, 4, 5, or 6.

[0043] In some embodiments, A is an optionally substituted 5-6 membered heterocyclyl ring.

[0044] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0045] In some embodiments, the cationic lipid is selected from DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA (MC3), COATSOME® SS-LC (formerly SS-18 / 4PE-13), COATSOME® SS-EC (formerly SS-33 / 4PE-15), COATSOME® SS-OC, COATSOME® SS-OP, di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L-319), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), or a mixture thereof.

[0046] In some embodiments, the cationic lipid is a compound of formula (II-1a): [ka] It is a compound of formula (II-2a). [ka]

[0047] In some embodiments, the cationic lipid is a compound of formula (II-1a), the structural lipid is cholesterol, the helper lipid is DSPC, and the PEG lipid is BRIJ™ S100.

[0048] In some embodiments, the cationic lipid is a compound of formula (II-1a), the structural lipid is cholesterol, the helper lipid is DSPC, and the PEG lipid is MYRJ™ S100, MYRJ™ S50, or MYRJ™ S40.

[0049] In some embodiments, the LNP comprises about 0.1% to about 2% molar PEG lipid, e.g., about 0.2% to about 0.8 mol%, about 0.4% to about 0.6 mol%, about 0.7% to about 1.3%, or about 1.2% to about 1.8% PEG lipid. In some embodiments, the LNP comprises about 0.2% to about 0.8%, or about 0.5% molar PEG lipid. In some embodiments, the LNP comprises about 1.2% to about 1.8%, or about 1.5% PEG lipid. In some embodiments, the LNP has a molar ratio of about 44% to about 54% cationic lipid, about 19% to about 25% helper lipid, about 24% to about 33% structural lipid, and about 0.2% to about 0.8% PEG lipid.

[0050] In some embodiments, the LNP comprises a compound of formula (II-1a), cholesterol, DSPC, and HO-PEG-CH(CH). 16 CH3, HO-PEG20-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 14 CH3, HO-PEG20-C 18 H 35 , HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3, HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG40-C(O)-CH2(CH2) 15 CH3, and HO-PEG50-C(O)-CH2(CH2) 15 and a PEG lipid selected from CH3, wherein the molar ratio of the compound of formula (II-1a):cholesterol:DSPC:PEG lipid is 49:28.5:22:0.5.

[0051] In some embodiments, the LNP comprises a compound of formula (II-1a), cholesterol, DSPC, and HO-PEG-CH(CH). 16 CH3, HO-PEG20-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 14 CH3, HO-PEG20-C 18 H 35 , HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13 CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3, HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG40-C(O)-CH2(CH2) 15 CH3, and HO-PEG50-C(O)-CH2(CH2) 15 and a PEG lipid selected from CH3, wherein the molar ratio of the compound of formula (II-1a):cholesterol:DSPC:PEG lipid is 49:27.5:22:1.5.

[0052] In some embodiments, the LNP comprises a compound of formula (II-1a), cholesterol, DSPC, and HO-PEG-CH(CH). 16 CH3, HO-PEG20-CH2(CH2) 16 CH3, HO-PEG20-CH2(CH2) 14 CH3, HO-PEG20-C 18 H 35, HO-PEG100-C(O)-CH2(CH2) 13 CH3, HO-PEG50-C(O)-CH2(CH2) 13 CH3, HO-PEG40-C(O)-CH2(CH2) 13 CH3, HO-PEG100-C(O)-CH2(CH2) 15 CH3, HO-PEG40-C(O)-CH2(CH2) 15 CH3, and HO-PEG50-C(O)-CH2(CH2) 15 and a PEG lipid selected from CH3, wherein the molar ratio of the compound of formula (II-1a):cholesterol:DSPC:PEG lipid is 49:38.5:11:1.5.

[0053] In some embodiments, the LNPs have a lipid nitrogen-to-phosphate (N:P) ratio of about 1 to about 25. In some embodiments, the LNPs have an N:P ratio of about 14. In some embodiments, hyaluronan is conjugated to the surface of the LNPs.

[0054] In one aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of lipid nanoparticles of the present disclosure. In some embodiments, the plurality of LNPs have an average diameter of about 50 nm to about 500 nm, about 150 nm to about 500 nm, about 200 nm to about 500 nm, about 300 nm to about 500 nm, about 350 nm to about 500 nm, about 400 nm to about 500 nm, about 425 nm to about 500 nm, about 450 nm to about 500 nm, or about 475 nm to about 500 nm. In some embodiments, the plurality of LNPs have an average diameter of about 50 nm to about 120 nm. In some embodiments, the plurality of LNPs have an average diameter of about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, or about 120 nm. In some embodiments, the plurality of LNPs have an average diameter of about 100 nm.

[0055] In some embodiments, the LNPs have an average zeta potential of about 40 mV to about −40 mV, about 20 mV to about −20 mV, about 10 mV to about −10 mV, about 5 mV to about −5 mV, or about 20 mV to about −40 mV. In some embodiments, the LNPs have an average zeta potential of less than about 5 mV, less than about 0 mV, less than about −5 mV, less than about −10 mV, less than about −20 mV, less than about −30 mV, less than about −35 mV, or less than about −40 mV. In some embodiments, the LNPs have an average zeta potential of about −50 mV to about −20 mV, about −40 mV to about −20 mV, about −30 mV to about −10 mV, about −20 mV to about 0 mV, about −15 mV to about 5 mV, or about −10 mV to about 10 mV. In some embodiments, the plurality of LNPs have an average zeta potential of about -30 mV, about -31 mV, about -32 mV, about -33 mV, about -34 mV, about -35 mV, about -36 mV, about -37 mV, about -38 mV, about -39 mV, or about -40 mV.

[0056] In some embodiments, administration of the pharmaceutical composition to a subject delivers the recombinant RNA polynucleotide to target cells in the subject, where the recombinant RNA polynucleotide produces an infectious oncolytic virus capable of lysing the target cells in the subject. In some embodiments, the target cells are cancerous cells. In some embodiments, the composition is formulated for intravenous and / or intratumoral delivery.

[0057] In some embodiments, the composition has a longer in vivo duration of therapeutic effect than that of a composition lacking the PEG lipid of Formula (A") and / or the ionizable lipid of Formula (I). In some embodiments, the composition has an in vivo duration of therapeutic effect of about 1 hour or more, about 2 hours or more, about 3 hours or more, about 4 hours or more, about 5 hours or more, about 6 hours or more, about 7 hours or more, about 8 hours or more, about 9 hours or more, about 10 hours or more, about 12 hours or more, about 14 hours or more, about 16 hours or more, about 18 hours or more, about 20 hours or more, about 25 hours or more, about 30 hours or more, about 35 hours or more, about 40 hours or more, about 45 hours or more, or about 50 hours or more. The composition has an in vivo half-life and / or AUC that is greater than or equal to a predetermined threshold.

[0058] In some embodiments, the encapsulation efficiency of synthetic RNA virus genome by LNP is at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100%. In some embodiments, the composition has a total lipid concentration of about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM. In some embodiments, the composition is formulated at a pH of about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, or about 6.

[0059] In some embodiments, the composition is formulated for multiple administration. In some embodiments, the subsequent administration is administered at least 3 days, at least 5 days, at least 7 days, at least 9 days, at least 11 days, at least 14 days, or at least 21 days after the first administration. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0060] In one aspect, the present disclosure provides a recombinant RNA molecule comprising a synthetic RNA viral genome encoding an oncolytic Coxsackievirus, wherein the Coxsackievirus is a CVA21 strain selected from the EF and KY strains. In some embodiments, the Coxsackievirus is a CVA21-KY strain, and the CVA21-KY strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:5. In some embodiments, the Coxsackievirus is a CVA21-EF strain, and the CVA21-EF strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:9. In some embodiments, the Coxsackievirus comprises a 5'UTR (IRES) sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:6 or 10. In some embodiments, the Coxsackievirus comprises a P1 sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7 or 11. In some embodiments, the Coxsackievirus comprises a 3D sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:8 or 12. In some embodiments, the synthetic RNA viral genome does not comprise a polynucleotide sequence having more than 95%, more than 90%, more than 85%, or more than 80% sequence identity to SEQ ID NO:1. In some embodiments, the recombinant RNA molecule does not comprise an RNA viral genome having 100% sequence identity to that of a wild-type Coxsackievirus.

[0061] In one aspect, the present disclosure provides a recombinant RNA molecule comprising a synthetic RNA viral genome encoding an oncolytic Coxsackievirus virus, wherein the Coxsackievirus is the CVA21 Kuykendall strain.

[0062] In one aspect, the present disclosure provides a recombinant RNA molecule comprising a synthetic RNA viral genome encoding a Seneca Valley virus (SVV), wherein the SVV comprises a chimeric SVV, and wherein the synthetic RNA viral genome comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:68.

[0063] In some embodiments, the recombinant RNA molecule further comprises a microRNA (miRNA) target sequence (miR-TS) cassette inserted into the polynucleotide sequence encoding the oncolytic virus, wherein the miR-TS cassette comprises one or more miRNA target sequences, and expression of one or more of the corresponding miRNAs in the cell inhibits replication of the encoded virus in the cell. In some embodiments, the one or more miRNAs are selected from miR-124, miR-1, miR-143, miR-128, miR-219, miR-219a, miR-122, miR-204, miR-217, miR-137, miR-142, and miR-126. In some embodiments, the miR-TS cassette comprises a. one or more copies of a miR-124 target sequence, one or more copies of a miR-1 target sequence, and one or more copies of a miR-143 target sequence; b. one or more copies of a miR-128 target sequence, one or more copies of a miR-219a target sequence, and one or more copies of a miR-122 target sequence; c. one or more copies of a miR-128 target sequence, one or more copies of a miR-204 target sequence, and one or more copies of a miR-219 target sequence, or d. Contains one or more copies of a miR-217 target sequence, one or more copies of a miR-137 target sequence, and one or more copies of a miR-126 target sequence.

[0064] In some embodiments, the recombinant RNA molecule can produce a replication-competent oncolytic virus when introduced into a cell by a non-viral delivery vehicle. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a mammalian cell present in a mammalian subject. In some embodiments, the one or more miR-TS cassettes are integrated into the 5' untranslated region (UTR) or 3' UTR of one or more viral genes. In some embodiments, the one or more miR-TS cassettes are integrated into the open reading frame (ORF), 5' untranslated region (UTR), or 3' UTR of one or more viral genes. In some embodiments, the recombinant RNA molecule is inserted into a nucleic acid vector. In some embodiments, the nucleic acid vector is a replicon.

[0065] In some embodiments, the synthetic RNA viral genome further comprises a heterologous polynucleotide encoding an exogenous payload protein. In some embodiments, the exogenous payload protein comprises or consists of an MLKL 4HB domain, a Gasdermin DN-terminal fragment, a Gasdermin EN-terminal fragment, an HMGB1 Box B domain, SMAC / Diablo, melittin, L-amino acid oxidase (LAAO), a disintegrin, TRAIL (TNFSF10), a nitroreductase, a reovirus FAST protein, leptin / FOSL2, α-1,3-galactosyltransferase, or adenosine deaminase 2 (ADA2). In some embodiments, the nitroreductase is NfsB or NfsA. In some embodiments, the reovirus FAST protein is ARV p14, BRV p15, or a p14-p15 hybrid. In some embodiments, the exogenous payload protein is a fluorescent protein, an enzyme protein, a cytokine, a chemokine, an antigen-binding molecule capable of binding to a cell surface receptor, or a ligand capable of binding to a cell surface receptor. In some embodiments, the cytokine is selected from GM-CSF, IFNγ, IL-2, IL-7, IL-12, IL-18, IL-21, and IL-36γ. In some embodiments, the ligand for the cell surface receptor is Flt3 ligand or TNFSF14. In some embodiments, the chemokine is selected from CXCL10, CCL4, CCL21, and CCL5. In some embodiments, the antigen binding molecule is capable of binding to and inhibiting an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor is PD-1. In some embodiments, the antigen binding molecule is capable of binding to a tumor antigen. In some embodiments, the antigen binding molecule is a bispecific T cell engager molecule (BiTE) or a bispecific light T cell engager molecule (LiTE).In some embodiments, the tumor antigen is a viral antigen selected from HBV-core (hepatitis B core antigen), HBV-pol, HbS-Ag, HPV E6, HPV E7, Merkel cell polyoma large T antigen, and Epstein-Barr virus antigens EBNA2 or BZLF1. In some embodiments, the tumor antigen is DLL3 or EpCAM.

[0066] In one aspect, the disclosure provides a recombinant DNA template comprising, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding an RNA molecule comprising a synthetic RNA viral genome, a polyA tail, and a 3' junction cleavage sequence.

[0067] In one aspect, the present disclosure provides a recombinant DNA molecule comprising, from 5' to 3', a promoter sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding an RNA molecule of the present disclosure, including a synthetic RNA viral genome, a polyA tail, and a 3' junction cleavage sequence.

[0068] In some embodiments, the recombinant DNA molecule comprises a leader sequence between the promoter sequence and the 5' junction cleavage sequence.

[0069] In one aspect, the disclosure provides a recombinant DNA molecule comprising, from 5' to 3', a promoter sequence, a leader sequence, a 5' junction cleavage sequence, a polynucleotide sequence encoding a recombinant RNA molecule comprising a synthetic RNA viral genome, a polyA tail, and a 3' junction cleavage sequence.

[0070] In some embodiments, the leader sequence is less than 100 bp in length. In some embodiments, the promoter sequence is a T7 promoter sequence. In some embodiments, the polyA tail is about 50-90 bp in length or about 65-75 bp in length. In some embodiments, the polyA tail is about 70 bp in length. In some embodiments, the polyA tail is about 10-50 bp, or 25-35 bp in length.

[0071] In some embodiments, the 5' junction cleavage sequence comprises or consists of a ribozyme sequence, and the 3' junction cleavage sequence comprises or consists of a ribozyme sequence. In some embodiments, the 5' ribozyme sequence is a hammerhead ribozyme sequence, and the 3' ribozyme sequence is a hepatitis delta virus ribozyme sequence. In some embodiments, the 5' junction cleavage sequence comprises or consists of an RNAse H primer binding sequence, and the 3' junction cleavage sequence comprises or consists of a restriction enzyme recognition sequence. In some embodiments, the 5' junction cleavage sequence comprises or consists of a ribozyme sequence, and the 3' junction cleavage sequence comprises or consists of a restriction enzyme recognition sequence. In some embodiments, the 5' ribozyme sequence comprises or consists of a hammerhead ribozyme sequence, a pistol ribozyme sequence, or a modified pistol ribozyme sequence.

[0072] In some embodiments, the 3' junction cleavage sequence comprises or consists of a type IIS restriction enzyme recognition sequence.

[0073] In some embodiments, the RNA molecule encodes the RNA viral genome of a Coxsackievirus (CVA). In some embodiments, the Coxsackievirus is the CVA21 strain. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 14 or 15. In some embodiments, the 5' junction cleavage sequence comprises or consists of a pistol ribozyme sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 18, and the P2 motif of the 5' ribozyme sequence has a polynucleotide sequence of "TTTT." In some embodiments, the 5' junction cleavage sequence comprises or consists of a pistol ribozyme sequence having at least 80%, at least 90%, or 100% sequence identity to SEQ ID NO: 17, and the P2 motif of the 5' ribozyme sequence has a polynucleotide sequence of "TTTA." In some embodiments, the 3' junction cleavage sequence comprises or consists of a BsmBI recognition sequence. In some embodiments, the 3' junction cleavage sequence comprises or consists of a BsaI recognition sequence. In some embodiments, the promoter sequence is a T7 promoter sequence, the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 15, the 5' junction cleavage sequence comprises or consists of a pistol ribozyme sequence according to SEQ ID NO: 18, the polyA tail is about 70 bp in length, and the 3' junction cleavage sequence comprises or consists of a BsmBI recognition sequence. In some embodiments, the promoter sequence is a T7 promoter sequence, the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 15, the 5' junction cleavage sequence comprises or consists of a pistol ribozyme sequence according to SEQ ID NO: 18, the polyA tail is about 70 bp in length, and the 3' junction cleavage sequence comprises or consists of a BsaI recognition sequence.

[0074] In some embodiments, the RNA molecule encodes the RNA viral genome of Seneca Valley Virus (SVV). In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 53-63. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58. In some embodiments, the 5' ribozyme sequence is a pistol ribozyme sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 64 or 65, and the P2 motif of the 5' ribozyme sequence has a polynucleotide sequence of "TCAA" or "TTAA." In some embodiments, the RNA virus genome comprises a 5' UTR (IRES) sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to nucleic acids 1-670 of SEQ ID NO: 68. In some embodiments, the 3' junction cleavage sequence comprises or consists of a SapI recognition sequence. In some embodiments, the promoter sequence is a T7 promoter sequence, the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 53, the 5' junction cleavage sequence comprises or consists of a pistol ribozyme sequence according to SEQ ID NO: 64, the polyA tail is approximately 70 bp in length, and the 3' junction cleavage sequence comprises or consists of a SapI recognition sequence. In some embodiments, the promoter sequence is a T7 promoter sequence, the leader sequence consists of a polynucleotide sequence according to SEQ ID NO: 58, the 5' junction cleavage sequence comprises or consists of a pistol-type ribozyme sequence according to SEQ ID NO: 64, the polyA tail is approximately 70 bp in length, and the 3' junction cleavage sequence comprises or consists of a SapI recognition sequence.

[0075] In some embodiments, the recombinant DNA molecule does not contain any additional nucleic acid within the region spanning the promoter sequence and the 3' junction cleavage sequence.

[0076] In one aspect, the present disclosure provides a method for producing a recombinant RNA molecule, comprising in vitro transcription of a DNA molecule of the present disclosure and purifying the resulting recombinant RNA molecule. In some embodiments, the recombinant RNA molecule comprises 5' and 3' ends that are native to the oncolytic virus encoded by the synthetic RNA viral genome.

[0077] In one aspect, the present disclosure provides a composition comprising an effective amount of a recombinant RNA molecule of the present disclosure and a carrier suitable for administration to a mammalian subject.

[0078] In one aspect, the present disclosure provides a particle comprising a recombinant RNA molecule of the present disclosure. In some embodiments, the particle is biodegradable. In some embodiments, the particle is selected from the group consisting of nanoparticles, exosomes, liposomes, and lipoplexes. In some embodiments, the exosomes are modified exosomes derived from intact or empty exosomes.

[0079] In one aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of particles of the present disclosure. In some embodiments, the plurality of particles has an average size of about 50 nm to about 500 nm, about 150 nm to about 500 nm, about 200 nm to about 500 nm, about 300 nm to about 500 nm, about 350 nm to about 500 nm, about 400 nm to about 500 nm, about 425 nm to about 500 nm, about 450 nm to about 500 nm, or about 475 nm to about 500 nm. In some embodiments, the plurality of particles has an average size of about 50 nm to about 120 nm. In some embodiments, the plurality of particles has an average size of about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, or about 120 nm. In some embodiments, the plurality of particles has an average size of about 100 nm.

[0080] In some embodiments, the plurality of particles has an average zeta potential of about 40 mV to about −40 mV, about 20 mV to about −20 mV, about 10 mV to about −10 mV, about 5 mV to about −5 mV, or about 20 mV to about −40 mV. In some embodiments, the plurality of particles has an average zeta potential of less than about 5 mV, less than about 0 mV, less than about −5 mV, less than about −10 mV, less than about −20 mV, less than about −30 mV, less than about −35 mV, or less than about −40 mV. In some embodiments, the plurality of particles has an average zeta potential of about −50 mV to about −20 mV, about −40 mV to about −20 mV, about −30 mV to about −10 mV, about −20 mV to about 0 mV, about −15 mV to about 5 mV, or about −10 mV to about 10 mV. In some embodiments, the plurality of particles has an average zeta potential of about -30 mV, about -31 mV, about -32 mV, about -33 mV, about -34 mV, about -35 mV, about -36 mV, about -37 mV, about -38 mV, about -39 mV, or about -40 mV.

[0081] In some embodiments, upon delivery of the composition to a subject, the encapsulated recombinant RNA molecule is delivered to a target cell, where the encapsulated recombinant RNA molecule produces an infectious virus capable of lysing the target cell.

[0082] In one aspect, the present disclosure provides inorganic particles comprising a recombinant RNA molecule of the present disclosure. In some embodiments, the inorganic particles are selected from the group consisting of gold nanoparticles (GNPs), gold nanorods (GNRs), magnetic nanoparticles (MNPs), magnetic nanotubes (MNTs), carbon nanohorns (CNHs), carbon fullerenes, carbon nanotubes (CNTs), calcium phosphate nanoparticles (CPNPs), mesoporous silica nanoparticles (MSNs), silica nanotubes (SNTs), or star-shaped hollow silica nanoparticles (SHNPs). In some embodiments, the average diameter of the particles is less than about 500 nm, between about 50 nm and 500 nm, between about 250 nm and about 500 nm, or about 350 nm.

[0083] In some embodiments, the LNPs of the present disclosure, the particles of the present disclosure, or the inorganic particles of the present disclosure further comprise a second recombinant RNA molecule encoding a payload molecule. In some embodiments, the second recombinant RNA molecule is a replicon.

[0084] In one aspect, the present disclosure provides a pharmaceutical composition comprising an LNP of the present disclosure, a particle of the present disclosure, or an inorganic particle of the present disclosure, wherein the composition is formulated for intravenous and / or intratumoral delivery. In some embodiments, the target cell is a cancerous cell.

[0085] In one aspect, the present disclosure provides a method for killing cancerous cells, comprising exposing cancerous cells to a particle of the present disclosure, a recombinant RNA molecule of the present disclosure, or a composition thereof under conditions sufficient to deliver the particle to the cancerous cells intracellularly, wherein the replication-competent virus produced by the encapsulated polynucleotide results in the killing of the cancerous cells. In some embodiments, the replication-competent virus is not produced in non-cancerous cells. In some embodiments, the method is carried out in vivo, in vitro, or ex vivo.

[0086] In one aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to a subject suffering from cancer an effective amount of a particle of the present disclosure, a recombinant RNA molecule of the present disclosure, or a composition thereof. In some embodiments, the particle or composition thereof is administered intravenously, intranasally, intratumorally, intraperitoneally, or as an inhalant. In some embodiments, the particle or composition thereof is administered intratumorally and / or intravenously. In some embodiments, the particle or composition thereof is administered repeatedly to the subject. In some embodiments, the subject is a mouse, rat, rabbit, cat, dog, horse, non-human primate, or human.

[0087] In some embodiments, the cancer is lung cancer, breast cancer, colon cancer, or pancreatic cancer, and the synthetic RNA viral genome comprises a polynucleotide sequence derived from the KY strain. In some embodiments, the cancer is bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer, or liver cancer, and the synthetic RNA viral genome comprises a polynucleotide sequence derived from the EF strain.

[0088] In some embodiments, the cancer is selected from lung cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, colorectal cancer, colon cancer, pancreatic cancer, liver cancer, renal cell carcinoma, gastric cancer, head and neck cancer, thyroid cancer, malignant glioma, glioblastoma, melanoma, B-cell chronic lymphocytic leukemia, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), Merkel cell carcinoma, diffuse large B-cell lymphoma (DLBCL), sarcoma, neuroblastoma, neuroendocrine carcinoma, rhabdomyosarcoma, medulloblastoma, bladder cancer, and marginal zone lymphoma (MZL). In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer, and liver cancer. In some embodiments, the cancer is renal cell carcinoma, lung cancer, or liver cancer. In some embodiments, the lung cancer is small cell lung cancer or non-small cell lung cancer (e.g., lung squamous cell carcinoma or lung adenocarcinoma). In some embodiments, the liver cancer is hepatocellular carcinoma (HCC) (e.g., hepatitis B virus-associated HCC). In some embodiments, the prostate cancer is treatment-emergent neuroendocrine prostate cancer. In some embodiments, the cancer is lung cancer, liver cancer, prostate cancer (e.g., CRPC-NE), bladder cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, neuroblastoma, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, medulloblastoma, neuroendocrine carcinoma, Merkel cell carcinoma, or melanoma. In some embodiments, the cancer is small cell lung cancer (SCLC) or neuroblastoma.

[0089] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of CVA21-EF virus.

[0090] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of CVA21-KY virus.

[0091] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of CVA21-Kuykendall virus.

[0092] In some embodiments, the virus is administered intratumorally and / or intravenously.

[0093] In some embodiments, the method further comprises administering to the subject an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the method further comprises administering engineered immune cells comprising an engineered antigen receptor.

[0094] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an oncolytic Coxsackievirus, the Coxsackievirus being the CVA21 strain, or a polynucleotide encoding CVA21, wherein the cancer is classified as susceptible to CVA21 infection based on expression of ICAM-1 and / or the percentage of ICAM-1-positive cancer cells.

[0095] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising: (a) determining the expression level of ICAM1 and / or the percentage of ICAM-1 positive cancer cells in the cancer; (b) classifying the cancer as susceptible to Coxsackievirus 21 (CVA21) infection based on the expression of ICAM-1 and / or the percentage of ICAM-1-positive cancer cells determined in (a); and (c) if the cancer is classified as susceptible to CVA21 infection in step (b), administering a therapeutically effective amount of CVA21 or a polynucleotide encoding CVA21 to the subject. The present invention provides a method comprising:

[0096] In one aspect, the disclosure provides a method of selecting a subject suffering from cancer for treatment with Coxsackievirus 21 (CVA21) or a polynucleotide encoding CVA21, comprising: (a) determining the expression level of ICAM-1 in the cancer and / or the percentage of ICAM-1-positive cancer cells; (b) classifying the cancer as susceptible to CVA21 infection based on the ICAM-1 expression level and / or percentage of ICAM1-positive cancer cells determined in (a); (c) if the cancer is classified as susceptible to CVA21 infection in (b), selecting the subject for treatment with CVA21 or a polynucleotide encoding CVA21; and (d) administering to the selected subject CVA21 or a polynucleotide encoding CVA21. The present invention provides a method comprising:

[0097] In some embodiments, the CVA21 strain is CVA21-KY. [Brief explanation of the drawings]

[0098] [Figure 1] 1 shows tumor volume in SK-MEL-28 tumor-bearing mice after intratumoral administration of CVA21-RNA molecules formulated with PBS or Lipofectamine, or intravenous administration of LNPs containing CVA21-Kuykendall strain RNA molecules (formulation ID: 70032-6C).

[0099] [Figure 2A] An in vitro transcription method for generating true 3' ends of picornaviruses using 3' type IIS restriction enzyme recognition sites is outlined. [Figure 2B]Electrophoresis of DNA digested with BsmBI or BsaI restriction enzymes is shown.

[0100] [Figure 3] 1 shows the RNase H approach to generating true 5' ends of picornaviruses using a 5' DNA primer and RNase H enzyme.

[0101] [Figure 4] A ribozyme approach to generating the true 5' end of picornaviruses is presented.

[0102] [Figure 5] A and B show hammerhead ribozymes for generating individual 5' ends. A shows a structural model of a minimal hammerhead ribozyme (HHR) that anneals to and cleaves the 5' end, as indicated by the arrow (SEQ ID NO: 75). B shows a structural model of a ribozyme with stabilized stem I (STBL) for cleaving the 5' end, as indicated by the arrow (SEQ ID NO: 76).

[0103] [Figure 6A] 1 shows a pistol ribozyme for generating individual 5' ends. FIG. 2 shows a schematic diagram of the characteristics of the wild-type pistol ribozyme (SEQ ID NO: 77). [Figure 6B] Figure 1 shows a pistol ribozyme for generating a distinct 5' end. Figure 2 shows a pistol ribozyme from P. polymyxa modeled by mFOLD with a tetraloop added to fuse the P3 strand. The dashed box indicates the region that was mutagenized to preserve the ribozyme fold in the context of the viral sequence. The "GUC" sequence shown in the dashed box was mutated to "UCA" to generate Pistol 1, and the "GUC" sequence was mutated to "TTA" to generate Pistol 2 (SEQ ID NO: 78). [Figure 6C] 1 shows a pistol-type ribozyme for generating individual 5'-ends. Sequence alignment of multiple pistol-type ribozyme variants and the location of the P2 motif are shown.

[0104] [Figure 7] A and B show the production of infectious CVA21 virus from an RNA polynucleotide. A shows the effect of the 5' UTR sequence on the production of infectious CVA21 from an RNA polynucleotide. B shows the production of infectious CVA21 from an RNA polynucleotide containing the 5' UTR of SEQ ID NO:2.

[0105] [Figure 8A] 1 shows the in vivo efficacy of CVA-LNP. [Figure 8B] 1 shows the in vivo effect of CVA-LNP. 2 shows changes in body weight over time. [Figure 8C] Shown are the results of H1299 cell lysis by CVA21 infection (left) and Western-based expression analysis of ICAM1 and DAF in the indicated cell lines (right). [Figure 8D] 1 is a chart showing mean ICAM-1 mRNA and protein expression in 130 human cell lines based on susceptibility / resistance to CVA21-KY infection. [Figure 8E] FIG. 1 shows the correlation between ICAM-1 expression and susceptibility to CVA21-KY infection.

[0106] [Figure 9] A schematic representation of the LNP / picornavirus RNA composition and mode of action is shown. LNP / picornavirus RNA is administered systemically, delivering picornavirus RNA genomes to permissive tumor cells, where they replicate to produce picornavirus virions. Picornaviral infection then spreads to neighboring tumor cells, eliciting tumor lysis and an antiviral immune response.

[0107] [Figure 10]The in vitro transcription process of CVA21-RNA and Neg-RNA is shown. Autocatalytic cleavage of CVA21-RNA by 5' and 3' ribozymes (Rib) generated CVA21-RNA with distinct 5' and 3' ends required for replication. In contrast, the Neg-RNA construct lacked the ribozyme sequence and was unable to replicate and produce virions.

[0108] [Figure 11A] A general outline of the use of a junction cleavage sequence to remove non-viral RNA polynucleotides from genomic transcripts in order to maintain distinct 5' and 3' native ends of the virus is provided. [Figure 11B] 1 shows a schematic diagram of the removal of non-viral RNA polynucleotides from genomic transcripts using a junction cleavage sequence to maintain distinct 5' and 3' native ends of the virus, where the 3' junction cleavage sequence contains a restriction enzyme recognition site.

[0109] [Figure 12A] A diagram summarizing the AUCs obtained from the cytotoxicity screen for the three CVA21 strains is shown. [Figure 12B] Relative IC50 values ​​for each of the 15 cell lines are shown.

[0110] [Figure 13] IC50 values ​​of the three CVA21 strains for each individual cell line in various cancer types are shown.

[0111] [Figure 14] Results are shown plotted as the ratio of AUC values ​​for EF and KY lines using lung and NSCLC cancer cell lines.

[0112] [Figure 15A] Results are shown plotted as the ratio of AUC values ​​for EF and KY strains using the mammary cell line. [Figure 15B] Results are shown plotted as the ratio of AUC values ​​for EF and KY strains using colon / GI cell lines. [Figure 15C] Results are shown plotted as the ratio of AUC values ​​for EF and KY strains using pancreatic cancer cell lines.

[0113] [Figure 16A] ICAM-1 expression on human lung cancer microarrays by immunohistochemistry is shown, including the percentage of tumor cells that are ICAM-1 positive and the H-score (the formula includes the percentage of cells expressing +1, +2, and +3 ICAM-1 intensity levels). [Figure 16B] Figure 1 shows the oncolytic efficacy of EF and KY lines against various lung adenocarcinoma cell lines. [Figure 16C] Figure 1 shows the oncolytic efficacy of EF and KY lines against various large cell lung cancer cell lines.

[0114] [Figure 17A] Results are shown plotted as the ratio of AUC values ​​for EF and KY strains using bladder cancer cell lines. [Figure 17B] Results are shown plotted as the ratio of AUC values ​​for EF and KY lines using renal carcinoma cell lines. [Figure 17C] Results are shown plotted as the ratio of AUC values ​​for EF and KY strains using liver cancer cell lines. [Figure 17D] Results are shown plotted as the ratio of AUC values ​​for EF and KY lines using ovarian cancer cell lines. [Figure 17E] Results are shown plotted as the ratio of AUC values ​​for EF and KY lines using GBM cancer cell lines.

[0115] [Figure 18A] Results are shown plotted as the ratio of AUC values ​​for EF and KY lines using breast cancer cell lines. [Figure 18B] Results are shown plotted as the ratio of AUC values ​​for EF and KY lines using renal cell carcinoma cell lines. [Figure 18C] Results are shown plotted as the ratio of AUC values ​​for the EF and KY strains using hepatocellular carcinoma cell lines.

[0116] [Figure 19] 1 is a table summarizing cell line susceptibility to KY and EF strains based on TCID50 values.

[0117] [Figure 20A] A plot of copy numbers of KY and EF strains in dissociated human tumor cells from three donors at 24 and 72 hours post-infection is shown. [Figure 20B] A plot of copy numbers of KY and EF strains in dissociated human tumor cells from three donors at 24 and 72 hours post-infection is shown.

[0118] [Figure 21] 1 shows the change in tumor size over time and calculated TGI percentages in animals treated with the indicated LNPs based on the NCI-H1299 xenograft model.

[0119] [Figure 22] 1 shows the change in tumor size over time and calculated TGI percentages in animals treated with the indicated LNPs based on the NCI-H2122 xenograft model.

[0120] [Figure 23] The change in tumor size over time and calculated TGI percentages in animals treated with the indicated LNPs based on a PC3 xenograft model are shown.

[0121] [Figure 24] Shown are changes in body weight (top panel) and liver chemistry (bottom panel) over time in animals receiving the indicated treatments.

[0122] [Figure 25A]Figure 1 shows the copy numbers of CVA21 strand RNA based on RT-qPCR in various tissues of animals treated with LNPs containing the indicated viral genomes. The copy numbers of CVA21 (-) strand RNA 48 hours after treatment are shown. [Figure 25B] Figure 1 shows the copy numbers of CVA21 strand RNA based on RT-qPCR in various tissues of animals treated with LNPs containing the indicated viral genomes. Figure 2 shows the copy numbers of CVA21 (-) strand RNA at 7 days post-treatment. [Figure 25C] Figure 1 shows the copy number of CVA21 strand RNA by RT-qPCR in various tissues of animals treated with LNPs containing the indicated viral genomes. Figure 2 shows a plot of the copy number of CVA21 (+) strand RNA by RT-qPCR in various tissues of animals treated with LNPs containing the indicated viral genomes 48 hours or 7 days post-infection.

[0123] [Figure 26] 1 shows the results of viral plaque assays of samples extracted from the indicated tissues.

[0124] [Figure 27A] Cell viability is shown for the indicated cell lines pretreated with increasing amounts of IFN before infection with the indicated virus strains. For calculation of the relative percentage of surviving cells, a value of 100% was set according to the mock-infected group, and a value of 0% was set according to the CVA21 virus-infected group without IFN pretreatment. Results are shown for the H1299 cell line. [Figure 27B] Cell viability is shown for the indicated cell lines pretreated with increasing amounts of IFN before infection with the indicated virus strains. For calculation of the relative percentage of surviving cells, a value of 100% was set according to the mock-infected group, and a value of 0% was set according to the CVA21 virus-infected group without IFN pretreatment. Results are shown for the H2122 cell line. [Figure 27C]Cell viability is shown for the indicated cell lines pretreated with increasing amounts of IFN before infection with the indicated virus strains. For calculation of the relative percentage of surviving cells, a value of 100% was set according to the mock-infected group, and a value of 0% was set according to the CVA21 virus-infected group without IFN pretreatment. Results are shown for the HFF cell line.

[0125] [Figure 28] A shows the relative ribavirin resistance frequency rates for the indicated virus strains. B shows the amount of viable recombinant virus recovered from the indicated assay groups.

[0126] [Figure 29] A shows the design of the template construct for the analysis of leader and 5' ribozyme sequences. B shows gel electrophoresis results of the indicated in vitro transcription products based on different leader sequence designs.

[0127] [Figure 30] Gel electrophoresis results of the indicated in vitro transcripts based on different 5' ribozyme designs are shown.

[0128] [Figure 31A] Figure 1 plots the change in tumor size over time in animals treated with the indicated LNPs containing RNA viral genomes derived from in vitro transcription of DNA templates of different designs (e.g., different polyA tail lengths and 5' ribozyme sequences) using the NCI-H1299 xenograft model. [Figure 31B] FIG. 1 shows a plot of the change in tumor size over time in animals treated with the indicated LNPs.

[0129] [Figure 32] The outline of the domain organization of the RNA virus genome of three CVA21 strains (EF, KY, and Kuykendall) and the nucleic acid start / end positions of selected regions are shown.

[0130] [Figure 33]

[0023] Figure 1 shows a non-limiting example of a CVA21 expression construct design and a schematic diagram of the corresponding in vitro transcription process to generate a synthetic RNA viral genome with precise 5' and 3' ends.

[0131] [Figure 34] FIG. 1 is a schematic diagram showing the domain organization of the SVV viral genome and the construction of chimeric viruses.

[0132] [Figure 35] Fluorescence microscopy images of NCI-H69AR cells infected with SVV-001 or SVV-IRES chimeric viruses carrying a GFP reporter gene are shown.

[0133] [Figure 36] Plots of fluorescence intensity (left) of NCI-H69AR and NCI-H69 cells infected with the indicated SVV virions with or without IFNα pretreatment are shown, along with representative fluorescence images (right) 12 h postinfection.

[0134] [Figure 37] Fluorescence microscopy images of NCI-H69AR cells infected with SVV-001 or SVV-P1 chimeric viruses carrying a GFP reporter gene are shown.

[0135] [Figure 38] A and B show fluorescence microscopy images of NCI-H69AR cells infected with SVV-001 or SVV-P3 chimeric viruses carrying a GFP reporter gene.

[0136] [Figure 39A] 1 shows the design of a template construct for analysis of the leader sequence and 5′ ribozyme sequence of the SVV template. [Figure 39B] 1 shows gel electrophoresis results of the indicated in vitro transcription products based on different leader sequence designs of the SVV template. [Figure 39C] The results of a viral plaque assay using H1299 cells are shown. [Figure 39D] Figure 1 shows RP-HPLC analysis of in vitro transcription products of viral genomes using different SVV leader sequences.

[0137] [Figure 40A]

[0023] Figure 1 shows the results of an in vivo dose titration efficacy study in which athymic nude mice bearing H446 tumors were treated with the indicated LNPs. Tumor size over time is shown. [Figure 40B] 1 shows the results of an in vivo dose titration efficacy study in which athymic nude mice bearing H446 tumors were treated with the indicated LNPs. 2 shows RT-qPCR measurements of SVV replication in tumor tissue from mice treated with 0.1 mg / kg of 96062-1 LNPs. [Figure 40C] 1 shows the results of an in vivo dose titration efficacy study in which athymic nude mice bearing H446 tumors were treated with the indicated LNPs. 1 shows a FISH assay for SVV replication.

[0138] [Figure 41] 1 shows tumor size over time in H446 tumor-bearing mice treated with LNPs.

[0139] [Figure 42] 1 shows the change in tumor size over time in animals treated with the indicated LNPs based on the H446 SCLC tumor model.

[0140] [Figure 43] 1 shows the change in tumor size over time in animals treated with the indicated LNPs or SVV virions in the presence or absence of anti-SVV neutralizing antibodies, based on the H446 SCLC tumor model.

[0141] [Figure 44]1 shows the change in tumor size over time in animals treated with the indicated LNPs based on the H82 SCLC tumor model.

[0142] [Figure 45] 1 shows the probability of survival over time in animals treated with the indicated LNPs based on the H82 SCLC orthotopic tumor model.

[0143] [Figure 46A] 1 is a chart showing quantitative analysis of tumor burden based on hDLL3 IHC. [Figure 46B] hDLL3 IHC images are shown.

[0144] [Figure 47A] 1 shows the results of SVV treatment of an SCLC PDX tumor model. 2 shows the change in tumor size over time in animals treated with the indicated LNPs based on an SCLC PDX tumor model. [Figure 47B] 1 shows the results of SVV treatment of an SCLC PDX tumor model. 1 shows the results of RT-qPCR measurements.

[0145] [Figure 48] 1 shows the change in tumor size over time in animals treated with the indicated LNPs containing SVV RNA viral genomes with polyA tails of different lengths.

[0146] [Figure 49A] Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. The numbers of NK, NKT, CD4, CD8, and Treg cells per mg of tumor are shown. [Figure 49B] Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. CD8 / Treg ratio is shown. [Figure 49C]Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. The number of CD8 T cells expressing CTLA-4 or PD-1 per mg of tumor is shown. [Figure 49D] Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. The number of CD8 T cells that are SLEC (CD127-KLRG1+) or MPEC (CD127+-KLRG1-) per mg of tumor is shown. [Figure 49E] Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. The ratio of M1 / M2 macrophages is shown. [Figure 49F] Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. Numbers of M1 and M2 macrophages are shown. [Figure 49G] Figure 1 shows immune cell infiltration and the effect of combined SVV / LNP and anti-PD1 treatment in the N1E-115 syngeneic neuroblastoma model. The number of CD45-PD-L1+ cells per mg of tumor is shown.

[0147] [Figure 50] 1 shows the change in tumor size over time in animals treated with the indicated LNPs based on the N1E-115 syngeneic neuroblastoma model.

[0148] [Figure 51] 1 shows the combined effect of SVV-LNP and anti-PD1 therapy in an animal bearing N1E-115 syngeneic neuroblastoma model.

[0149] [Figure 52A] 1 is a chart depicting results from an H446 mouse tumor model showing tumor growth upon repeated dosing with an LNP composition of the present disclosure. [Figure 52B] 1 is a chart showing weight changes in an H446 mouse tumor model upon administration of an LNP composition.

[0150] [Figure 53A] 1 is a chart depicting the results of a PK study in mice of LNP compositions containing PEG2k-DPG, PEG2k-DMG, or BRIJ™ S100 as the PEG lipid. [Figure 53B] 1 is a chart depicting the results of a PK study in mice of LNP compositions containing PEG2k-DPG, PEG2k-DMG, or BRIJ™ S100 as the PEG lipid.

[0151] [Figure 54A] 1 shows UV A280 absorbance profiles of CVA21 viral genomes with various polyA tail lengths using oligo-dT chromatography. [Figure 54B] 1 shows UV A280 absorbance profiles of SVV viral genomes with various polyA tail lengths using oligo-dT chromatography.

[0152] [Figure 55] Figures A-C show particle characteristics of CAT4 and CAT5 LNP compositions. A shows particle size, B shows polydispersity index determined by dynamic light scattering experiments for CAT4 and CAT5 LNP compositions made with various RNA acidification buffers, and C shows encapsulation efficiency of these LNP compositions measured by RiboGreen.

[0153] [Figure 56] Figures A-C show particle characteristics of LNP compositions containing the indicated CAT lipids. A shows particle size, B shows the polydispersity index determined by dynamic light scattering experiments of the LNP compositions, and C shows the encapsulation efficiency of these LNP compositions as measured by RiboGreen.

[0154] [Figure 57] 1 shows a schematic representation of the formulation process for LNP formulations.

[0155] [Figure 58A]1 shows RNA levels measured by NanoLuc luciferase activation. 1 shows RNA levels measured by NanoLuc luciferase activation 96 hours after dosing with each LNP formulation. [Figure 58B] 1 shows RNA levels measured by NanoLuc luciferase activation. 1 shows RNA levels measured by NanoLuc luciferase activation 96 hours after dosing with each LNP formulation. [Figure 58C] 1 shows RNA levels measured by NanoLuc luciferase activation. 1 shows RNA levels measured by NanoLuc luciferase activation 96 hours after dosing with each LNP formulation. [Figure 58D] 1 shows RNA levels measured by NanoLuc luciferase activation. 1 shows RNA levels measured by NanoLuc luciferase activation 72 hours after dosing with each LNP formulation. [Figure 58E] 1 shows RNA levels measured by NanoLuc luciferase activation. 1 shows RNA levels measured by NanoLuc luciferase activation 72 hours after dosing with each LNP formulation. [Figure 58F] 1 shows RNA levels measured by NanoLuc luciferase activation. 1 shows RNA levels measured by NanoLuc luciferase activation 72 hours after dosing with each LNP formulation.

[0156] [Figure 59A] Tumor volume (left) and body weight change (right) over the treatment period for mice treated with each LNP formulation are shown. Results are shown for CAT 1, 2, 3, 4, and 5 LNP formulations. [Figure 59B] The tumor volume (left) and body weight change (right) over the treatment period for mice treated with each LNP formulation are shown. Results are shown for LNP formulations CAT6, 7, 8, 9, and 10. [Figure 59C]The tumor volume (left) and body weight change (right) of mice treated with each LNP formulation over the treatment period are shown. Results for CAT11, 12, 13, 14, 15, and 16 are shown. [Figure 59D] Tumor volume (left) and body weight change (right) over the treatment period for mice treated with each LNP formulation are shown. Results are shown for CAT 17, 19, 20, 25, 31, and 7 LNP formulations. [Figure 59E] The tumor volume (left) and body weight change (right) of mice treated with each LNP formulation over the treatment period are shown. Results are shown for LNP formulations CAT18, 21, 22, 23, 26, 28, 29, 32, and 34.

[0157] [Figure 60A] Figure 1 shows in vivo RNA levels and the effect on tumor volume and body weight for the indicated LNP formulations, as measured by luminescence generated by NanoLuc luciferase activation 72 hours after dosing with each LNP formulation. [Figure 60B] The effects on in vivo RNA levels and tumor volume and body weight are shown for the indicated LNP formulations. Tumor volume (right) and body weight change (left) over the treatment period for mice treated with each LNP formulation are shown.

[0158] [Figure 61] A to D show the concentration of ionizable lipids (SS-OC) contained in LNP in the plasma of treated mice measured by LC-MS. A shows the results for OC / Brij on a Q1W2 dosing schedule. B shows the results for OC / Brij on a Q2W2 dosing schedule. C shows the results for OC / DMG on a Q2W2 dosing schedule. D shows the results for OC / DPG on a Q2W2 dosing schedule.

[0159] [Figure 62A]1 shows the concentration of ionizable lipids (SS-OC or CAT7) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for OC / Brij on a Q2W2 dosing schedule. [Figure 62B] 1 shows the concentration of ionizable lipids (SS-OC or CAT7) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / DMG_6 on a Q2W2 dosing schedule. [Figure 62C] 1 shows the concentration of ionizable lipids (SS-OC or CAT7) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / DMG_3 on a Q2W2 dosing schedule. [Figure 62D] 1 shows the concentration of ionizable lipids (SS-OC or CAT7) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / DMG_5 on a Q2W2 dosing schedule. [Figure 62E] 1 shows the concentration of ionizable lipids (SS-OC or CAT7) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / DMG1_1 on a Q2W2 dosing schedule. [Figure 62F] 1 shows the concentration of ionizable lipids (SS-OC or CAT7) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / CHM6_4 on a Q2W2 dosing schedule.

[0160] [Figure 63A] 1 shows the concentration of ionizable lipids (SS-OC, CAT7, or CAT11) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / Brij. [Figure 63B] 1 shows the concentration of ionizable lipids (SS-OC, CAT7, or CAT11) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results for OC / DMG are shown. [Figure 63C] 1 shows the concentration of ionizable lipids (SS-OC, CAT7, or CAT11) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results are shown for CAT7 / CHM6_4. [Figure 63D] 1 shows the concentration of ionizable lipids (SS-OC, CAT7, or CAT11) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results for CAT11 / DMG are shown. [Figure 63E] 1 shows the concentration of ionizable lipids (SS-OC, CAT7, or CAT11) contained in LNPs in the plasma of treated mice as measured by LC-MS. Results for CAT11 / Birj are shown.

[0161] [Figure 64A] IgM levels measured by ELISA assay in mice treated with each LNP formulation are shown at the indicated time points. [Figure 64B] IgM levels measured by ELISA assay in mice treated with each LNP formulation are shown at the indicated time points.

[0162] [Figure 65A] IgG levels measured by ELISA assay in mice treated with each LNP formulation at the indicated time points are shown. [Figure 65B] IgG levels measured by ELISA assay in mice treated with each LNP formulation at the indicated time points are shown.

[0163] [Figure 66] A and B show plasma levels of mRNA BiTE (A) or hEPO (B) measured by ECL assay.

[0164] [Figure 67] 1 shows the A-optimal design of a screening experiment for LNPs containing CAT7.

[0165] [Figure 68] 1 shows a predictive profiler based on an experimental design run of LNPs containing CAT7 and modeled using the Self-Validated Ensemble Modeling (SVEM) method. DETAILED DESCRIPTION OF THE INVENTION

[0166] There is a need in the art for selecting specific oncolytic virus strains with appropriate tropism and high potency in specific cancer types. Different virus strains, even from the same species, vary greatly in efficacy and toxicity in killing cancer cells. Chimeric viruses can be generated by replacing portions of the viral genome of one virus strain with the corresponding portion of a different strain. In some embodiments, such chimeric viruses may be more effective in killing cancer cells and / or have other advantageous properties. Furthermore, production of such viruses or compositions containing the corresponding viral genome requires optimization of vector template design and manufacturing processes (e.g., expression, purification, encapsulation, and storage). In some embodiments, the present disclosure provides viral genome and template vector designs for in vitro viral expression. In some embodiments, the viral genome is replicable. The present disclosure also provides viral genomes that can be encapsulated in non-immunogenic particles, such as lipid nanoparticles, polymer nanoparticles, or exosomes, that can be repeatedly administered to a subject. In some embodiments, the particles further encapsulate a polynucleotide encoding a payload molecule. Thus, the present disclosure enables safe and effective systemic delivery of recombinant polynucleotide vectors, providing a method for the treatment and prevention of a wide range of proliferative disorders (eg, cancer).

[0167] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein, including, but not limited to, patents, patent applications, papers, books, and articles, are expressly incorporated herein by reference in their entirety for all purposes. If one or more of the incorporated documents or portions of documents defines a term that conflicts with the definition of that term in this application, the definition set forth in this application shall control. However, mention of any references, papers, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0168] definition As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include every integer value within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the listed components, unless otherwise indicated. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously. As used herein, "plurality" can refer to one or more components (e.g., one or more miRNA target sequences). In this application, the use of "or" means "and / or" unless otherwise indicated.

[0169] As used in this application, the terms "about" and "approximately" are used as equivalents. Any numbers used in this application, whether about or not, are intended to cover all normal variations recognized by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 10% in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except when such number exceeds 100% of the possible values). In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 10% in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except when such number exceeds 100% of the possible values).

[0170] "Decrease" or "reducing" refers to a decrease or reduction of at least 5%, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%, of a particular value compared to a reference value. A decrease or reduction of a particular value can also be expressed as a fold change in the value compared to the reference value, e.g., a decrease of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, or more times compared to the reference value.

[0171] "Increase" refers to an increase of at least 5%, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100, 200, 300, 400, 500%, or more, of a particular value compared to a reference value. An increase in a particular value can also be expressed as a fold change in the value compared to the reference value, e.g., an increase of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more, compared to the reference level.

[0172] The term "sequence identity" refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same and in the same relative positions. As such, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, one sequence typically serves as a reference sequence to which test sequences are compared. The term "reference sequence" refers to the molecule to which a test sequence is compared. Unless otherwise specified, the term "sequence identity" in the claims refers to sequence identity calculated by Clustal Omega® version 1.2.4 using default parameters.

[0173] The term "derived from" refers to a polypeptide or polynucleotide sequence that comprises all or a portion of a reference polypeptide or polynucleotide sequence. For example, an RNA polynucleotide encoding an SVV or CVA genome described herein can comprise a polynucleotide sequence that is derived from all or a portion of a reference SVV or CVA genome (e.g., a naturally occurring or modified SVV or CVA genome). A polypeptide or polynucleotide sequence that is "derived from" a reference polypeptide or polynucleotide sequence also includes a polypeptide and / or polynucleotide sequence that contains another amino acid or nucleic acid mutation (e.g., a substitution, deletion, and / or insertion) relative to the reference polypeptide or polynucleotide sequence.

[0174] "Complementary" refers to the ability of pairing through base stacking and specific hydrogen bonding between two sequences containing naturally occurring or non-naturally occurring (e.g., modified as described above) bases (nucleotides) or their analogs. For example, if a base at a certain position in a nucleic acid can hydrogen bond with a base at a corresponding position in a target, these bases are considered complementary to each other at that position. Nucleic acids can contain universal bases or inert abasic spacers that do not contribute positively or negatively to hydrogen bonding. Base pairs can include both standard Watson-Crick base pairs and non-Watson-Crick base pairs (e.g., wobble base pairs and Hoogsteen base pairs). In the case of complementary base pairs, it is understood that adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), cytosine-type bases (C) are complementary to guanosine-type bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole will hybridize with and be considered complementary to any A, C, U, or T. Nichols et al., Nature, 1994;369:492-493, and Loakes et al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005;33(19):6258-6267.

[0175] An "expression cassette" or "expression construct" refers to a polynucleotide sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.

[0176] The term "subject" includes animals, such as, for example, mammals. In some embodiments, a mammal is a primate. In some embodiments, a mammal is a human. In some examples, the subject is a livestock animal, such as a cattle, sheep, goat, dairy cow, pig, or a domesticated animal, such as a dog or cat. In some embodiments (e.g., particularly in a research context), the subject is a rodent (e.g., a mouse, rat, hamster), rabbit, primate, or pig, such as an inbred pig. The terms "subject" and "patient" are used interchangeably herein.

[0177] "Administering," as used herein, refers to introducing an agent or composition into a subject or bringing a composition into contact with cells and / or tissues.

[0178] As used herein, "treatment" refers to the delivery of an agent or composition to a subject to affect a physiological outcome. In some embodiments, treatment refers to the treatment of a disease in a mammal, e.g., a human, and includes (a) inhibiting the disease, i.e., arresting the onset of the disease or preventing the progression of the disease, (b) alleviating the disease, i.e., causing regression of the disease state, and (c) curing the disease.

[0179] The term "effective amount" refers to the amount of an agent or composition required to produce a particular physiological effect (e.g., the amount required to increase, activate, and / or enhance a particular physiological effect). The effective amount of a particular agent can be expressed in various ways based on the properties of the agent, such as mass / volume, number of cells / volume, particles / volume, (mass of agent) / (mass of subject), number of cells / (mass of subject), or particles / (mass of subject). The effective amount of a particular agent is expressed as the half-maximal effective concentration (EC 50 ), which refers to the concentration of a drug that produces a specific physiological response whose magnitude is midway between the basal level and the maximal response level.

[0180] A "population" of cells refers to any number of cells greater than one, but preferably at least 1 x 10 3cells, at least 1 x 10 4 cells, at least 1 x 10 5 cells, at least 1 x 10 6 cells, at least 1 x 10 7 cells, at least 1 x 10 8 cells, at least 1 x 10 9 cells, at least 1 x 10 10 A population of cells can refer to an in vitro population (e.g., a population of cells in culture) or an in vivo population (e.g., a population of cells present in a particular tissue).

[0181] "Effector function" refers to the function of an immune cell involved in generating, maintaining, and / or enhancing an immune response against a target cell or target antigen.

[0182] The terms "microRNA," "miRNA," and "miR" are used interchangeably herein to refer to small, non-coding endogenous RNAs, approximately 21-25 nucleotides in length, that regulate gene expression by directing their target messenger RNAs (mRNAs) for degradation or translational repression.

[0183] As used herein, the term "composition" refers to a formulation of a recombinant RNA molecule or particle-encapsulated recombinant RNA molecule described herein that can be administered or delivered to a subject or cell.

[0184] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0185] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, and / or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans and / or domestic animals.

[0186] The term "replicative viral genome" refers to a viral genome that encodes all viral genes necessary for viral replication and the production of infectious viral particles.

[0187] The term "oncolytic virus" refers to a virus that has been modified to preferentially infect cancer cells or that naturally preferentially infects cancer cells.

[0188] The term "vector" is used herein to refer to a nucleic acid molecule capable of transporting, encoding, or carrying another nucleic acid molecule.

[0189] The terms "corresponding to" or "corresponding to," as used herein with respect to an amino acid or nucleic acid position(s), refer to a position(s) in a first polypeptide / polynucleotide sequence that aligns with a given amino acid / nucleic acid in a reference polypeptide / polynucleotide sequence when the first and reference polypeptide / polynucleotide sequences are aligned. Alignment is performed by one of skill in the art using software designed for this purpose, e.g., Clustal Omega version 1.2.4, using the default parameters for that version.

[0190] The term "encapsulation efficiency" or "EE%" refers to the percentage of target molecules (e.g., synthetic RNA virus genomes) that are successfully encapsulated into LNPs. The encapsulation efficiency is calculated by the following formula: (EE%) = (Wt / Wi) × 100% where Wt is the total amount of drug in the LNP suspension and Wi is the total amount of drug initially added during preparation. As an illustrative example, if 97 mg of target molecule are encapsulated in the LNPs out of a total of 100 mg of target molecule initially provided in the composition, the encapsulation efficiency can be given as 97%.

[0191] The term "lipid nitrogen-to-phosphate ratio" or "(N:P)" refers to the ratio of positively chargeable lipid amine groups to nucleic acid phosphate groups in a lipid nanoparticle.

[0192] The term "half-life" refers to the pharmacokinetic properties of a molecule (e.g., a molecule encapsulated in a lipid nanoparticle). Half-life can be expressed as the time required for 50% of a known amount of a molecule to be eliminated from a subject's body (e.g., a human patient or other mammal) or a specific compartment thereof after its administration in vivo through biological processes (e.g., metabolism, excretion, accelerated blood clearance, etc.), as measured, for example, in serum (i.e., circulating half-life) or other tissues. Generally, an increase in half-life results in an increase in the mean residence time (MRT) of the administered molecule in the circulation.

[0193] The term "accelerated blood clearance" or "ABC" refers to the phenomenon in which certain pharmaceutical agents (e.g., PEG-containing LNPs) are rapidly eliminated from the blood upon second and subsequent administration. ABC has been observed for many lipid delivery vehicles, including liposomes and LNPs.

[0194] As used herein, the term " ratio " when used in relation to lipid composition (for example, as percentage of total lipid content) refers to molar ratio unless clearly indicated otherwise.The molar ratio as percentage of total lipid content can also be expressed by " mol% ".For example, " 49:22:28.5:0.5 mol% " means the molar ratio of 49:22:28.5:0.5.

[0195] As used herein, the term "aliphatic" or "aliphatic group" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation; or a non-aromatic monocyclic or bicyclic hydrocarbon (also referred to herein as "carbocyclic," "alicyclic," or "cycloalkyl") that is fully saturated or contains one or more units of unsaturation, but has a single point of attachment to the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-4 aliphatic carbon atoms. In still other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a non-aromatic monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0196] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having a specified number of carbon atoms. In some embodiments, alkyl refers to a branched or unbranched saturated hydrocarbon group having three carbon atoms (C3). In some embodiments, alkyl refers to a branched or unbranched saturated hydrocarbon group having six carbon atoms (C6). In some embodiments, the term "alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, and hexyl.

[0197] As used herein, the term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH) n -, where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0198] The term "aryl," used alone or as part of a larger moiety, such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring systems having a total of 5 to 14 ring members, in which at least one ring in the system is aromatic, and in which each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present disclosure, "aryl" refers to aromatic ring systems, including, but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. When the term "aryl" is used herein, its scope also includes groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0199] The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety such as "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms, with 6, 10, or 14 pi electrons shared in a cyclic arrangement, and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of a basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently are optionally substituted.

[0200] The term "haloaliphatic" refers to an aliphatic group substituted with one or more halogen atoms.

[0201] The term "haloalkyl" refers to a straight or branched chain alkyl group that is substituted with one or more halogen atoms.

[0202] The term "halogen" means F, Cl, Br, or I.

[0203] As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or . +It can be NR (such as in the case of T-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. Heterocyclyl groups can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions independently are optionally substituted.

[0204] A heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. Heterocyclyl groups can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions independently are optionally substituted.

[0205] As described herein, compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each of the group's substitutable positions, and when multiple positions in any given structure may be substituted with multiple substituents selected from a specified group, the substituents may be the same or different at each position. Combinations of substituents envisioned by the present disclosure preferably result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that remains substantially unchanged when subjected to conditions that allow for the production, detection, and, in certain embodiments, recovery, purification, and use of the compounds for one or more of the purposes disclosed herein.

[0206] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH) 0-4 R ° ;-(CH2) 0-4 OR ° ;-O(CH2) 0-4 R ° , -O-(CH2) 0-4 C(O)OR ° ;-(CH2) 0-4 CH(OR ° )2;-(CH2) 0-4 SR ° ;R ° may be substituted with -(CH2) 0-4 Ph;R ° may be substituted with -(CH2) 0-4 O(CH2) 0-1 Ph;R ° -CH=CHPh; R ° may be substituted with -(CH2) 0-4 O(CH2) 0-1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R° )2;-(CH2) 0-4 N(R ° )C(O)R ° ;-N(R ° )C(S)R ° ;-(CH2) 0-4 N(R ° )C(O)NR ° 2;-N(R ° )C(S)NR ° 2;-(CH2) 0-4 N(R ° )C(O)OR ° ;-N(R ° )N(R ° )C(O)R ° ;-N(R ° )N(R ° )C(O)NR ° 2;-N(R ° )N(R ° )C(O)OR ° ;-(CH2) 0-4 C(O)R ° ;-C(S)R ° ;-(CH2) 0-4 C(O)OR ° ;-(CH2) 0-4 C(O)SR ° ;-(CH2) 0-4 C(O)OSiR ° 3;-(CH2) 0-4 OC(O)R ° ;-OC(O)(CH2) 0-4 SR ° 、SC(S)SR ° ;-(CH2) 0-4 SC(O)R ° ;-(CH2) 0-4 C(O)NR ° 2;-C(S)NR ° 2;-C(S)SR ° ;-SC(S)SR ° 、-(CH2) 0-4 OC(O)NR ° 2;-C(O)N(OR ° )R ° ;-C(O)C(O)R ° ;-C(O)CH2C(O)R ° ;-C(NOR ° )R° ;-(CH2) 0-4 SSR ° ;-(CH2) 0-4 S(O)2R ° ;-(CH2) 0-4 S(O)2OR ° ;-(CH2) 0-4 OS(O)2R ° ;-S(O)2NR ° 2;-(CH2) 0-4 S(O)R ° ;-N(R ° )S(O)NR ° 2;-N(R ° )S(O)2R ° ;-N(OR ° )R ° ;-C(NH)NR ° 2;-P(O)2R ° ;-P(O)R ° 2;-OP(O)R ° 2;-OP(O)(OR ° )2;SiR ° 3;-(C 1-4 Linear or branched alkylene)ON(R ° )2; or -(C 1-4 Linear or branched alkylene)C(O)ON(R ° )2, where each R ° are optionally substituted as defined below and independently represent hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (a 5- to 6-membered heteroaryl ring), or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, regardless of the above definitions, R ° two independent occurrences of together with their intervening atom(s) form a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0207] R ° (or R °Suitable monovalent substituents on the ring formed by two independent occurrences of -(CH) together with an intervening atom are independently halogen, -(CH) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2;-O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● , -(C 1-4 Linear or branched alkylene)C(O)OR ● , or -SSR ● where each R ● is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 R is independently selected from Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. ° Suitable divalent substituents on a saturated carbon atom of include ═O and ═S.

[0208] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group are ═O, ═S, ═NNR * 2, =NNHC(O)R * , =NNHC(O)OR* , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S-, where R * Each independent occurrence of is hydrogen, optionally substituted as defined below, C 1-6 A suitable divalent substituent attached to the adjacent substitutable carbon of an "optionally substituted" group is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. * 2) 2-3 O-, where R * Each independent occurrence of is hydrogen, optionally substituted as defined below, C 1-6 It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0209] R * Suitable substituents on the aliphatic groups are halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0210] A suitable substituent on a substitutable nitrogen of an "optionally substituted" group is -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † where each R † are independently hydrogen, optionally substituted C as defined below, 1-6 an aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, regardless of the above definitions, R † two independent occurrences of together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0211] R † Suitable substituents on the aliphatic groups are, independently, halogen, —R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0212] As used herein, the term "partially unsaturated" refers to a ring moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0213] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and 2-hydroxy-ethanesulfonate. Examples of the salts include phonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0214] Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N(C1-4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0215] "Pharmaceutically acceptable derivative" means any non-toxic salt, ester, salt of an ester, or other derivative of a compound of the present disclosure which, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the present disclosure, or an active metabolite or residue thereof.

[0216] The term "tertiary amine" is used to describe an amine (nitrogen atom) bound to three carbon-containing groups, each of which is covalently bonded to an amine group through its carbon atom. Tertiary amines may be protonated or may be complexed with a Lewis acid.

[0217] The recitation of a list of chemical groups in any definition of a variable herein includes that definition of that variable as any single group or combination of listed groups. The recitation of an embodiment of a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. Unless otherwise indicated, structures depicted herein are also intended to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of that structure, e.g., R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E stereoisomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the disclosed compounds are within the scope of the disclosure. Unless otherwise indicated, all tautomeric forms of the disclosed compounds are within the scope of the disclosure.

[0218] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0219] Synthetic RNA virus genome In some embodiments, the present disclosure provides recombinant RNA molecules encoding oncolytic viruses (e.g., RNA genomes). Such recombinant RNA molecules are referred to herein as "synthetic viral genomes" or "synthetic RNA viral genomes." In such embodiments, the synthetic RNA viral genomes can produce infectious and lytic viruses when introduced into cells via a non-viral delivery vehicle, and do not require the presence of additional exogenous genes or proteins within the cell to replicate and produce infectious viruses. Rather, endogenous translation mechanisms within the host cell mediate the expression of viral proteins from the synthetic RNA viral genome. The expressed viral proteins then mediate viral replication and assembly into infectious viral particles (which may include capsid proteins, envelope proteins, and / or membrane proteins) comprising the RNA viral genome. Thus, the RNA polynucleotides described herein (i.e., synthetic RNA viral genomes), when introduced into a host cell, produce viruses capable of infecting another host cell. In some embodiments, the oncolytic virus is a picornavirus (see schematic in Figure 9). In some embodiments, the picornavirus is CVA21. In some embodiments, the picornavirus is SVV.

[0220] In some embodiments, the synthetic viral genome is provided as a recombinant ribonucleic acid (RNA) (i.e., a synthetic RNA viral genome). In some embodiments, the synthetic RNA viral genome comprises one or more nucleic acid analogs. Examples of nucleic acid analogs include 2'-O-methyl substituted RNA, 2'-O-methoxy-ethyl bases, 2' fluoro bases, locked nucleic acids (LNA), unlocked nucleic acids (UNA), bridged nucleic acids (BNA), morpholinos, and peptide nucleic acids (PNA). In some embodiments, the synthetic RNA viral genome is a replicon, an RNA viral genome encoding a transgene, an mRNA molecule, or a circular RNA molecule (circRNA). In some embodiments, the synthetic RNA viral genome comprises a single-stranded RNA (ssRNA) viral genome. In some embodiments, the single-stranded genome can be a positive-sense or negative-sense genome.

[0221] In some embodiments, the recombinant RNA molecule is a circular RNA molecule (circRNA). CircRNA molecules lack free ends necessary for exonuclease-mediated degradation, thus extending the half-life of the RNA molecule and enabling more stable protein production over time (see, e.g., Wesselhoeft et al., Engineering circular RNA for potent and stable translation in eukaryotic cells. Nature Communications. (2018) 9:2629). To produce a functional RNA virus from a circRNA molecule, the circular construct must be "pryed open" once it enters the cell so that a linear RNA genome with appropriate 3' and 5' natural ends can be produced. Therefore, in some embodiments, the recombinant RNA molecule encoding an oncolytic virus is provided as a circRNA molecule and further comprises one or more additional RNA sequences that facilitate linearization of the circRNA molecule inside the cell. Examples of such additional RNA sequences include siRNA target sites, miRNA target sites, and guide RNA target sites. Corresponding siRNA, miRNA, or gRNA can be combined with the circRNA molecule. Alternatively, miRNA target sites may be selected based on the expression of cognate miRNAs in target cells, such that cleavage of the circRNA molecule and initial expression of the encoded oncolytic virus is restricted to target cells expressing the specific miRNA.

[0222] The synthetic RNA viral genomes described herein encode oncolytic viruses, examples of which are known in the art and include, but are not limited to, picornaviruses (e.g., coxsackieviruses), polioviruses, measles viruses, vesicular stomatitis viruses, orthomyxoviruses, and maraba viruses. In some embodiments, the oncolytic virus encoded by the synthetic RNA viral genome is a Picornaviridae virus, such as a Coxsackievirus, a poliovirus (including chimeric polioviruses such as PVS-RIPO and other chimeric picornaviruses), or a Seneca Valley virus, or any virus of chimeric origin from any of a number of picornaviruses; a Arenaviridae virus, such as a Lassa virus; a Retroviridae virus, such as a murine leukemia virus; a Orthomyxoviridae virus, such as influenza A virus; a Paramyxoviridae virus, such as a Newcastle disease virus or measles virus; a Reoviridae virus, such as a mammalian orthoreovirus; a Togaviridae virus, such as a Sindbis virus; or a Rhabdoviridae virus, such as a vesicular stomatitis virus (VSV) or a Maraba virus.

[0223] Positive-sense single-stranded RNA virus In some embodiments, the synthetic RNA virus genome described herein encodes a single-stranded RNA (ssRNA) virus genome. In some embodiments, the ssRNA virus is a positive-sense ssRNA (+-sense ssRNA) virus. Exemplary +-sense ssRNA viruses include members of the Picornaviridae family (e.g., including Coxsackieviruses, Polioviruses, and Seneca Valley Virus (SVV), SVV-A), Coronaviridae family (e.g., alphacoronaviruses such as HCoV-229E and HCoV-NL63, and betacoronaviruses such as HCoV-HKU1, HCoV-OC3, and MERS-CoV), Retroviridae family (e.g., murine leukemia viruses), and Togaviridae family (e.g., alphaviruses such as Semliki Forest virus, Sindbis virus, Ross River virus, or Chikungunya virus). Additional exemplary genera and species of positive-sense ssRNA viruses are listed in Table 1 below. [Table 1-1] [Table 1-2]

[0224] In some embodiments, the recombinant RNA molecules described herein encode a picornavirus selected from a coxsackievirus, a poliovirus, and a Seneca Valley virus (SVV). In some embodiments, the recombinant RNA molecules described herein encode a coxsackievirus. In some aspects of this embodiment, the recombinant RNA molecule encodes a coxsackievirus and comprises the 5' UTR sequence of SEQ ID NO: 2 (see, e.g., Brown et al., "Complete Genomic Sequencing Shows that Polioviruses and Members of Human Enterovirus Species C Are Closely Related in the Noncapsid Coding Region." Journal of Virology, (2003) 77:16, pp. 8973-8984. GenBank Accession No. AF546702). In such embodiments, the 5'UTR sequence of SEQ ID NO:2 unexpectedly increases the production of functional Coxsackievirus compared to other previously reported 5'UTR sequences (see, e.g., Newcombe et al., Cellular receptor interactions of C-cluster human group A coxsackieviruses Journal of General Virology (2003), 84, 3041-3050. GenBank accession number AF465515). In some aspects of this embodiment, the recombinant RNA molecule encodes a Coxsackievirus and comprises the sequence of SEQ ID NO:1.

[0225] In some embodiments, the synthetic RNA viral genome described herein encodes a Coxsackievirus. In some embodiments, the Coxsackievirus is selected from CVB3, CVA21, and CVA9. Exemplary Coxsackievirus nucleic acid sequences are provided under GenBank Reference No. M33854.1 (CVB3), GenBank Reference No. KT161266.1 (CVA21), and GenBank Reference No. D00627.1 (CVA9). In some embodiments, the synthetic RNA viral genome described herein encodes a modified CVA21 virus comprising SEQ ID NO: 1, which is the Kuykendall (Kuyk) strain. In some embodiments, the sequence of the Kuykendall strain viral genome is according to GenBank Accession No. AF465515.1 or AF546702.1. In some embodiments, the synthetic RNA viral genome described herein encodes a chimeric Coxsackievirus. In some embodiments, the synthetic RNA viral genome described herein encodes a CVA21 strain selected from the CVA21-EF and CVA21-KY strains. In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain. An exemplary sequence of the EF strain viral genome is provided by GenBank Accession No. EF015029.1. In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-KY strain. An exemplary sequence of the KY strain viral genome is provided by GenBank Accession No. KY284011.1. As shown in Figures 11-26, the EF and KY strains provide superior therapeutic efficacy over the Kuykendall laboratory strain and previously reported synthetic picornavirus compositions.

[0226] The domain organization of the three CVA21 strains (EF, KY, and Kuykendall) is shown in Figure 32, and the sequence identity between various regions of these three strains is shown in Table 2 below. [Table 2]

[0227] One or more specific regions in the viral genome of the CVA21 EF or KY strain may contribute to the beneficial therapeutic effects observed in the EF and KY strains over the Kuykendall laboratory strain. In some embodiments, the one or more specific regions are selected from the group consisting of the 5'UTR (IRES) region, the P1 region, and the 3D region. The nucleic acid locations of each of these specific regions in the viral strains described herein are as follows: (a) The 5'UTR (IRES) region of CVA21-Kuykendall encompasses nucleic acids 1 to 713 of SEQ ID NO: 1. The 5'UTR (IRES) region of CVA21-KY encompasses nucleic acids 1 to 713 of SEQ ID NO: 5. The 5'UTR (IRES) region of CVA21-EF encompasses nucleic acids 1 to 748 of SEQ ID NO: 9. (b) The P1 region of CVA21-Kuykendall encompasses nucleic acids 714 to 3350 of SEQ ID NO: 1. The P1 region of CVA21-KY encompasses nucleic acids 714 to 3350 of SEQ ID NO: 5. The P1 region of CVA21-EF encompasses nucleic acids 749 to 3385 of SEQ ID NO: 9. (c) The 3D region of CVA21-Kuykendall encompasses nucleic acids 5952-7340 of SEQ ID NO: 1. The 3D region of CVA21-KY encompasses nucleic acids 5952-7340 of SEQ ID NO: 5. The 3D region of CVA21-EF encompasses nucleic acids 5987-7375 of SEQ ID NO: 9.

[0228] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-KY strain. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a polynucleotide sequence according to SEQ ID NO:5. In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:5 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a polynucleotide sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:1 (including all ranges and subranges therebetween).

[0229] In some embodiments, a synthetic RNA viral genome described herein encodes a CVA21-KY strain and comprises a 5' UTR (IRES) sequence according to SEQ ID NO:6 (corresponding to nucleic acids 1-713 of SEQ ID NO:5). In some embodiments, a synthetic RNA viral genome described herein encodes a CVA21-KY strain and comprises a 5' UTR (IRES) sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:6 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-KY strain comprises a 5'UTR (IRES) sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:2 (including all ranges and subranges therebetween).

[0230] In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a P1 sequence according to SEQ ID NO:7 (corresponding to nucleic acids 714-3350 of SEQ ID NO:5). In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a P1 sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:7 (including all ranges and subranges therebetween). In some embodiments, a synthetic RNA viral genome encoding the CVA21-KY strain comprises a P1 sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:3 (including all ranges and subranges therebetween).

[0231] In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a 3D sequence according to SEQ ID NO:8 (corresponding to nucleic acids 5952-7340 of SEQ ID NO:5). In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-KY strain and comprises a 3D sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:8 (including all ranges and subranges therebetween). In some embodiments, a synthetic RNA viral genome encoding the CVA21-KY strain comprises a 3D sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:4 (including all ranges and subranges therebetween).

[0232] In some embodiments, the synthetic RNA viral genome described herein encodes the CVA21-EF strain. In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a polynucleotide sequence according to SEQ ID NO:9. In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:9 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a polynucleotide sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:1 (including all ranges and subranges therebetween).

[0233] In some embodiments, a synthetic RNA viral genome described herein encodes a CVA21-EF strain and comprises a 5' UTR (IRES) sequence according to SEQ ID NO: 10 (corresponding to nucleic acids 1-748 of SEQ ID NO: 9). In some embodiments, a synthetic RNA viral genome described herein encodes a CVA21-EF strain and comprises a 5' UTR (IRES) sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 10 (including all ranges and subranges therebetween). In some embodiments, the synthetic RNA viral genome encoding the CVA21-EF strain comprises a 5'UTR (IRES) sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO:2 (including all ranges and subranges therebetween).

[0234] In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a P1 sequence according to SEQ ID NO: 11 (corresponding to nucleic acids 749-3385 ​​of SEQ ID NO: 9). In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a P1 sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 11 (including all ranges and subranges therebetween). In some embodiments, a synthetic RNA viral genome encoding the CVA21-EF strain comprises a P1 sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO: 3 (including all ranges and subranges therebetween).

[0235] In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a 3D sequence according to SEQ ID NO: 12 (corresponding to nucleic acids 5987-7375 of SEQ ID NO: 9). In some embodiments, a synthetic RNA viral genome described herein encodes the CVA21-EF strain and comprises a 3D sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 12 (including all ranges and subranges therebetween). In some embodiments, a synthetic RNA viral genome encoding the CVA21-EF strain comprises a 3D sequence that is less than 95%, less than 90%, less than 85%, or less than 80% identical to SEQ ID NO: 4 (including all ranges and subranges therebetween).

[0236] In some embodiments, the CVA21 RNA virus genome described herein does not contain the nucleotide sequence CGUCUC (SEQ ID NO: 83) or GAGACG (SEQ ID NO: 84). The corresponding complementary DNA sequences, CGTCTC (SEQ ID NO: 85) and GAGACG (SEQ ID NO: 86), are BsmBI restriction enzyme recognition sites.

[0237] In some embodiments, the CVA21 RNA virus genome described herein does not contain the nucleotide sequence GGUCUC (SEQ ID NO: 87) or GAGACC (SEQ ID NO: 88). The corresponding complementary DNA sequences, GGTCTC (SEQ ID NO: 89) and GAGACC (SEQ ID NO: 90), are BsaI restriction enzyme recognition sites.

[0238] In some embodiments, the synthetic RNA viral genome described herein encodes a Seneca Valley Virus (SVV). In some embodiments, the SVV is selected from wild-type SVV (such as SVV-001, SEQ ID NO: 25), or a mutant or chimeric SVV (such as SVV-001-S177A encoded by SEQ ID NO: 26, or SVV-IRES-2-S177A encoded by SEQ ID NO: 68 or SEQ ID NO: 73).

[0239] In some embodiments, the SVV is an SVV-S177 mutant. In some embodiments, the SVV is an SVV-S177A mutant. As used herein with respect to an SVV viral genome, the term "S177 mutant" refers to an SVV viral genome encoding a VP2 protein containing a mutation at amino acid S177 of the wild-type protein (amino acid numbering according to the VP2 protein encoded by SEQ ID NO:25). Thus, the term "S177A mutant" refers to an SVV mutant having an amino acid substitution of S177A in the VP2 protein. In SEQ ID NO:25, the VP2 S177 residue is encoded by the codon "UCU" at nucleic acid positions 1645-1647. Thus, the SVV-S177 mutant contains a nucleic acid mutation within the region corresponding to nucleic acid positions 1645-1647 of SEQ ID NO:25. In some embodiments, the SVV-S177A mutant contains the codon sequence "GCU," "GCC," "GCA," or "GCG" in the region corresponding to nucleic acid positions 1645-1647 of SEQ ID NO:25. In some embodiments, the SVV-S177A variant comprises the codon sequence "GCG" in the region corresponding to nucleic acid positions 1645-1647 of SEQ ID NO:25.

[0240] In some embodiments, the SVV RNA viral genome described herein does not comprise the nucleotide sequence GCUCUUC (SEQ ID NO:79) or GAAGAGC (SEQ ID NO:80). The corresponding complementary DNA sequences, GCTCTTC (SEQ ID NO:81) and GAAGAGC (SEQ ID NO:82), are SapI restriction enzyme recognition sites. In some embodiments, a wild-type SVV RNA viral genome comprises SEQ ID NO:79 at a position corresponding to nucleic acids 1504-1510 and / or nucleic acids 5293-5299 of SEQ ID NO:25. In some embodiments, an SVV RNA viral genome of the disclosure comprises at least one nucleotide substitution relative to SEQ ID NO:79 within the region corresponding to nucleic acids 1504-1510 and / or nucleic acids 5293-5299 of SEQ ID NO:25. In some embodiments, the at least one nucleotide substitution is a silent mutation that does not change the amino acid encoded by the corresponding region of DNA. In some embodiments, an SVV RNA viral genome of the disclosure comprises a cytidine (“C”) at a position corresponding to nucleic acids 1509 and / or 5298 of SEQ ID NO:25.

[0241] In some embodiments, the SVV RNA viral genome described herein does not contain the nucleotide sequence GGUCUC (SEQ ID NO: 87) or GAGACC (SEQ ID NO: 88). The corresponding complementary DNA sequences, GGTCTC (SEQ ID NO: 89) and GAGACC (SEQ ID NO: 90), are BsaI restriction enzyme recognition sites.

[0242] In some embodiments, the synthetic RNA viral genomes described herein encode chimeric picornaviruses (e.g., encode a virus that includes one portion, such as a capsid protein or IRES, from a first picornavirus and another portion, such as a nonstructural gene, such as a protease or polymerase, from a second picornavirus). In some embodiments, the synthetic RNA viral genomes described herein encode chimeric SVVs.

[0243] In some embodiments, the synthetic RNA viral genomes described herein encode SVV that include one or more specific regions derived from an SVV strain selected from the group consisting of SVV-001 (SEQ ID NO:25 or SEQ ID NO:72 (GenBank Identification No. DQ641257.1)), SVA / BRA / MG2 / 2015 (SEQ ID NO:69; GenBank Identification No. KR063108.1), SVA / Canada / MB / NCFAD-104 / 2015 (SEQ ID NO:70; GenBank Identification No. KY486156.1), and SVV-MN15-308 (SEQ ID NO:71; GenBank Identification No. KU359214.1). In some embodiments, the one or more specific regions are selected from the group consisting of the 5'UTR (IRES) region, the P1 region, and the P3 region. The nucleic acid locations of each of these specific regions for the viral strains described herein are as follows: (a) The 5'UTR (IRES) region of SVV-001 includes nucleic acids 1 to 668 of SEQ ID NO: 25. The 5'UTR (IRES) region of SVA / BRA / MG2 / 2015 includes nucleic acids 1 to 656 of SEQ ID NO: 69. The 5'UTR (IRES) region of SVA / Canada / MB / NCFAD-104 / 2015 includes nucleic acids 1 to 612 of SEQ ID NO: 70. The 5'UTR (IRES) region of SVV-MN15-308 includes nucleic acids 1 to 610 of SEQ ID NO: 71. (b) The P1 region of SVV-001 encompasses nucleic acids 669 to 3477 of SEQ ID NO: 25. The P1 region of SVA / BRA / MG2 / 2015 encompasses nucleic acids 657 to 3465 of SEQ ID NO: 69. The P1 region of SVA / Canada / MB / NCFAD-104 / 2015 encompasses nucleic acids 613 to 3421 of SEQ ID NO: 70. The P1 region of SVV-MN15-308 encompasses nucleic acids 611 to 3419 of SEQ ID NO: 71. (c) The P3 region of SVV-001 encompasses nucleic acids 4855-7212 of SEQ ID NO: 25. The P3 region of SVA / BRA / MG2 / 2015 encompasses nucleic acids 4843-7200 of SEQ ID NO: 69. The P3 region of SVA / Canada / MB / NCFAD-104 / 2015 encompasses nucleic acids 4799-7156 of SEQ ID NO: 70. The P3 region of SVV-MN15-308 encompasses nucleic acids 4797-7154 of SEQ ID NO: 71.

[0244] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a 5'UTR (IRES) region (nucleic acids 1-656 of SEQ ID NO:69) from SVA / BRA / MG2 / 2015. In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 1-656 of SEQ ID NO:69. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVA / BRA / MG2 / 2015, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0245] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a 5'UTR (IRES) region (nucleic acids 1-612 of SEQ ID NO:70) from SVA / Canada / MB / NCFAD-104 / 2015. In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 1-612 of SEQ ID NO:70. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVA / Canada / MB / NCFAD-104 / 2015, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0246] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a 5'UTR (IRES) region from SVV-MN15-308 (nucleic acids 1-610 of SEQ ID NO:71). In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 1-610 of SEQ ID NO:71. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVV-MN15-308, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0247] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a P1 region (nucleic acids 657-3465 of SEQ ID NO:69) from SVA / BRA / MG2 / 2015. In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 657-3465 of SEQ ID NO:69. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVA / BRA / MG2 / 2015, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0248] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a P1 region (nucleic acids 613-3421 of SEQ ID NO:70) from SVA / Canada / MB / NCFAD-104 / 2015. In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 613-3421 of SEQ ID NO:70. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVA / Canada / MB / NCFAD-104 / 2015, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0249] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a P1 region from SVV-MN15-308 (nucleic acids 611-3419 of SEQ ID NO:71). In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 611-3419 of SEQ ID NO:71. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVV-MN15-308, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0250] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a P3 region (nucleic acids 4843-7200 of SEQ ID NO: 69) from SVA / BRA / MG2 / 2015. In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 4843-7200 of SEQ ID NO: 69. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVA / BRA / MG2 / 2015, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0251] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a P3 region (nucleic acids 4799-7156 of SEQ ID NO:70) from SVA / Canada / MB / NCFAD-104 / 2015. In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 4799-7156 of SEQ ID NO:70. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVA / Canada / MB / NCFAD-104 / 2015, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0252] In some embodiments, the synthetic RNA viral genome described herein encodes an SVV that includes a P3 region from SVV-MN15-308 (nucleic acids 4797-7154 of SEQ ID NO: 71). In some embodiments, the synthetic RNA viral genome encoding the SVV comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to nucleic acids 4797-7154 of SEQ ID NO: 71. In some embodiments, the remainder of the SVV viral genome, other than one or more regions derived from SVV-MN15-308, is derived from SVV-001 and comprises a polynucleotide sequence having at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to the corresponding region of SEQ ID NO: 25. In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant).

[0253] In some embodiments, the synthetic RNA viral genome described herein encodes a chimeric SVV comprising a 5'UTR (IRES) region from SVA / Canada / MB / NCFAD-104 / 2015 (SEQ ID NO: 70) and the remainder of the viral genome from SVV-001 (SEQ ID NO: 25). In some embodiments, the SVV is an SVV-S177 mutant (e.g., an S177A mutant). In some embodiments, the synthetic RNA viral genome has at least 80%, at least 85%, 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 99.8%, at least 99.9%, or 100% sequence identity (including all ranges and subranges therebetween) to SEQ ID NO: 68.

[0254] In some embodiments, the synthetic RNA viral genome is engineered and comprises less than 100% sequence identity with that of a wild-type virus (e.g., wild-type CVA21 or wild-type SVV). In some embodiments, the synthetic RNA viral genome comprises less than 99.9%, less than 99.8%, less than 99.7%, less than 99.6%, less than 99.5%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, or less than 90% sequence identity with that of the corresponding wild-type virus.

[0255] In some embodiments, the synthetic RNA virus genome comprises a microRNA (miRNA) target sequence (miR-TS) cassette, wherein the miR-TS cassette comprises one or more miRNA target sequences, and expression of one or more of the corresponding miRNAs in a cell inhibits replication of the encoded oncolytic virus in the cell. In some embodiments, the one or more miRNAs are selected from miR-124, miR-1, miR-143, miR-128, miR-219, miR-219a, miR-122, miR-204, miR-217, miR-137, miR-142, and miR-126. In some embodiments, the miR-TS cassette comprises one or more copies of the miR-124 target sequence, one or more copies of the miR-1 target sequence, and one or more copies of the miR-143 target sequence. In some embodiments, the miR-TS cassette comprises one or more copies of the miR-128 target sequence, one or more copies of the miR-219a target sequence, and one or more copies of the miR-122 target sequence. In some embodiments, the miR-TS cassette comprises one or more copies of the miR-128 target sequence, one or more copies of the miR-204 target sequence, and one or more copies of the miR-219 target sequence. In some embodiments, the miR-TS cassette comprises one or more copies of the miR-217 target sequence, one or more copies of the miR-137 target sequence, and one or more copies of the miR-126 target sequence.

[0256] In some embodiments, the synthetic RNA viral genome comprises one or more miR-TS cassettes integrated into the 5' untranslated region (UTR) or 3' UTR of one or more essential viral genes. In some embodiments, the synthetic RNA viral genome comprises one or more miR-TS cassettes integrated into the 5' untranslated region (UTR) or 3' UTR of one or more non-essential genes. In some embodiments, the synthetic RNA viral genome comprises one or more miR-TS cassettes integrated into the 5' or 3' of one or more essential viral genes.

[0257] In some embodiments, the synthetic RNA viral genome comprises a heterologous polynucleotide encoding a payload molecule. In such embodiments, the synthetic RNA viral genome drives the production of infectious oncolytic virus and expression of the payload molecule. In some embodiments, expression of the payload molecule can increase the therapeutic efficacy of the oncolytic virus. In some embodiments, the payload molecule is selected from IL-12, GM-CSF, CXCL10, IL-36γ, CCL21, IL-18, IL-2, CCL4, CCL5, anti-CD3-anti-FAP BiTE, an antigen binding molecule that binds to DLL3, or an antigen binding molecule that binds to EpCAM. In some embodiments, the payload molecule comprises or consists of the MLKL 4HB domain. In some embodiments, the payload molecule comprises or consists of a Gasdermin DN-terminal fragment. In some embodiments, the payload molecule comprises or consists of a Gasdermin EN-terminal fragment. In some embodiments, the payload molecule comprises or consists of an HMGB1 Box B domain. In some embodiments, the payload molecule comprises or consists of SMAC / Diablo. In some embodiments, the payload molecule comprises or consists of melittin. In some embodiments, the payload molecule comprises or consists of L-amino acid oxidase (LAAO). In some embodiments, the payload molecule comprises or consists of a disintegrin. In some embodiments, the payload molecule comprises or consists of TRAIL (TNFSF10). In some embodiments, the payload molecule comprises or consists of a nitroreductase (e.g., E. coli NfsB or NfsA). In some embodiments, the payload molecule comprises or consists of a reovirus FAST protein (e.g., ARV p14, BRV p15, or a p14-p15 hybrid). In some embodiments, the payload molecule comprises or consists of leptin / FOSL2. In some embodiments, the payload molecule comprises or consists of α-1,3-galactosyltransferase.In some embodiments, the payload molecule comprises or consists of adenosine deaminase 2 (ADA2). In some embodiments, the payload molecule comprises or consists of a cytokine selected from IL-36γ, IL-7, IL-12, IL-18, IL-21, IL2, or IFNγ. Further description regarding types of payload molecules suitable for use in these embodiments is provided below.

[0258] Methods for producing recombinant RNA virus genomes In some embodiments, the present disclosure provides recombinant DNA molecules encoding the synthetic RNA viral genomes described herein. Such recombinant DNA molecules are referred to herein as "DNA templates" or "recombinant DNA templates." In some embodiments, the recombinant DNA molecules are used as templates for in vitro transcription of the encoded synthetic RNA viral genomes. In some embodiments, the recombinant DNA molecule (e.g., the DNA template) comprises, from 5' to 3', one or more of the following elements: (i) a promoter, (ii) a 5' leader sequence, (iii) a 5' junction cleavage sequence, (iv) a DNA polynucleotide sequence encoding a synthetic RNA genome, (v) a polyA tail, and / or (vi) a 3' junction cleavage sequence. In some embodiments, the recombinant DNA molecule (e.g., the DNA template) encoding a recombinant RNA molecule comprises each of the following elements: (i) a promoter, (ii) a 5' leader sequence, (iii) a 5' junction cleavage sequence, (iv) a DNA polynucleotide sequence encoding a synthetic RNA genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence. Each of these elements is described in detail below. The description provided for each individual element is such that a particular embodiment of each element may be incorporated into a final recombinant DNA molecule (e.g., a DNA template), e.g., a particular leader sequence disclosure may be combined with a particular 5' junction cleavage sequence disclosure, etc.

[0259] In some embodiments, the recombinant DNA molecule (e.g., DNA template) does not contain additional nucleic acid between the two flanking elements, but may contain additional nucleic acid upstream of the promoter sequence or downstream of the 3' junction cleavage sequence. In some embodiments, the promoter sequence is a T7 promoter sequence. In some embodiments, the T7 promoter sequence comprises or consists of SEQ ID NO:91.

[0260] In some embodiments, the synthetic RNA virus genomes described herein are produced in vitro using one or more recombinant DNA templates comprising a polynucleotide encoding the synthetic RNA virus genome. In other words, the recombinant DNA template is a vector comprising a polynucleotide encoding the synthetic RNA virus genome. The term "vector" is used herein to refer to a nucleic acid molecule that can transport, encode, or carry another nucleic acid molecule. The nucleic acid to be transported is typically inserted into a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within a cell and / or may contain sequences sufficient to allow integration into host cell DNA. In some embodiments, the recombinant RNA molecules encoding the oncolytic viruses described herein are produced using one or more DNA vectors.

[0261] In some embodiments, the synthetic RNA viral genomes described herein are produced by introducing a recombinant DNA molecule (e.g., a DNA template) containing a polynucleotide encoding the recombinant RNA molecule into a suitable host cell in vitro (e.g., by transfection, transduction, electroporation, etc.). Suitable host cells include insect and mammalian cell lines. The host cells are cultured for an appropriate period of time to allow expression of the polynucleotide and production of the synthetic RNA viral genome. The synthetic RNA viral genome is then isolated from the host cell and formulated (e.g., encapsulated into particles) for therapeutic use. A schematic diagram of the in vitro synthesis of a CVA21 RNA viral genome containing 3' and 5' ribozymes is shown in Figure 10. The same schematic applies to the synthesis of RNA viral genomes (e.g., CVA21 or SVV viral genomes) using other combinations of junction cleavage sequences (see, e.g., Figure 11A). If the 3' junction cleavage sequence contains or consists of a restriction enzyme recognition site, the recombinant DNA molecule (e.g., a DNA template) can be digested with the corresponding restriction enzyme prior to the in vitro transcription process, as shown in Figure 11B.

[0262] T7 promoter In some embodiments, the recombinant DNA molecule (e.g., the DNA template) comprises a T7 promoter. In some embodiments, the T7 promoter comprises or consists of the polynucleotide sequence of SEQ ID NO: 91. In some embodiments, the T7 promoter comprises or consists of the polynucleotide sequence of SEQ ID NO: 91 containing up to 1, 2, 3, or 4 mutations.

[0263] In some embodiments, the T7 promoter is located immediately before the leader sequence, with no additional nucleotides between them. In some embodiments, the T7 promoter is located immediately before the 5' junction cleavage sequence, with no additional nucleotides between them. In some embodiments, the viral genome encodes CVA21 or SVV.

[0264] Junction cleavage sequence In some embodiments, recombinant RNA molecules comprising the synthetic RNA virus genomes described herein require distinct 5' and 3' ends native to the virus. RNA transcripts produced in vitro by T7 RNA polymerase or by mammalian RNA Pol II contain mammalian 5' and 3' UTRs and do not contain the distinct native ends necessary for the production of infectious RNA viruses. For example, T7 RNA polymerase requires a guanosine residue at the 5' end of the template polynucleotide to initiate transcription. However, SVV begins with a uridine residue at its 5' end. Therefore, the T7 leader sequence required for in vitro transcription of SVV transcripts must be removed to generate the native 5' SVV end required for the production of functional, infectious SVV. Therefore, in some embodiments, recombinant DNA molecules (e.g., DNA templates) suitable for use in producing the synthetic RNA virus genomes described herein require additional non-viral 5' and 3' sequences that enable the generation of distinct 5' and 3' ends native to the virus. Such sequences are referred to herein as junction cleavage sequences (JCS). In some embodiments, the junction cleavage sequence acts to cleave T7 RNA polymerase- or Pol II-encoded RNA transcripts at the junction of the viral RNA and mammalian mRNA sequences, such that non-viral RNA polynucleotides are removed from the transcript to maintain distinct 5' and 3' ends of the virus (see schematic diagram in Figure 11A). In some embodiments, the junction cleavage sequence acts to generate appropriate ends during linearization of a DNA plasmid encoding a synthetic viral genome (e.g., use of a 3' restriction enzyme recognition sequence to generate appropriate 3' ends during linearization of a plasmid template and prior to in vitro transcription of a synthetic RNA genome).

[0265] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) suitable for use in producing a synthetic RNA viral genome described herein comprises at least one 5' junction cleavage sequence and at least one 3' junction cleavage sequence. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) suitable for use in producing a synthetic RNA viral genome described herein comprises one or more 5' junction cleavage sequences and at least one 3' junction cleavage sequence. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) suitable for use in producing a synthetic RNA viral genome described herein comprises at least one 5' junction cleavage sequence and one or more 3' junction cleavage sequences. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) suitable for use in producing a synthetic RNA viral genome described herein comprises one or more 5' junction cleavage sequences and one or more 3' junction cleavage sequences. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) suitable for use in producing a synthetic RNA viral genome described herein comprises two 5' junction cleavage sequences and at least one 3' junction cleavage sequence. In some embodiments, recombinant DNA molecules (e.g., DNA templates) suitable for use in producing the synthetic RNA viral genomes described herein comprise at least one 5' junction cleavage sequence and two 3' junction cleavage sequences.

[0266] The nature of the junction cleavage sequence and the removal of non-viral RNA from viral genome transcripts can be achieved by various methods. For example, in some embodiments, the junction cleavage sequence is the target of an RNA interference (RNAi) molecule. As used herein, "RNA interference molecule" refers to an RNA polynucleotide that mediates the degradation of a target mRNA sequence via an endogenous gene silencing pathway (e.g., Dicer and the RNA-induced silencing complex (RISC)). Exemplary RNA interference agents include microRNAs (miRNAs), artificial miRNAs (amiRNAs), short hairpin RNAs (shRNAs), and small interfering RNAs (siRNAs). Furthermore, any system for cleaving RNA transcripts at specific sites currently known in the art or defined in the future can be used to generate distinct ends native to the virus.

[0267] In some embodiments, the RNAi molecule is an miRNA. miRNA refers to a naturally occurring small non-coding RNA molecule approximately 18-25 nucleotides in length that is at least partially complementary to a target mRNA sequence. In animals, miRNA genes are transcribed into primary miRNAs (pri-miRNAs), which are double-stranded and form stem-loop structures. Pri-miRNAs are then cleaved in the nucleus by the microprocessor complex, which contains class 2 RNase III, Drosha, and the microprocessor subunit, DCGR8, to form 70-100 nucleotide precursor miRNAs (pre-miRNAs). The pre-miRNAs form hairpin structures and are transported to the cytoplasm, where they are processed by the RNase III enzyme, Dicer, into approximately 18-25 nucleotide duplexes. Although either strand of the duplex can function as a functional miRNA, typically one strand of the miRNA is degraded, and only one strand is loaded onto Argonaute (AGO) nuclease to generate an effector RNA-induced silencing complex (RISC) in which the miRNA and its mRNA target interact (Wahid et al., 1803:11, 2010, 1231-1243). In some embodiments, the 5' and / or 3' junction cleavage sequence is a miRNA target sequence.

[0268] In some embodiments, the RNAi molecule is an artificial miRNA (amiRNA) derived from a Pol II transcript with an embedded synthetic miRNA (see, e.g., Liu et al., Nucleic Acids Res (2008) 36:9; 2811-2834; Zeng et al., Molecular Cell (2002), 9; 1327-1333; Fellman et al., Cell Reports (2013) 5; 1704-1713). In some embodiments, the 5' and / or 3' junction cleavage sequence is an amiRNA target sequence.

[0269] In some embodiments, the RNAi molecule is an siRNA molecule. siRNA refers to a double-stranded RNA molecule typically about 21-23 nucleotides in length. Double-stranded siRNA molecules are processed in the cytoplasm by association with a multiprotein complex called the RNA-induced silencing complex (RISC), during which the "passenger" sense strand is enzymatically cleaved from the double strand. The antisense "guide" strand contained in the activated RISC then guides the RISC to the corresponding mRNA by sequence complementarity, and AGO nuclease cleaves the target mRNA, resulting in specific gene silencing. In some embodiments, the siRNA molecule is derived from an shRNA molecule. shRNAs are single-stranded artificial RNA molecules about 50-70 nucleotides in length that form a stem-loop structure. Expression of the shRNA in cells is achieved by introducing a DNA polynucleotide encoding the shRNA via a plasmid or viral vector. The shRNA is then transcribed into a product that mimics the stem-loop structure of the pre-miRNA, and after nuclear export, the hairpin is processed by Dicer to form a double-stranded siRNA molecule, which is then further processed by RISC to mediate target gene silencing. In some embodiments, the 5' and / or 3' junction cleavage sequence is an siRNA target sequence.

[0270] In some embodiments, the junction cleavage sequence is a guide RNA (gRNA) target sequence. In such embodiments, the gRNA is designed and introduced using a Cas endonuclease (e.g., Cas13) with RNase activity to mediate cleavage of the viral genome transcript at the precise junction site. In some embodiments, the 5' and / or 3' junction cleavage sequence is a gRNA target sequence.

[0271] In some embodiments, the junction cleavage sequence is a sequence encoding a pri-miRNA. When a polynucleotide (e.g., a recombinant RNA molecule) encoding a viral genome is transcribed, these sequences form a pri-miRNA stem-loop structure, which is cleaved in the nucleus by Drosha, cleaving the transcript at the precise junction site. In some embodiments, the 5' and / or 3' junction cleavage sequence is a pri-mRNA target sequence.

[0272] In some embodiments, the junction cleavage sequence is a primer binding sequence that promotes cleavage by the endoribonuclease RNAse H. In such embodiments, primers that anneal to the 5' and / or 3' junction cleavage sequence are added to an in vitro reaction along with an RNAse H enzyme. RNAse H specifically hydrolyzes the phosphodiester bonds of RNA hybridized to DNA, thus enabling cleavage of the synthetic RNA genome intermediate at the precise junction cleavage sequence to produce the required 5' and 3' natural ends.

[0273] In some embodiments, the junction cleavage sequence comprises or consists of a restriction enzyme recognition site, resulting in the generation of distinct ends of the viral transcript during linearization of the plasmid-templated runoff RNA synthesis using T7 RNA polymerase. In some embodiments, the junction cleavage sequence is a Type IIS restriction enzyme recognition site. Type IIS restriction enzymes constitute a specific group of enzymes that recognize asymmetric DNA sequences and cleave at a defined distance outside their recognition sequence, typically within 1 to 20 nucleotides. Exemplary Type IIS restriction enzymes include AcuI, AlwI, BaeI, BbsI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBi, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BstI, CaspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboI, MlyI, MmeI, MnlL, NmeAIII, PleI, SapI, and SfaNI. The recognition sequences for these Type IIS restriction enzymes are known in the art. See the New England Biolabs website at neb.com / tools-and-resources / selection-charts / type-iis-restriction-enzymes. In some embodiments, the junction cleavage sequence comprises a SapI restriction enzyme recognition site. In some embodiments, the junction cleavage sequence comprises a BsmBI restriction enzyme recognition site. In some embodiments, the junction cleavage sequence comprises a BsaI restriction enzyme recognition site. Those of skill in the art will understand that because cleavage sites for Type IIS restriction enzymes are typically outside of (e.g., offset by 1-5 nucleotides) the enzyme's recognition site, the corresponding junction cleavage sequence may also include additional nucleotide(s) required by the corresponding restriction enzyme to create distinct ends of the viral transcript (e.g., a polyA tail at the 3' end).

[0274] In some embodiments, the junction cleavage sequence encodes a ligand-induced self-cleaving ribozyme, referred to as an "aptazyme." An aptazyme is a ribozyme sequence that contains an aptamer domain specific for a ligand. Binding of the aptamer domain to the ligand triggers activation of the ribozyme's enzymatic activity, resulting in cleavage of the RNA transcript. Exemplary aptazymes include theophylline-dependent aptazymes (e.g., hammerhead ribozymes linked to theophylline-dependent aptamers as described in Auslander et al., Mol BioSyst. (2010) 6, 807-814), tetracycline-dependent aptazymes (e.g., Zhong et al., eLife 2016;5:e18858 DOI:10.7554 / eLife.18858; Win and Smolke, PNAS (2007) 104;14283-14288; Whittmann and Suess, Mol Biosyt (2011) 7;2419-2427; Xiao et al., Chem&Biol (2008) 15;125-1137; and Beilstein et al., ACS Syn Biol (2015) 4; 526-534), a hammerhead ribozyme linked to a Tet-dependent aptamer, a guanine-dependent aptazyme (e.g., a hammerhead ribozyme linked to a guanine-dependent aptamer as described in Nomura et al., Chem Commun., (2012) 48(57); 7215-7217). In some embodiments, the 5' and / or 3' junction cleavage sequence is a sequence encoding an aptazyme.

[0275] In some embodiments, the junction cleavage sequence is a target sequence for an RNAi molecule (e.g., an siRNA molecule, an shRNA molecule, an miRNA molecule, or an amiRNA molecule), a gRNA molecule, or an RNAse H primer. In such embodiments, the junction cleavage sequence is at least partially complementary to the sequence of the RNAi molecule, the gRNA molecule, or the primer molecule. Methods of sequence alignment for comparing and determining percent sequence identity and percent complementarity are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology), by Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. This can be done using algorithms known in the art, including the BLAST and BLAST 2.0 algorithms described in, respectively, U.S. Pat. No. 6,237,163 and U.S. Pat. No. 6,237,163 and U.S. Pat. No. 6,237,163. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0276] In some embodiments, the 5' junction cleavage sequence and the 3' junction cleavage sequence are from the same group (e.g., both are RNAi target sequences, both are sequences encoding ribozymes, etc.). For example, in some embodiments, the junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, shRNA, amiRNA, or miRNA target sequence) and is incorporated into the 5' and 3' ends of a polynucleotide encoding a viral genome (e.g., a recombinant RNA molecule). In such embodiments, the 5' and 3' RNAi target sequences can be the same (i.e., the target of the same siRNA, amiRNA, or miRNA) or different (i.e., the 5' sequence is the target of one siRNA, shmiRNA, or miRNA, and the 3' sequence is the target of another siRNA, amiRNA, or miRNA). In some embodiments, the junction cleavage sequence is a guide RNA target sequence and is incorporated into the 5' and 3' ends of a polynucleotide encoding a viral genome (e.g., a recombinant RNA molecule). In such embodiments, the 5' and 3' gRNA target sequences can be the same (i.e., targets for the same gRNA) or different (i.e., the 5' sequence is a target for one gRNA and the 3' sequence is a target for another gRNA). In some embodiments, the junction cleavage sequences are sequences encoding a pri-mRNA and are incorporated at the 5' and 3' ends of a polynucleotide sequence encoding a viral genome (e.g., a recombinant RNA molecule). In some embodiments, the junction cleavage sequences are sequences encoding a ribozyme and are incorporated immediately 5' and 3' to a polynucleotide sequence encoding a viral genome (e.g., a recombinant RNA molecule).

[0277] In some embodiments, the 5' junction cleavage sequence and the 3' junction cleavage sequence are from the same group but are different variants or types. For example, in some embodiments, the 5' and 3' junction cleavage sequences can be target sequences of an RNAi molecule, where the 5' junction cleavage sequence is an siRNA target sequence and the 3' junction cleavage sequence is an miRNA target sequence (or vice versa). In some embodiments, the 5' and 3' junction cleavage sequences can be sequences encoding ribozymes, where the 5' junction cleavage sequence is a sequence encoding a hammerhead ribozyme and the 3' junction cleavage sequence is a sequence encoding a hepatitis delta virus ribozyme.

[0278] In some embodiments, the 5' junction cleavage sequence and the 3' junction cleavage sequence are different types. For example, in some embodiments, the 5' junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, amiRNA, or miRNA target sequence), and the 3' junction cleavage sequence is a ribozyme sequence, an aptazyme sequence, a pri-miRNA sequence, or a gRNA target sequence. In some embodiments, the 5' junction cleavage sequence is a ribozyme sequence, and the 3' junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, amiRNA, or miRNA target sequence), an aptazyme sequence, a sequence encoding a pri-miRNA, or a gRNA target sequence. In some embodiments, the 5' junction cleavage sequence is an aptazyme sequence, and the 3' junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, amiRNA, or miRNA target sequence), a ribozyme sequence, a pri-miRNA sequence, or a gRNA target sequence. In some embodiments, the 5' junction cleavage sequence is a pri-miRNA sequence and the 3' junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, amiRNA, or miRNA target sequence), a ribozyme sequence, an aptazyme sequence, or a gRNA target sequence. In some embodiments, the 5' junction cleavage sequence is a gRNA target sequence and the 3' junction cleavage sequence is an RNAi target sequence (e.g., an siRNA, amiRNA, or miRNA target sequence), a ribozyme sequence, a pri-miRNA sequence, or an aptazyme sequence.

[0279] Exemplary placement of the junction cleavage sequence relative to the polynucleotide encoding the synthetic viral genome is shown in Tables 3 and 4 below. [Table 3] [Table 4-1] [Table 4-2]

[0280] In some embodiments, the junction cleavage sequence encodes a ribozyme that mediates self-cleavage of the synthetic RNA genome intermediate to produce the native, distinct 5' and / or 3' ends required for the production of the final synthetic viral RNA genome and subsequent infectious RNA virus. Exemplary ribozymes include hammerhead ribozymes (e.g., the hammerhead ribozyme shown in Figure 5A), Varkud satellite (VS) ribozyme, hairpin ribozyme, GIR1 branched ribozyme, glmS ribozyme, twister ribozyme, twister sister ribozyme (e.g., twister sister 1 or twister sister 2), pistol ribozyme (e.g., the pistol ribozyme shown in Figures 6A-6C, the Env25 pistol ribozyme, or the Alistipes Putredinis pistol ribozyme), hatchet ribozyme, and hepatitis delta virus ribozyme. In some embodiments, the 5' and / or 3' junction cleavage sequence is a sequence encoding a ribozyme.

[0281] In some embodiments, the 5' junction cleavage sequence comprises or consists of a ribozyme sequence, hi some embodiments, the 5' ribozyme sequence is selected from a hammerhead ribozyme sequence, a pistol ribozyme sequence, or a twister sister ribozyme sequence.

[0282] In some embodiments, the 5' junction cleavage sequence comprises or consists of a 5' pistol ribozyme sequence. In some embodiments, the 5' pistol ribozyme sequence is derived from P. polymyxa. In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity (including all ranges and subranges therebetween) to any one of SEQ ID NOS: 16-19 and 23-24. In some embodiments, the 5' pistol ribozyme sequence comprises a P2 motif, shown in Figures 6A and 6C, which is 4 nucleotides in length and located in a region corresponding to nucleic acid positions 27-30 of SEQ ID NOS: 16-19 and 23-24. In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to SEQ ID NO: 17, and the polynucleotide sequence of the P2 motif is "TTTA". In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to SEQ ID NO: 18, and the polynucleotide sequence of the P2 motif is "TTTT".In some embodiments, the 5' pistol ribozyme sequence from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19 (including all ranges and subranges therebetween), and the polynucleotide sequence of the P2 motif is "TTGT". In some embodiments, the 5' pistol ribozyme sequence is incorporated into a recombinant DNA molecule for in vitro transcription of a Coxsackievirus (e.g., CVA21) RNA virus genome.

[0283] In some embodiments, the 5' junction cleavage sequence comprises or consists of a 5' pistol ribozyme sequence. In some embodiments, the 5' pistol ribozyme sequence is derived from P. polymyxa. In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64 or 65 (including all ranges and subranges therebetween). In some embodiments, the 5' pistol ribozyme sequence comprises a P2 motif that is 4 nucleotides in length and is located in a region corresponding to nucleic acid positions 27-30 of SEQ ID NO: 64 or 65. In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to SEQ ID NO:64, and the polynucleotide sequence of the P2 motif is "TCAA". In some embodiments, the 5' pistol ribozyme sequence derived from P. polymyxa comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to SEQ ID NO:65, and the polynucleotide sequence of the P2 motif is "TTAA". In some embodiments, a 5' pistol-type ribozyme sequence is incorporated into a recombinant DNA molecule for in vitro transcription of the SVV (eg, SVV-IRES-2) RNA viral genome.

[0284] In some embodiments, the 5' junction cleavage sequence comprises or consists of an Env25 pistol ribozyme. In some embodiments, the DNA sequence encoding the Env25 pistol ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 96 (including all ranges and sub-ranges therebetween). In some embodiments, the Env25 pistol ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 100 (including all ranges and sub-ranges therebetween).

[0285] In some embodiments, the 5' junction cleavage sequence comprises or consists of an Alistipes Putredinis pistol ribozyme. In some embodiments, the DNA sequence encoding the Alistipes Putredinis pistol ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 97 (including all ranges and sub-ranges therebetween). In some embodiments, the Alistipes Putredinis pistol ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101 (including all ranges and sub-ranges therebetween).

[0286] In some embodiments, the 5' junction cleavage sequence comprises or consists of a Twister Sister 1 ribozyme. In some embodiments, the DNA sequence encoding the Twister Sister 1 ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 98 (including all ranges and sub-ranges therebetween). In some embodiments, the Twister Sister 1 ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 102 (including all ranges and sub-ranges therebetween).

[0287] In some embodiments, the 5' junction cleavage sequence comprises or consists of a Twister Sister 2 ribozyme. In some embodiments, the DNA sequence encoding the Twister Sister 2 ribozyme comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 99 (including all ranges and sub-ranges therebetween). In some embodiments, the Twister Sister 2 ribozyme RNA sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103 (including all ranges and sub-ranges therebetween).

[0288] Leader sequence In some embodiments, the recombinant DNA molecule (e.g., the DNA template) comprises a leader sequence between the promoter sequence and the 5' junction cleavage sequence. In some embodiments, the presence of the leader sequence facilitates or ensures proper folding of the downstream 5' junction cleavage sequence (e.g., a 5' ribozyme sequence).

[0289] In some embodiments, the leader sequence is about 5 bp, about 10 bp, about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, or about 100 bp in length, including all ranges and subranges therebetween. In some embodiments, the leader sequence is at least 5 bp, at least 10 bp, at least 15 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, at least 40 bp, at least 45 bp, at least 50 bp, at least 55 bp, at least 60 bp, at least 65 bp, at least 70 bp, at least 75 bp, at least 80 bp, at least 85 bp, at least 90 bp, at least 95 bp, or at least 100 bp in length, including all ranges and subranges therebetween. In some embodiments, the leader sequence is less than 5 bp, less than 10 bp, less than 15 bp, less than 20 bp, less than 25 bp, less than 30 bp, less than 35 bp, less than 40 bp, less than 45 bp, less than 50 bp, less than 55 bp, less than 60 bp, less than 65 bp, less than 70 bp, less than 75 bp, less than 80 bp, less than 85 bp, less than 90 bp, less than 95 bp, or less than 100 bp in length, including all ranges and subranges therebetween. In some embodiments, the leader sequence is about 50-70 bp, about 40-60 bp, about 60-80 bp, about 40-80 bp, about 30-70 bp, about 50-90 bp, about 30-90 bp, about 20-60 bp, or about 60-100 bp in length, including all ranges and subranges therebetween. In some embodiments, the leader sequence is about 57 bp or about 55-60 bp in length.

[0290] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having 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%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to any one of SEQ ID NOs: 13-15. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having 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%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to SEQ ID NO: 15. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 15. In some embodiments, the leader sequence is followed or immediately followed by a 5' pistol ribozyme sequence (e.g., a pistol ribozyme from P. Polymyxa or a variant thereof). In some embodiments, the leader sequence is incorporated into a recombinant DNA molecule (e.g., a DNA template) for in vitro transcription of the CVA21 RNA viral genome.

[0291] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having 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%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to any one of SEQ ID NOs: 53-63. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having 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%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to any one of SEQ ID NOs: 53-60 and 62-63. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to any one of SEQ ID NOs: 53-60 and 62-63.

[0292] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having 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%, or 100% sequence identity (including all ranges and sub-ranges therebetween) to SEQ ID NO: 53. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 53.

[0293] In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence having 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%, or 100% (including all ranges and sub-ranges therebetween) sequence identity to SEQ ID NO: 58. In some embodiments, the leader sequence comprises or consists of a polynucleotide sequence according to SEQ ID NO: 58.

[0294] In some embodiments, the leader sequence is followed or immediately followed by a 5' pistol ribozyme sequence (e.g., a pistol ribozyme according to SEQ ID NO: 64 or 65 or a variant thereof). In some embodiments, the leader sequence is incorporated into a recombinant DNA molecule (e.g., a DNA template) for in vitro transcription of the SVV RNA viral genome.

[0295] Poly A tail In some embodiments, the recombinant DNA molecule (e.g., the DNA template) includes a sequence encoding a polyA tail. In some embodiments, the polyA tail is added to the 3' end of the synthetic RNA viral genome. In some embodiments, the polyA tail is 2-500 bp in length (i.e., 2-500 pA). In some embodiments, the polyA tail is 2-100, 2-150, 2-200, 2-250, 2-300, 2-400, or 2-500 bp in length, including all ranges and subranges therebetween. In some embodiments, the polyA tail is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 bp in length, including all ranges and subranges therebetween. In some embodiments, the polyA tail is about 10-30, 20-40, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 90-110, 100-120, 110-130, 120-140, 130-150, 140-160, 150-170, 160-180, 170-190, or 180-200 bp in length, including all ranges and subranges therebetween. In some embodiments, the polyA tail is about 65-75, 60-80, 55-85, 50-90, 45-95, or 40-100 bp in length, including all ranges and subranges therebetween. In some embodiments, the polyA tail is about 70 bp in length. In some embodiments, a longer polyA tail (e.g., about 70 bp in length) improves the loading capacity of the synthetic RNA viral genome in oligo-dT chromatography compared to a corresponding synthetic RNA viral genome with a shorter polyA tail (e.g., about 30 bp in length).In some embodiments, the loading capacity is improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, or at least 10-fold compared to a synthetic RNA viral genome with a polyA tail about 30 bp in length.

[0296] Non-limiting examples of recombinant DNA molecule design In some embodiments, the synthetic RNA viral genomes described herein are produced in vitro by in vitro RNA transcription (see, e.g., the schematics in Figures 10, 11A, 11B, and 33). The synthetic RNA viral genomes are then purified and formulated for therapeutic use (e.g., encapsulated in lipid nanoparticles).

[0297] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a ribozyme sequence, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises or consists of a ribozyme sequence. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' hammerhead ribozyme sequence (e.g., a wild-type HHR or a modified HHR as provided in Figures 5A and 5B), (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' hepatitis delta virus ribozyme sequence.

[0298] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a ribozyme sequence, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises a restriction enzyme recognition site. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' hammerhead ribozyme sequence (e.g., a wild-type HHR or a modified HHR as provided in Figures 5A and 5B), (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises or consists of a SapI restriction enzyme recognition site.

[0299] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a ribozyme sequence, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises or consists of a restriction enzyme recognition site. In some embodiments, a DNA template comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' hammerhead ribozyme sequence (e.g., a wild-type HHR or modified HHR as provided in Figures 5A and 5B), (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises or consists of a BsaI restriction enzyme recognition site.

[0300] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence (e.g., the pistol 1 or pistol 2 ribozyme sequence shown in Figure 6B), (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0301] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence (e.g., the pistol 1 or pistol 2 ribozyme sequence shown in Figure 6B), (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0302] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a 5' RNAse H primer binding site, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises a restriction enzyme recognition site. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', a polynucleotide that comprises: (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a 5' RNAse H primer binding site, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises or consists of a SapI restriction enzyme recognition site.

[0303] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a 5' RNAse H primer binding site, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises a restriction enzyme recognition site. In some embodiments, a recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', a polynucleotide that comprises: (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a 5' junction cleavage sequence that comprises or consists of a 5' RNAse H primer binding site, (iii) a polynucleotide encoding a synthetic RNA viral genome, and (iv) a 3' junction cleavage sequence that comprises or consists of a BsaI restriction enzyme recognition site.

[0304] In some embodiments, the synthetic RNA viral genome is a Coxsackievirus (CVA) genome. In some embodiments, the Coxsackievirus is the CVA21 strain. In some embodiments, the CVA21 strain is the EF strain. In some embodiments, the CVA21 strain is the KY strain.

[0305] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) an optional leader sequence, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence (e.g., a pistol ribozyme derived from P. Polymyxa or a variant thereof), (iv) a polynucleotide encoding a synthetic RNA viral genome, (v) a polyA tail (e.g., about 20-80 bp in length, or about 30-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a restriction enzyme recognition site (e.g., for BsmBI or BsaI restriction enzymes).

[0306] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence (e.g., SEQ ID NO: 14 or 15), (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence (e.g., a pistol ribozyme derived from P. Polymyxa or a variant thereof), (iv) a polynucleotide encoding the CVA21 synthetic RNA virus genome, (v) a polyA tail (e.g., about 20 to 80 bp in length, or about 30 to 70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsmBI restriction enzyme recognition site.

[0307] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence (e.g., SEQ ID NO: 14 or 15), (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence (e.g., a pistol ribozyme derived from P. Polymyxa or a variant thereof), (iv) a polynucleotide encoding the CVA21 synthetic RNA virus genome, (v) a polyA tail (e.g., about 20 to 80 bp in length, or about 30 to 70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0308] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence according to SEQ ID NO: 15, (iii) a 5' junction cleavage sequence that comprises, or consists of, a 5' pistol ribozyme sequence, (iv) a polynucleotide encoding a CVA21 synthetic RNA viral genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence that comprises, or consists of, a BsmBI restriction enzyme recognition site, wherein the combination of the 5' pistol ribozyme sequence and the polyA tail is selected from one of embodiments E1-E68 provided in Table 5A below.

[0309] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence according to SEQ ID NO: 15, (iii) a 5' junction cleavage sequence that comprises, or consists of, a 5' pistol ribozyme sequence, (iv) a polynucleotide encoding the CVA21 synthetic RNA viral genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence that comprises, or consists of, a BsaI restriction enzyme recognition site, wherein the combination of the 5' pistol ribozyme sequence and the polyA tail is selected from one of embodiments E1-E68 provided in Table 5A below.

[0310] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence according to SEQ ID NO: 14, (iii) a 5' junction cleavage sequence that comprises, or consists of, a 5' pistol ribozyme sequence, (iv) a polynucleotide encoding a CVA21 synthetic RNA viral genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence that comprises, or consists of, a BsmBI restriction enzyme recognition site, wherein the combination of the 5' pistol ribozyme sequence and the polyA tail is selected from one of embodiments E1-E68 provided in Table 5A below.

[0311] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence according to SEQ ID NO: 14, (iii) a 5' junction cleavage sequence that comprises, or consists of, a 5' pistol ribozyme sequence, (iv) a polynucleotide encoding the CVA21 synthetic RNA viral genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence that comprises, or consists of, a BsaI restriction enzyme recognition site, wherein the combination of the 5' pistol ribozyme sequence and the polyA tail is selected from one of embodiments E1-E68 provided in Table 5A below. [Table 5A-1] [Table 5A-2]

[0312] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 15, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 18, (iv) a polynucleotide encoding the CVA21 synthetic RNA viral genome, (v) a polyA tail (e.g., a polyA tail of about 70 bp, about 60-80 bp, or about 50-90 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsmBI restriction enzyme recognition site.

[0313] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 15, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 18, (iv) a polynucleotide encoding the CVA21 synthetic RNA virus genome, (v) a polyA tail (e.g., a polyA tail of about 70 bp, about 60-80 bp, or about 50-90 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0314] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) includes a DNA polynucleotide that encodes a CVA21-KY strain viral genome and that comprises or consists of a sequence according to SEQ ID NO: 20. In some embodiments, the DNA polynucleotide encoding the CVA21-KY strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20 (including all ranges and subranges therebetween).

[0315] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) includes a DNA polynucleotide that encodes a CVA21-KY strain viral genome and that comprises or consists of a sequence according to SEQ ID NO: 93. In some embodiments, the DNA polynucleotide encoding the CVA21-KY strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 93 (including all ranges and subranges therebetween).

[0316] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) includes a DNA polynucleotide that encodes a CVA21-EF strain viral genome and that comprises or consists of a sequence according to SEQ ID NO: 21. In some embodiments, the DNA polynucleotide encoding the CVA21-EF strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21 (including all ranges and subranges therebetween).

[0317] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) includes a DNA polynucleotide that encodes a CVA21-EF strain viral genome and that comprises or consists of a sequence according to SEQ ID NO: 95. In some embodiments, the DNA polynucleotide encoding the CVA21-EF strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 95 (including all ranges and subranges therebetween).

[0318] In some embodiments, a recombinant DNA molecule (e.g., a DNA template) includes a DNA polynucleotide that encodes a CVA21-Kuykendall strain viral genome and that comprises or consists of a sequence according to SEQ ID NO: 22. In some embodiments, the DNA polynucleotide encoding the CVA21-Kuykendall strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 (including all ranges and subranges therebetween).

[0319] In some embodiments, the recombinant DNA molecule (e.g., DNA template) includes a DNA polynucleotide that encodes a CVA21-Kuykendall strain viral genome and that comprises or consists of a sequence according to SEQ ID NO: 94. In some embodiments, the DNA polynucleotide encoding the CVA21-Kuykendall strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 94 (including all ranges and subranges therebetween).

[0320] In some embodiments, the synthetic RNA viral genome is a Seneca Valley Virus (SVV) genome.

[0321] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) an optional leader sequence, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence (e.g., a pistol ribozyme derived from P. Polymyxa or a variant thereof), (iv) a polynucleotide encoding a synthetic RNA viral genome, (v) a polyA tail (e.g., about 20-80 bp in length, or about 30-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a restriction enzyme recognition site (e.g., for SapI or BsaI restriction enzyme).

[0322] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence (e.g., any one of SEQ ID NOs: 53-60 and 62-63), (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol-type ribozyme sequence (e.g., according to SEQ ID NO: 64 or 65, or a variant thereof), (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail (e.g., about 20-80 bp in length, or about 30-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0323] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence (e.g., any one of SEQ ID NOs: 53-60 and 62-63), (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol-type ribozyme sequence (e.g., according to SEQ ID NO: 64 or 65, or a variant thereof), (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail (e.g., about 20-80 bp in length, or about 30-70 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0324] In some embodiments, the recombinant DNA molecule (e.g., the DNA template) comprises a 3' SapI restriction site after the polyA tail and does not comprise a SapI restriction site within the polynucleotide encoding the SVV synthetic RNA viral genome.

[0325] In some embodiments, the recombinant DNA molecule (e.g., the DNA template) comprises a 3' BsaI restriction site after the polyA tail and does not comprise a BsaI restriction site within the polynucleotide encoding the SVV synthetic RNA viral genome.

[0326] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence according to any one of SEQ ID NOs: 53-60 and 62-63, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence, (iv) a polynucleotide encoding an SVV synthetic RNA viral genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site, wherein the combination of leader sequence, 5' pistol ribozyme sequence, and polyA tail is selected from one of embodiments S1 to S340 provided in Table 5B below.

[0327] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a promoter sequence (e.g., a T7 polymerase promoter), (ii) a leader sequence according to any one of SEQ ID NOs: 53-60 and 62-63, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence, (iv) a polynucleotide encoding an SVV synthetic RNA viral genome, (v) a polyA tail, and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site, wherein the combination of the leader sequence, 5' pistol ribozyme sequence, and polyA tail is selected from one of embodiments S1 to S340 provided in Table 5B below. [Table 5B-1] [Table 5B-2] [Table 5B-3] [Table 5B-4] [Table 5B-5] [Table 5B-6] [Table 5B-7] [Table 5B-8] [Table 5B-9] [Table 5B-10] [Table 5B-11]

[0328] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 53, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 64, (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail (e.g., a polyA tail of about 70 bp, about 60-80 bp, or about 50-90 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0329] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 53, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 64, (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail (e.g., a polyA tail of about 70 bp, about 60-80 bp, or about 50-90 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0330] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 58, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 64, (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail (e.g., a polyA tail of about 70 bp, about 60-80 bp, or about 50-90 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0331] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 58, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 64, (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail (e.g., a polyA tail of about 70 bp, about 60-80 bp, or about 50-90 bp in length), and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0332] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 58, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol ribozyme sequence according to SEQ ID NO: 64, (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail of approximately 70 bp in length, and (vi) a 3' junction cleavage sequence comprising or consisting of a SapI restriction enzyme recognition site.

[0333] In some embodiments, the recombinant DNA molecule (e.g., a DNA template) comprises, from 5' to 3', (i) a T7 polymerase promoter sequence, (ii) a leader sequence according to SEQ ID NO: 58, (iii) a 5' junction cleavage sequence comprising or consisting of a 5' pistol-type ribozyme sequence according to SEQ ID NO: 64, (iv) a polynucleotide encoding the SVV synthetic RNA viral genome, (v) a polyA tail of approximately 70 bp in length, and (vi) a 3' junction cleavage sequence comprising or consisting of a BsaI restriction enzyme recognition site.

[0334] In some embodiments, the recombinant DNA molecule (e.g., DNA template) includes a DNA polynucleotide that encodes an SVV viral genome and that comprises or consists of a sequence according to SEQ ID NO: 52. In some embodiments, the DNA polynucleotide encoding the SVV strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52 (including all ranges and subranges therebetween).

[0335] In some embodiments, the recombinant DNA molecule (e.g., DNA template) includes a DNA polynucleotide that encodes an SVV viral genome and that comprises or consists of a sequence according to SEQ ID NO: 92. In some embodiments, the DNA polynucleotide encoding the SVV strain viral genome comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92 (including all ranges and subranges therebetween).

[0336] Exemplary embodiments of DNA templates encoding the SVV or CVA genome are provided in Table 18 below. [Table 18]

[0337] Particles containing synthetic RNA genomes In some embodiments, the synthetic RNA genomes described herein are encapsulated in a "particle." As used herein, particle refers to a non-tissue-derived composition of matter, such as a liposome, lipoplex, nanoparticle, nanocapsule, microparticle, microsphere, lipid particle, exosome, or vesicle. In certain embodiments, the particle is non-proteinaceous and non-immunogenic. In such embodiments, encapsulation of the synthetic RNA genomes described herein enables delivery of viral genomes without inducing a systemic antiviral immune response and reduces the effects of neutralizing antiviral antibodies. Furthermore, encapsulation of the synthetic RNA genomes described herein protects the genome from degradation and facilitates introduction into target host cells. In some embodiments, the present disclosure provides nanoparticles comprising the synthetic RNA genomes described herein. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles further comprise a second RNA molecule encoding a payload molecule.

[0338] In some embodiments, the particles are biodegradable in the subject. In such embodiments, multiple doses of particles can be administered to the subject without particle accumulation in the subject. Examples of suitable particles include polystyrene particles, poly(lactic-co-glycolic acid) PLGA particles, polypeptide-based cationic polymer particles, cyclodextrin particles, chitosan, N,N,N-trimethylchitosan particles, lipid-based particles, poly(β-amino ester) particles, low-molecular-weight polyethyleneimine particles, polyphosphoester particles, disulfide-crosslinked polymer particles, polyamidoamine particles, polyethyleneimine (PEI) particles, and PLURIONICS-stabilized polypropylene sulfide particles.

[0339] In some embodiments, the polynucleotides described herein are encapsulated in inorganic particles. In some embodiments, the inorganic particles are gold nanoparticles (GNPs), gold nanorods (GNRs), magnetic nanoparticles (MNPs), magnetic nanotubes (MNTs), carbon nanohorns (CNHs), carbon fullerenes, carbon nanotubes (CNTs), calcium phosphate nanoparticles (CPNPs), mesoporous silica nanoparticles (MSNs), silica nanotubes (SNTs), or star-shaped hollow silica nanoparticles (SHNPs).

[0340] Preferably, the particles described herein are nanoscopic in size to enhance solubility, avoid potential complications caused by phagocytic clearance and in vivo aggregation, and promote pinocytosis. In some embodiments, the particles have an average diameter of less than about 1000 nm. In some embodiments, the particles have an average diameter of less than about 500 nm. In some embodiments, the particles have an average diameter of about 30 to about 100 nm, about 50 to about 100 nm, or about 75 to about 100 nm. In some embodiments, the particles have an average diameter of about 30 to about 75 nm, or about 30 to about 50 nm. In some embodiments, the particles have an average diameter of about 100 to about 500 nm. In some embodiments, the particles have an average diameter of about 200 to 400 nm. In some embodiments, the particles have an average size of about 350 nm.

[0341] Exosomes In some embodiments, the synthetic RNA genomes described herein are encapsulated in exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after multivesicular bodies fuse with the plasma membrane of a parent cell (e.g., the cell from which the exosomes are released, also referred to herein as the donor cell). The surface of exosomes comprises a lipid bilayer derived from the parent cell's plasma membrane and may further comprise membrane proteins expressed on the parent cell surface. In some embodiments, exosomes may also contain cytosol from the parent cell. Exosomes are produced by many different cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs), and have been identified in plasma, urine, bronchoalveolar lavage fluid, intestinal epithelial cells, and tumor tissue. Because the composition of exosomes depends on the parent cell type from which they are derived, there are no "exosome-specific" proteins. However, many exosomes contain proteins associated with the intracellular vesicles from which they originated in the parent cell (e.g., proteins associated with and / or expressed by endosomes and lysosomes). For example, exosomes can be enriched in antigen-presenting molecules such as major histocompatibility complexes I and II (MHC-I and MHC-II), tetraspanins (e.g., CD63), several heat shock proteins, cytoskeletal components such as actin and tubulin, proteins involved in intracellular membrane fusion, cell-cell interaction molecules (e.g., CD54), signaling proteins, and cytosolic enzymes.

[0342] Exosomes can mediate the transfer of cellular proteins from one cell (e.g., a parent cell) to a target or recipient cell by fusing the exosome membrane with the target cell's plasma membrane. Therefore, modifying the material encapsulated by exosomes provides a mechanism by which exogenous agents, such as the polynucleotides described herein, can be introduced into target cells. Exosomes modified to contain one or more exogenous agents (e.g., the polynucleotides described herein) are referred to herein as "modified exosomes." In some embodiments, modified exosomes are produced by the introduction of an exogenous agent (e.g., a polynucleotide described herein) and then introduced into a parent cell. In such embodiments, the exogenous nucleic acid is introduced into a parent exosome-producing cell such that the exogenous nucleic acid itself, or a transcript of the exogenous nucleic acid, is incorporated into modified exosomes produced from the parent cell. The exogenous nucleic acid can be introduced into the parent cell by means known in the art, such as transduction, transfection, transformation, electroporation, and / or microinjection of the exogenous nucleic acid.

[0343] In some embodiments, modified exosomes are produced by directly introducing a synthetic RNA genome described herein into exosomes. In some embodiments, the synthetic RNA genome described herein is introduced into intact exosomes. "Intact exosomes" refer to exosomes that contain proteins and / or genetic material derived from the parent cell that produces them. Methods for obtaining intact exosomes are known in the art (see, e.g., Alvarez-Erviti L. et al., Nat Biotechnol. 2011 Apr;29(4):34-5; Ohno S, et al., Mol Ther 2013 Jan;21(1):185-91; and European Patent Publication No. 2010663).

[0344] In certain embodiments, the synthetic RNA genome is introduced into empty exosomes. "Empty exosomes" refer to exosomes lacking proteins and / or genetic material (e.g., DNA or RNA) derived from parental cells. Methods for producing empty exosomes (e.g., lacking genetic material derived from parental cells) are known in the art and include UV exposure, mutation / deletion of endogenous proteins that mediate nucleic acid loading into exosomes, and electroporation and chemical treatment to open pores in the exosome membrane so that endogenous genetic material can exit the exosome through the opened pores. In some embodiments, empty exosomes are produced by opening exosomes by treating them with an aqueous solution having a pH of about 9 to about 14 to obtain exosome membranes, removing intravesicular components (e.g., intravesicular proteins and / or nucleic acids), and reassembling the exosome membrane to form empty exosomes. In some embodiments, intravesicular components (e.g., intravesicular proteins and / or nucleic acids) are removed by ultracentrifugation or density gradient ultracentrifugation. In some embodiments, the membranes are reassembled by sonication, mechanical vibration, extrusion through a porous membrane, electric current, or a combination of one or more of these techniques. In certain embodiments, the membranes are reassembled by sonication.

[0345] In some embodiments, loading intact or empty exosomes with the synthetic RNA genomes described herein to produce modified exosomes can be achieved using conventional molecular biology techniques, such as in vitro transformation, transfection, and / or microinjection. In some embodiments, an exogenous agent (e.g., a polynucleotide described herein) is directly introduced into intact or empty exosomes by electroporation. In some embodiments, an exogenous agent (e.g., a polynucleotide described herein) is directly introduced into intact or empty exosomes by lipofection (e.g., transfection). Lipofection kits suitable for use in producing exosomes according to the present disclosure are known in the art and commercially available (e.g., FuGENE® HD Transfection Reagent from Roche and LIPOFECTAMINE™ 2000 from Invitrogen). In some embodiments, an exogenous agent (e.g., a polynucleotide described herein) is directly introduced into intact or empty exosomes by transformation using heat shock. In such embodiments, exosomes isolated from parent cells are treated with Ca to permeabilize the exosome membrane. 2+ The exosomes are cooled in the presence of divalent cations, such as HCl (in CaCl2). The exosomes can then be incubated with exogenous nucleic acid and briefly heat-shocked (e.g., at 42°C for 30-120 seconds). In certain embodiments, loading of empty exosomes with exogenous agents (e.g., polynucleotides described herein) can be achieved by mixing or co-incubating the agent with the exosome membrane after removal of intravesicular components. Thus, modified exosomes reassembled from the exosome membrane incorporate the exogenous agent into the intravesicular space. Additional methods for producing exosome-encapsulated nucleic acids are known in the art (see, e.g., U.S. Patent Nos. 9,889,210, 9,629,929, and 9,085,778; International PCT Publication Nos. WO 2017 / 161010 and WO 2018 / 039119).

[0346] Exosomes can be obtained from a number of different parent cells, including cell lines, bone marrow-derived cells, and cells derived from primary patient samples. Exosomes released from parent cells can be isolated from the supernatant of parent cell cultures by means known in the art. For example, the physical properties of exosomes can be exploited to separate exosomes from culture media or other sources, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc.), density (e.g., conventional or gradient centrifugation), and Svedberg constant (e.g., sedimentation with or without external forces). Alternatively, or in addition, isolation can be based on one or more biological properties and may include methods that utilize surface markers (e.g., precipitation, reversible binding to solid phases, FACS separation, specific ligand binding, nonspecific ligand binding, etc.). Analysis of exosome surface proteins can be determined by flow cytometry using fluorescently labeled antibodies against exosome-associated proteins such as CD63. Additional markers for characterizing exosomes are described in International PCT Publication No. WO 2017 / 161010. In still further contemplated methods, exosomes may be fused using chemical and / or physical methods, including PEG-induced fusion and / or ultrasonic fusion.

[0347] In some embodiments, exosomes can be isolated using size exclusion chromatography. In some embodiments, exosomes can be further isolated after chromatographic separation (in one or more chromatographic fractions) by centrifugation techniques, as is widely known in the art. In some embodiments, exosome isolation can involve a combination of methods, including, but not limited to, previously described differential centrifugation (see, e.g., Raposo, G. et al., J. Exp. Med. 183, 1161-1172 (1996)), ultracentrifugation, size-based membrane filtration, concentration, and / or rate-zonal centrifugation.

[0348] In some embodiments, the exosome membrane comprises one or more of phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. Additionally, the membrane may comprise one or more polypeptides and one or more polysaccharides, such as glycans. Exemplary exosome membrane compositions and methods for modifying the relative amounts of one or more membrane components are described in International PCT Publication No. WO 2018 / 039119.

[0349] In some embodiments, the particles are exosomes and have a diameter of about 30 to about 100 nm, about 30 to about 200 nm, or about 30 to about 500 nm. In some embodiments, the particles are exosomes and have a diameter of about 10 nm to about 100 nm, about 20 nm to about 100 nm, about 30 nm to about 100 nm, about 40 nm to about 100 nm, about 50 nm to about 100 nm, about 60 nm to about 100 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 100 nm to about 200 nm, about 100 nm to about 150 nm, about 150 nm to about 200 nm, about 100 nm to about 250 nm, about 250 nm to about 500 nm, or about 10 nm to about 1000 nm. In some embodiments, the particles are exosomes and have a diameter of about 20 nm to 300 nm, about 40 nm to 200 nm, about 20 nm to 250 nm, about 30 nm to 150 nm, or about 30 nm to 100 nm.

[0350] compound Compounds of formula (I) In various embodiments, provided herein are compounds of formula (I): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: A is -N(CHR N1 )(CH2R N2 ), or a 4- to 7-membered heterocyclyl ring containing at least one N, wherein the 4- to 7-membered heterocyclyl ring is 3 and optionally substituted with Each X is independently -O-, -N(R 1 )-, or -N(R 2 )- and R 1 is an optionally substituted C1-C 31 selected from the group consisting of aliphatic and steroidal; R 2 is an optionally substituted C1-C 31 selected from the group consisting of aliphatic and steroidal; R 3 is an optionally substituted C1-C6 aliphatic; R N1 and R N2 are each independently hydrogen, hydroxy-C1-C6 alkyl, C2-C6 alkenyl, or C3-C7 cycloalkyl; L 1 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 alkenylene chains, L 2 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 alkenylene chains, L 3 is a bond, an optionally substituted C1-C6 alkylene chain, or a divalent optionally substituted C3-C7 cycloalkylene.

[0351] In some embodiments, when A is —N(CH 3 )(CH 3 ) and X is O, L 3 is not a C1-C6 alkylene chain.

[0352] In some embodiments, the present disclosure provides a compound of formula (Ia): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein m is 0, 1, 2, 3, 4, 5, or 6.

[0353] In some embodiments, the present disclosure provides a compound of formula (Ib): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein n is 0, 1, 2, or 3; and m is 0, 1, 2, 3, 4, 5, or 6.

[0354] In some embodiments, the present disclosure provides a compound of formula (I-bi): [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0355] In some embodiments, the present disclosure provides a compound of formula (I-bii): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein m is 0, 1, 2, or 3; p and q are each 0, 1, 2, or 3; and q+p is 3 or less.

[0356] In some embodiments, the present disclosure provides a compound of formula (I-biii): [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0357] In some embodiments, the present disclosure provides a compound of formula (Ic): [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0358] In some embodiments, A is —N(CHR N1 )(CH2R N2) or an optionally substituted 4- to 7-membered heterocyclyl ring containing at least one N.

[0359] In some embodiments, A is —N(CHR N1 )(CH2R N2 In some embodiments, R N1 and R N2 are each independently selected from hydrogen, hydroxy-C1-C3 alkylene, C2-C4 alkenyl, or C3-C4 cycloalkyl.

[0360] In some embodiments, R N1 and R N2 are each independently hydrogen, -CHCH=CH, -CHCHOH, [ka] In some embodiments, R N1 and R N2 and are the same. In some embodiments, R N1 and R N2 Each is hydrogen. In some embodiments, R N1 and R N2 are each C-C alkenyl, e.g., —CHCH═CH. In some embodiments, R N1 and R N2 are each hydroxy-C1-C3 alkylene, e.g., —CH2CH2OH. In some embodiments, R N1 and R N2 In some embodiments, R N1 and R N2 is hydrogen and the other is C-C cycloalkyl. In some embodiments, R N1 and R N2 One of them is hydrogen and the other is [ka] is.

[0361] In some embodiments, A is an optionally substituted 4- to 7-membered heterocyclyl ring containing at least one N. In some embodiments, A is an optionally substituted 4- to 7-membered heterocyclyl ring containing only one N. In some embodiments, A is an unsubstituted 4- to 7-membered heterocyclyl ring containing at least one N. In some embodiments, A is an unsubstituted 4- to 7-membered heterocyclyl ring containing only one N. In some embodiments, A is an optionally substituted 5- to 6-membered heterocyclyl ring containing at least one N. In some embodiments, A is an unsubstituted 5- to 6-membered heterocyclyl ring containing at least one N.

[0362] In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N, and the N atom of A is a tertiary amine.

[0363] In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N and further containing one or more S. In some embodiments, A is an optionally substituted 4-7 membered heterocyclyl ring containing at least one N and further containing only one S.

[0364] In some embodiments, A is selected from the group consisting of azetidine, pyrrolidine, piperidine, azepane, and thiomorpholine. In some embodiments, A is selected from the group consisting of pyrrolidine and piperidine.

[0365] In some embodiments, L 1 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20 In some embodiments, L is selected from the group consisting of: 2 is an optionally substituted C1-C 20 Alkylene chains and divalent optionally substituted C-C 20In some embodiments, L is selected from the group consisting of: 1 is an optionally substituted C1-C 20 In some embodiments, L 2 is an optionally substituted C1-C 20 It is an alkylene chain.

[0366] In some embodiments, L 1 and L 2 and are the same. In some embodiments, L 1 and L 2 is different.

[0367] In some embodiments, L 1 is an optionally substituted C1-C 10 In some embodiments, L 2 is an optionally substituted C1-C 10 In some embodiments, L 1 is an optionally substituted C1-C5 alkylene chain. In some embodiments, L 2 is an optionally substituted C1-C5 alkylene chain.

[0368] In some embodiments, L 1 and L 2 Each is -CHCHCHCH-. In some embodiments, L 1 and L 2 Each is -CHCHCH-. In some embodiments, L 1 and L 2 are each -CH2CH2-.

[0369] In some embodiments, L 3 is a bond, an optionally substituted C-C alkylene chain, or a divalent optionally substituted C-C cycloalkylene. 3 is a bond. In some embodiments, L 3is an optionally substituted C1-C6 alkylene chain. In some embodiments, L 3 is an optionally substituted C1-C3 alkylene chain. In some embodiments, L 3 is an unsubstituted C1-C3 alkylene chain. In some embodiments, L 3 is -CH-. In some embodiments, L 3 is -CHCH-. In some embodiments, L 3 is -CH2CH2CH2-. In some embodiments, L 3 is a divalent C-C cycloalkylene. In some embodiments, L 3 teeth [ka] is.

[0370] In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 2 to 10. In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 2 to 8. In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 2 to 5. In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 2 to 4. In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 2. In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 3. In some embodiments, the number of carbon atoms between S and N of A in the thiolate of Formula (I) is 4.

[0371] In some embodiments, R 1 is an optionally substituted C1-C 31 In some embodiments, R is selected from the group consisting of aliphatic and optionally substituted steroidyl. 2 is an optionally substituted C1-C 31 In some embodiments, R is selected from the group consisting of aliphatic and optionally substituted steroidyl.1 is an optionally substituted C1-C 31 In some embodiments, R 2 is an optionally substituted C1-C 31 In some embodiments, R 1 is an optionally substituted C5-C 25 In some embodiments, R 2 is an optionally substituted C5-C 25 In some embodiments, R 1 is optionally substituted C 10 -C 20 In some embodiments, R 2 is optionally substituted C 10 -C 20 In some embodiments, R 1 is optionally substituted C 10 -C 20 In some embodiments, R 2 is optionally substituted C 10 -C 20 In some embodiments, R 1 is the unsubstituted C 10 -C 20 In some embodiments, R 2 is the unsubstituted C 10 -C 20 It is alkyl.

[0372] In some embodiments, R 1 is optionally substituted C 14 -C 16 In some embodiments, R 2 is optionally substituted C 14 -C 16 In some embodiments, R 1 is the unsubstituted C 14 -C 16 In some embodiments, R 2 is the unsubstituted C 14 -C16 It is alkyl.

[0373] In some embodiments, R 1 is an optionally substituted branched C3-C 31 In some embodiments, R 2 is an optionally substituted branched C3-C 31 In some embodiments, R 1 is an optionally substituted branched C 10 -C 20 In some embodiments, R 2 is an optionally substituted branched C 10 -C 20 In some embodiments, R 1 is an optionally substituted branched C 14 -C 16 In some embodiments, R 2 is an optionally substituted branched C 14 -C 16 In some embodiments, R 1 is a substituted branched C3-C 31 In some embodiments, R 2 is a substituted branched C3-C 31 In some embodiments, R 1 is a substituted branched C 10 -C 20 In some embodiments, R 2 is a substituted branched C 10 -C 20 In some embodiments, R 1 is a substituted branched C 14 -C 16 In some embodiments, R 2 is a substituted branched C 14 -C 16 It is alkyl.

[0374] In some embodiments, R 1 and R2 is the same.

[0375] In some embodiments, R 1 and R 2 In some embodiments, R 1 is an optionally substituted C6-C 20 alkenyl, and R 2 is optionally substituted C 10 -C 20 In some embodiments, R 1 is C6-C 20 alkenyl, and R 2 is a branched C 10 -C 20 It is alkyl.

[0376] In some embodiments, A contains at least one N and 0 to 6 R 3 In some embodiments, R is a 4- to 7-membered heterocyclyl ring optionally substituted with 3 is an optionally substituted C1-C6 aliphatic. In some embodiments, R 3 is an optionally substituted C1-C3 aliphatic. In some embodiments, R 3 is an optionally substituted C1-C6 alkyl. In some embodiments, R 3 is an optionally substituted C1-C3 alkyl. In some embodiments, R 3 is unsubstituted C1-C6 alkyl. In some embodiments, R 3 is unsubstituted C1-C3 alkyl. In some embodiments, R 3 is an optionally substituted C1-C6 alkenyl. In some embodiments, R 3 is an optionally substituted C1-C3 alkenyl. In some embodiments, R 3 is unsubstituted C1-C6 alkenyl. In some embodiments, R 3 is unsubstituted C1-C3 alkenyl.

[0377] In some embodiments, R 3 is substituted with 1 to 3 C-C cycloalkyl. In some embodiments, R 3 is substituted with one C-C cycloalkyl. In some embodiments, R 3 is substituted with cyclopropanyl. In some embodiments, R 3 is substituted with 1 to 3 -OH. In some embodiments, R 3 is substituted with one -OH.

[0378] In some embodiments, m is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0379] In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 1 or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0380] In some embodiments, the compound of Formula (I) is a compound selected from Table 21, or a pharmaceutically acceptable salt or solvate thereof. [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5]

[0381] Compound of formula (A) In various embodiments, provided herein are compounds of formula (A): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1 is -[(CH2) 0-3 -C(O)O] 1-3 -, -(CH2) 0-3 -C(O)O-(CH2) 1-3 -OC(O)- or -C(O)N(H)-; R P1 is C5-C 25 Alkyl or C5-C 25 is alkenyl, R P2 is hydrogen or -CH3.

[0382] In some embodiments, Formula (A) is HO—(CHCHO) n -C(O)N(H)-(CH2) 17 Not CH3.

[0383] In some embodiments, L P1 is —CHC(O)O—, —CHCHC(O)O—, —CHC(O)OCHC(O)O—, —CHC(O)OCHCHOC(O)—, or —C(O)N(H)—.

[0384] In some embodiments, the PEG lipid is a compound of Formula (Aa), Formula (Ab), Formula (Ac), Formula (Ad), or Formula (Ae): [ka] or a pharmaceutically acceptable salt thereof.

[0385] In some embodiments, R P1 is C6-C 24 , C 10 -C 20 , C 10 -C 18 , C 10 -C 16 , C 10 -C 14 , C 10 -C 12 , C 12 -C 20 , C 12 -C 18 , C 12 -C 16 , C 12 -C 14 , C 14 -C 20 , C 14 -C 18 , C 14 -C 16 , C 16 -C 20 , C 16 -C 18 , or C 18 -C 20 In some embodiments, R P1 is C 14 -C 18 In some embodiments, R P1 is C 14 -C 16 In some embodiments, R P1 is C 15 -C 17 In some embodiments, R P1 is C 16 -C 18 In some embodiments, R P1 are C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C21 , C 22 , C 23 , or C 24 In some embodiments, R P1 is C6-C 24 , C 10 -C 20 , C 10 -C 18 , C 10 -C 16 , C 10 -C 14 , C 10 -C 12 , C 12 -C 20 , C 12 -C 18 , C 12 -C 16 , C 12 -C 14 , C 14 -C 20 , C 14 -C 18 , C 14 -C 16 , C 16 -C 20 , C 16 -C 18 , or C 18 -C 20 In some embodiments, R is alkenyl. P1 is C 14 -C 18 In some embodiments, R is alkenyl. P1 is C 14 - 16 In some embodiments, R is alkenyl. P1 is C 15 -C 17 In some embodiments, R is alkenyl. P1 is C 16 - 18 In some embodiments, R is alkenyl. P1 are C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C18 , C 19 , C 20 , C 21 , C 22 , C 23 , or C 24 It is alkenyl.

[0386] In some embodiments, R P2 is hydrogen. In some embodiments, R P2 is -CH3.

[0387] In some embodiments, n is, on average, 10 to 200, 10 to 180, 10 to 160, 10 to 140, 10 to 120, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 10 to 20, 20 to 200, 20 to 180, 20 to 160, 20 to 140, 20 to 120, 20 to 100, 20 to 80, 20 to 60, 20 to 40, 40 to 200, 40 to 180, 40 to 160, 40 to 140, 40 to 120, 40 to 100, 40 to 80, 40 to 60, 60 to 200, 60 to 60 0-180, 60-160, 60-140, 60-120, 60-100, 60-80, 80-200, 80-180, 80-160, 80-140, 80-120, 80-100, 100-200, 100-180, 100-160, 100-140, 100-120, 120-200, 120-180, 120-160, 120-140, 140-200, 140-180, 140-160, 160-200, 160-180, or 180-200. In some embodiments, n is, on average, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some embodiments, n is, on average, about 20. In some embodiments, n is, on average, about 40. In some embodiments, n is, on average, about 45. In some embodiments, n is, on average, about 50. In some embodiments, n is, on average, about 68. In some embodiments, n is, on average, about 75. In some embodiments, n is, on average, about 100.

[0388] In some embodiments, the compound of Formula (A) is HO-(CH2CH2O) n -CH2C(O)O-(CH2) 17 CH3 [n is about 45 on average], H3CO-(CH2CH2O) n -CH2C(O)O-(CH2) 17 CH3 [n is about 45 on average], HO-(CH2CH2O) n -CH2C(O)O-(CH2) 15 CH3 [n is about 45 on average], HO-(CH2CH2O) n -CH2C(O)O-(CH2) 13 CH3 [n is about 45 on average], and HO-(CH2CH2O) n -C(O)N(H)-(CH2) 17 CH3 [n is about 45 on average] or a pharmaceutically acceptable salt thereof.

[0389] Alternative Embodiments In alternative embodiments, the compounds described herein may contain one or more isotopic substitutions. For example, hydrogen is 2 H (D or deuterium) or 3 H (T or tritium), and carbon can be, for example, 13 C or 14 C, and oxygen may be, for example, 18 O, and nitrogen may be, for example, 15 In other embodiments, specific isotopes (e.g., 3 H, 13 C. 14 C. 18 O, or 15N) may represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, 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 99%, or at least 99.9% of the total isotopic abundance of the elements occupying a particular site on the compound.

[0390] lipid nanoparticles In certain embodiments, the synthetic RNA virus genomes described herein are encapsulated in lipid nanoparticles (LNPs). In certain embodiments, the LNPs comprise one or more lipids, such as triglycerides (e.g., tristearin), diglycerides (e.g., glycerol bahenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). In some embodiments, the LNPs comprise one or more cationic lipids, one or more structural lipids, and one or more helper lipids. In some embodiments, the LNPs comprise one or more cationic lipids, cholesterol, and one or more neutral lipids.

[0391] In some embodiments, the compounds of the present disclosure are used to form nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise a PEG lipid, an ionizable lipid, a helper lipid, and a structural lipid. In some embodiments, the LNPs described herein are formulated for delivery of a therapeutic agent to a subject in need thereof. In some embodiments, the LNPs described herein are formulated for delivery of a nucleic acid molecule to a subject in need thereof.

[0392] The lipid composition in an LNP significantly affects the therapeutic use and efficacy of a particular LNP. For example, LNP formulations such as SS-OC / cholesterol / DSPC / PEG2k-DPG typically exhibit increased clearance rates upon repeated intravenous (IV) administration in, for example, mice, non-human primates (NHPs), and / or humans, and exhibit significantly shorter in vivo circulation times after a second dose than after a first dose. Reduced circulation times can adversely affect the delivery efficiency of LNPs, presumably by reducing LNP exposure to targets. Thus, while such formulations may be useful for delivering drugs that do not require multiple administrations, their use for delivering drugs that require subsequent administrations may be limited by this reduced circulation time.

[0393] There remains a need for LNP formulations that exhibit tunable circulation and exposure to target cells, for example, sustained circulation and consistent exposure, upon repeated administration in vivo. The present disclosure provides such LNP formulations by incorporating ionizable lipids and / or PEG-lipids of the present disclosure into the lipid formulation of the LNP. The sustained circulation of the LNPs of the present disclosure upon repeated administration results in sustained therapeutic effects of the synthetic RNA viral genome encapsulated therein.

[0394] In some embodiments, without the ionizable lipids and / or PEG lipids of the present disclosure, the body may eliminate the LNPs before the synthetic RNA viral genome is released, resulting in rapid clearance of the LNPs and their components upon repeated dosing, reducing the efficiency of delivery of subsequent doses of the encapsulated synthetic RNA viral genome. In some embodiments, the ionizable lipids and / or PEG lipids of the present disclosure, when incorporated into the LNPs, slow the clearance of the LNPs upon repeated dosing, allowing for the release of the encapsulated synthetic RNA viral genome and sustained therapeutic effect.

[0395] Polyethylene glycol (PEG) lipids In some embodiments, the PEG lipids of the present disclosure comprise a hydrophilic head group and a hydrophobic lipid tail. In some embodiments, the hydrophilic head group is a PEG moiety. In some embodiments, the PEG lipids of the present disclosure comprise a mono-lipid tail. In some embodiments, the PEG lipids of the present disclosure comprise a mono-alkyl lipid tail, a mono-alkenyl lipid tail, a mono-alkynyl lipid tail, or a mono-acyl lipid tail. In some embodiments, the mono-lipid tail comprises an ether group, a carbonyl group, or an ester group. In some embodiments, the PEG lipids of the present disclosure may comprise a polyoxyethylene alkyl ether, a polyoxyethylene alkenyl ether, or a polyoxyethylene alkynyl ether (such molecules are also known as BRIJ™ or Brij molecules). In some embodiments, the PEG lipids of the present disclosure may comprise a polyoxyethylene alkyl ester, a polyoxyethylene alkenyl ester, or a polyoxyethylene alkynyl ester (such molecules are also known as MYRJ™ molecules).

[0396] In some embodiments, the PEG lipid may comprise a di-acyl lipid tail.

[0397] In some embodiments, the PEG lipid is a compound of formula (A) [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein the variables are defined herein.

[0398] In some embodiments, the PEG lipid is a compound of formula (A'): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1’ is a bond, -C(O)-, -[(CH2) 0-3 -C(O)O] 1-3 -, -(CH2)0-3 -C(O)O-(CH2) 1-3 -OC(O)- or -C(O)N(H)-; R P1’ is C5-C 25 Alkyl or C5-C 25 is alkenyl, R P2’ is hydrogen or -CH3.

[0399] In some embodiments, L P1’ is a bond, —C(O)—, —CHC(O)O—, —CHCHC(O)O—, —CHC(O)OCHC(O)O—, —CHC(O)OCHCHOC(O)—, or —C(O)N(H)—. In some embodiments, R P1’ is R P1 In some embodiments, R P2’ is R P2 is.

[0400] In some embodiments, the PEG lipid is a compound of formula (A"): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, L P1” is a bond, -[(CH2) 0-3 -C(O)O] 1-3 -, -(CH2) 0-3 -C(O)O-(CH2) 1-3 -OC(O)- or -C(O)N(H)-; R P1” is C5-C 25 Alkyl or C5-C 25 is alkenyl, R P2” is hydrogen or -CH3.

[0401] In some embodiments, L P1”is a bond, —CHC(O)O—, —CHCHC(O)O—, —CHC(O)OCHC(O)O—, —CHC(O)OCHCHOC(O)—, or —C(O)N(H)—.

[0402] In some embodiments, the PEG lipid is a compound of formula (A"-a), formula (A"-b), formula (A"-c), formula (A"-cd), formula (A"-e), or formula (A"-f): [ka] or a pharmaceutically acceptable salt thereof.

[0403] In some embodiments, R P1” is R P1 In some embodiments, R P2” is R P2 is.

[0404] In some embodiments, the PEG lipid is a compound of formula (A″-f1): [ka] or a pharmaceutically acceptable salt thereof.

[0405] In some embodiments, the PEG lipid is a compound of formula (A″-f2): [ka] or a pharmaceutically acceptable salt thereof.

[0406] In some embodiments, the PEG lipid is a compound of formula (A″-f3): [ka] or a pharmaceutically acceptable salt thereof.

[0407] In some embodiments, the PEG lipid of the present disclosure is a compound of formula (B): [ka] or a pharmaceutically acceptable salt thereof, wherein: n is an integer between 10 and 200, inclusive, R B1 is C5-C 25 Alkyl or C5-C 25 It is alkenyl.

[0408] In some embodiments, R B1 is R P1 is.

[0409] In some embodiments, the PEG lipid is a compound of formula (Ba): [ka] or a pharmaceutically acceptable salt thereof.

[0410] In some embodiments, the PEG lipid is a compound of formula (Bb): [ka] or a pharmaceutically acceptable salt thereof.

[0411] In some embodiments, n is, on average, 10 to 200, 10 to 180, 10 to 160, 10 to 140, 10 to 120, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 10 to 20, 20 to 200, 20 to 180, 20 to 160, 20 to 140, 20 to 120, 20 to 100, 20 to 80, 20 to 60, 20 to 40, 40 to 200, 40 to 180, 40 to 160, 40 to 140, 40 to 120, 40 to 100, 40 to 80, 40 to 60, 60 to 200, 60 to 60 0-180, 60-160, 60-140, 60-120, 60-100, 60-80, 80-200, 80-180, 80-160, 80-140, 80-120, 80-100, 100-200, 100-180, 100-160, 100-140, 100-120, 120-200, 120-180, 120-160, 120-140, 140-200, 140-180, 140-160, 160-200, 160-180, or 180-200. In some embodiments, n is, on average, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some embodiments, n is, on average, about 20. In some embodiments, n is, on average, about 40. In some embodiments, n is, on average, about 45. In some embodiments, n is, on average, about 50. In some embodiments, n is, on average, about 68. In some embodiments, n is, on average, about 75. In some embodiments, n is, on average, about 100.

[0412] In some embodiments, the PEG lipid comprises a PEG moiety having an average molecular weight of about 500 to about 10,000 daltons. In some embodiments, the PEG lipid has a molecular weight of about 500 to about 5,000 daltons, about 500 to about 4,000 daltons, about 500 to about 3,000 daltons, about 500 to about 2,000 daltons, about 500 to about 1,000 daltons, about 500 to about 800 daltons, about 500 to about 600 daltons, about 600 to about 5,000 daltons, about 600 to about 4,000 daltons, about 600 to about 3,000 daltons, about 600 to about 2,000 daltons, about 600 to about 1,000 daltons, about 600 to about 800 daltons, about 800 to about 5,000 daltons, about 800 to about 4,000 daltons, about 800 to about 3,000 daltons, or about The PEG moiety has an average molecular weight of 800 to about 2,000 daltons, about 800 to about 1,000 daltons, about 1,000 to about 5,000 daltons, about 1,000 to about 4,000 daltons, about 1,000 to about 3,000 daltons, about 1,000 to about 2,000 daltons, about 2,000 to about 5,000 daltons, about 2,000 to about 4,000 daltons, about 2,000 to about 3,000 daltons, about 3,000 to about 5,000 daltons, about 3,000 to about 4,000 daltons, about 5,000 to about 10,000 daltons, about 5,000 to about 7,500 daltons, or about 7,500 to about 10,000 daltons. In some embodiments, the PEG portion of the PEG lipid has an average molecular weight of about 1,500 to about 2,500 daltons. In some embodiments, the PEG portion of the PEG lipid has an average molecular weight of about 1,000 to about 5,000 daltons. In some embodiments, the PEG lipid comprises a PEG portion having an average molecular weight of about 500, about 600, about 800, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, or about 10,000 daltons.In some embodiments, the PEG lipid comprises a PEG moiety having an average molecular weight of at least 500, 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, or at least 10,000 daltons. In some embodiments, the PEG-lipid comprises a PEG moiety having an average molecular weight of 500 daltons or less, 1,000 daltons or less, 1,500 daltons or less, 2,000 daltons or less, 2,500 daltons or less, 3,000 daltons or less, 3,500 daltons or less, 4,000 daltons or less, 4,500 daltons or less, 5,000 daltons or less, 6,000 daltons or less, 7,000 daltons or less, 8,000 daltons or less, 9,000 daltons or less, or 10,000 daltons or less. All values ​​are inclusive of all endpoints.

[0413] In some embodiments, the PEG lipid is polyoxyethylene (100) stearyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (20) oleyl ether, polyoxyethylene (20) stearyl ether, or a mixture thereof. In some embodiments, the PEG lipid is polyoxyethylene (100) stearate, polyoxyethylene (50) stearate, polyoxyethylene (40) stearate, polyoxyethylene palmitate, or a mixture thereof.

[0414] In some embodiments of the present disclosure, the PEG-lipid is [ka] (BRIJ (trademark) S100), CAS number 9005-00, rational formula C 18 H 37 (OCH2CH2) nOH, where n is 100. BRIJ™ S100 is also known colloquially as polyoxyethylene (100) stearyl ether. Thus, in some embodiments, the PEG lipid is HO-PEG-CH(CH). 16 It is CH3.

[0415] In some embodiments of the present disclosure, the PEG-lipid is [ka] (BRIJ™ C20), CAS number 9004-95-9, rational formula C 16 H 33 (OCH2CH2) n OH, where n is 20. BRIJ™ C20 is also known as BRIJ™ 58, commonly known as polyethylene glycol hexadecyl ether, polyoxyethylene (20) cetyl ether. Thus, in some embodiments, the PEG lipid is HO-PEG20-CH2(CH2). 14 It is CH3.

[0416] In some embodiments of the present disclosure, the PEG-lipid is [ka] (BRIJ™ O20), CAS number 9004-98-2, rational formula C 18 H 35 (OCH2CH2) n OH, where n is 20. BRIJ™ O20 is also known colloquially as polyoxyethylene (20) oleyl ether. Thus, in some embodiments, the PEG lipid is HO-PEG20-C 18 H 35 is.

[0417] In some embodiments of the present disclosure, the PEG-lipid is [ka] (BRIJ (trademark) S20), CAS number 9005-00-9, rational formula C 18 H 37 (OCH2CH2) n OH, where n is 20. BRIJ™ S20 is also known colloquially as polyethylene glycol octadecyl ether or polyoxyethylene (20) stearyl ether. Thus, in some embodiments, the PEG lipid is HO-PEG20-CH2(CH2). 16 It is CH3.

[0418] In some embodiments of the present disclosure, the PEG-lipid is [ka] (MYRJ(trademark) S100), CAS number 9004-99-3, rational formula C 17 H 35 C(O)(OCH2CH2) n OH, where n is 100. MYRJ™ S100 is also known colloquially as polyoxyethylene (100) stearate. Thus, in some embodiments, the PEG lipid is HO-PEG-CH(CH). 15 It is CH3.

[0419] In some embodiments of the present disclosure, the PEG-lipid is [ka] (MYRJ(trademark) S50), CAS number 9004-99-3, rational formula C 17 H 35 C(O)(OCH2CH2) n OH, where n is 50. MYRJ™ S50 is also known colloquially as polyoxyethylene (50) stearate. Thus, in some embodiments, the PEG lipid is HO-PEG50-CH2(CH2). 15 It is CH3.

[0420] In some embodiments of the present disclosure, the PEG-lipid is [ka] (MYRJ(trademark) S40), CAS number 9004-99-3, rational formula C 17 H 35 C(O)(OCH2CH2) n OH, where n is 40. MYRJ™ S40 is also known colloquially as polyoxyethylene (40) stearate. Thus, in some embodiments, the PEG lipid is HO-PEG40-CH2(CH2). 15 It is CH3.

[0421] In some embodiments of the present disclosure, the PEG-lipid is [ka] is.

[0422] In some embodiments of the present disclosure, the PEG-lipid is [ka] is.

[0423] In some embodiments of the present disclosure, the PEG lipid is PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, PEG-cholesterol (l-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctadecyl]-2-methyl-1,3-dioxaoctadecyl]-2 ... nyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-di The PEG lipid may be stearoyl-sn glycerol, methoxypolyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), or l,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In some embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA. In still other embodiments, the PEG lipid may be PEG2k-C-DMA. In some embodiments, the PEG lipid may be PEG2k-DSA. In other embodiments, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.

[0424] In some embodiments, PEG-lipids with a single lipid tail of the present disclosure (e.g., PEG-lipids of formula (A), (A'), (A"), or (B)) may reduce accelerated blood clearance (ABC) upon administration and / or repeated administration of LNP compositions of the present disclosure. In some embodiments, PEG-lipids with a single lipid tail of the present disclosure may reduce or deplete PEG-specific antibodies (e.g., anti-PEG IgM) produced by a subject's immune system upon administration and / or repeated administration of LNP compositions of the present disclosure.

[0425] In some embodiments, the PEG lipid comprises a poly(ethylene)glycol chain up to 5 kDa in length covalently attached to one or more C6-C20 alkyl-containing lipids. In some embodiments, the PEG lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-amine). In some embodiments, the PEG lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-PEG5K), 1,2-dipalmitoyl-rac-glycerol methoxypolyethylene glycol-2000 (DPG-PEG2K), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-5000 (DSG-PEG5K), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DSG-PEG2K), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-5000 (DMG-PEG5K), and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DMG-PEG2K). In some embodiments, the PEG lipid is DSPE-PEG5K. In some embodiments, the PEG lipid is DPG-PEG2K. In some embodiments, the PEG lipid is DSG-PEG2K. In some embodiments, the PEG lipid is DMG-PEG2K. In some embodiments, the PEG lipid is DSG-PEG5K. In some embodiments, the PEG lipid is DMG-PEG5K.

[0426] In some embodiments, the PEG-lipid is a cleavable PEG-lipid. Examples of cleavable PEG derivatives include those modified with peptide bonds (Kulkarni et al. (2014). Mmp-9-responsive PEG cleavable nanovesicles for efficient delivery of chemotherapeutics to pancreatic cancer. Mol Pharmaceutics 11:2390-9; Lin et al. (2015). Drug / dye-loaded, multifunctional PEG-chitosan-iron oxide nanocomposites for methotraxate synergistically self-targeted cancer therapy and dual model imaging. ACS Appl Mater Interfaces 7:11908-20.), and those modified with disulfide keys (Yan et al. (2014). A method to accelerate the gelation of disulfide-crosslinked hydrogels. Chin J Polym Sci 33:118-27; Wu & Yan (2015). Copper nanopowder catalyzed These include cross-coupling of diaryl disulfides with aryl iodides in PEG-400. Synlett 26:537-42), vinyl ether bond, hydrazone bond (Kelly et al. (2016). Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells. Org Biomol Chem 14:9278-86.), and ester bond (Xu et al. (2008)).Esterase-catalyzed dePEGylation of pH-sensitive vesicles modified with cleavable PEG-lipid derivatives. J Control Release 130:238-45). See also Fang et al. (2017) Cleaveable PEGylation: a strategy for overcoming the "PEG dilemma" in efficient drug delivery. Drug Delivery 24:2,22-32.

[0427] In some embodiments, the PEG lipid is an activated PEG lipid. Exemplary activated PEG lipids include PEG-NH2, PEG-MAL, PEG-NHS, and PEG-ALD. Such functionalized PEG lipids are useful in conjugating targeting moieties to lipid nanoparticles (e.g., by binding antigen-binding molecules, peptides, glycans, etc.) to direct the particles to specific target cells or tissues. In some embodiments, a functionalized moiety (e.g., -NH2, -MAL, -NHS, -ALD) is added to the free end of the PEG moiety of the PEG lipids of the present disclosure (e.g., BRIJ™ or MYRJ™ family PEG lipids).

[0428] cationic lipids In some embodiments, the LNPs provided herein comprise one or more cationic lipids. "Cationic lipid" and "ionizable lipid" are used interchangeably herein.

[0429] Cationic lipids refer to any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DODAC), and N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DODAC). Cationic lipids that are positively charged at sub-physiological pH include, but are not limited to, DODAP, DODMA, and DMDMA. In some embodiments, the cationic lipid is C 18 They contain an alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0430] In some embodiments, the cationic lipid comprises a protonatable tertiary amine head group. Such lipids are referred to herein as ionizable lipids. Ionizable lipids refer to lipid species that comprise an ionizable amine head group and typically comprise a pKa of less than about 7. Thus, in an acidic pH environment, the ionizable amine head group becomes protonated, allowing the ionizable lipid to preferentially interact with negatively charged molecules (e.g., nucleic acids, such as the recombinant polynucleotides described herein), thereby facilitating nanoparticle assembly and encapsulation. Thus, in some embodiments, the ionizable lipid can increase the loading of nucleic acids into lipid nanoparticles. In an environment with a pH greater than about 7 (e.g., a physiological pH of about 7.4), the ionizable lipid comprises a neutral charge. When particles comprising the ionizable lipid are taken up into the low pH environment of an endosome (e.g., pH < 7), the ionizable lipid reprotonates and associates with the anionic endosomal membrane, facilitating the release of the contents encapsulated by the particle. In some embodiments, the LNP comprises an ionizable lipid, for example, a 7.SS cleavable pH-responsive lipid mimic (such as the COATSOME® SS series).

[0431] In some embodiments, the cationic lipid of the LNP is DLinDMA, DLin-KC2-DMA, DLin-MC3-DMA (MC3), COATSOME® SS-LC (formerly SS-18 / 4PE-13), COATSOME® SS-EC (formerly SS-33 / 4PE-15), COATSOME® SS-OC, COATSOME® SS-OP, di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L-319), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), or a mixture thereof.

[0432] In some embodiments, the cationic lipid of the LNP is a compound of formula (I): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein the variables are defined herein.

[0433] In some embodiments, the cationic lipid of the present disclosure is a compound selected from Table 21, or a pharmaceutically acceptable salt thereof.

[0434] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-1): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: R la and R 1b are each independently a C1-C8 aliphatic or —O(C1-C8 aliphatic)—, where the O atom, if present, is attached to the piperidine ring; X a and X b are each independently -C(O)O- * , -OC(O)- * , -C(O)N(R x 1 )- * , -N(R x 1 )C(O)- * , -O(C=O)N(R x 1 )- * , -N(R x 1 )(C=O)O- * , or -O-, where - * are R 2a or R 2b indicates the point of attachment to R x 1 each occurrence is independently selected from hydrogen and optionally substituted C1-C4 alkyl; R 2a and R 2b are each independently a sterol residue, a fat-soluble vitamin residue, or C 13 -C23 It is aliphatic.

[0435] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-2): [ka] or a pharmaceutically acceptable salt or solvate thereof, wherein: R 1a’ and R 1b’ are each independently C1-C8 alkylene or —O(C1-C8 alkylene), where the O atom, if present, is attached to the piperidine ring; Y a’ and Y b’ are each independently -C(O)O- * , -OC(O)- * , -C(O)N(R x 1 )- * , -N(R x 1 )C(O)- * , -O(C=O)N(R x 1 )- * , -N(R x 1 )(C=O)O- * , -N(R x 1 )C(O)N(R x 1 )-, or -O-, where - * is R 2a or R 2b indicates the point of attachment to R x 1 each occurrence is independently selected from hydrogen and optionally substituted C1-C4 alkyl; Z a’ and Z b’ are each independently an optionally substituted arylene-C0-C8 alkylene or an optionally substituted arylene-C0-C8 heteroalkylene, where the alkylene or heteroalkylene group is each independently selected from Y a’ and Y b’ is bound to R 2a’ and R 2b’ are each independently a sterol residue, a fat-soluble vitamin residue, or C 12 -C 22 It is aliphatic.

[0436] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-1a) (COATSOME® SS-OC) or a compound of formula (II-2a) (COATSOME® SS-OP). [ka]

[0437] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-1a) (COATSOME® SS-OC). COATSOME® SS-OC is also known as SS-18 / 4PE-16.

[0438] In some embodiments, the cationic lipid of the LNP is a compound of formula (II-2a) (COATSOME® SS-OP).

[0439] In some embodiments, the cationic lipid of the LNP is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0440] Helper lipids In some embodiments, the LNPs described herein comprise one or more helper lipids. The term "helper lipid" refers to a lipid that can increase the delivery of LNPs to a target, e.g., a cell. Without wishing to be bound by any particular theory, it is contemplated that the helper lipid may enhance the stability and / or fusogenicity of the lipid nanoparticle. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a phospholipid substitute or surrogate. In some embodiments, the helper lipid is an alkylresorcinol.

[0441] In some embodiments, the helper lipid is phosphatidylcholine (PC). In some embodiments, the helper lipid is not phosphatidylcholine (PC). In some embodiments, the helper lipid is a phospholipid or phospholipid substitute. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids or phospholipid substitutes, or a combination thereof. Generally, phospholipids comprise a phosphate head group and one or more fatty acid tails. In some embodiments, the phospholipid can comprise one or more multiple (e.g., double or triple) bonds (i.e., one or more unsaturations). In some embodiments, the helper lipid is non-cationic.

[0442] The phosphate head group can be selected from, for example, the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0443] The fatty acid tail may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0444] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidiglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids such as sphingomyelin.

[0445] In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0446] In some embodiments, the non-cationic helper lipid is a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) substitute.

[0447] In some embodiments, phospholipid can facilitate fusion with membrane.For example, cationic phospholipid can interact with one or more negatively charged phospholipids of membrane (for example, cell membrane or intracellular membrane).Fusion of phospholipid with membrane can allow one or more components of lipid-containing composition to pass through membrane, for example, allow one or more components to be delivered to cell.

[0448] In some embodiments, the phosphate head group can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid tail can be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0449] In some embodiments, the phospholipid is a compound according to formula (III): [ka] where R prepresents a phosphate head group, and R1 and R2 represent fatty acid tails, with or without unsaturation, which may be the same or different. The phosphate head group may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid tail may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-naturally occurring species, including naturally occurring species with modifications and substitutions such as branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids may be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, the alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azides. Such reactions can be useful in functionalizing the lipid bilayer of LNPs to facilitate membrane penetration or cell recognition, or in conjugating LNPs to useful components such as targeting or imaging moieties (e.g., dyes).

[0450] In some embodiments, the LNP comprises one or more non-cationic helper lipids (e.g., neutral lipids). Exemplary neutral helper lipids include (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), (1,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), ceramide, and sphingomyelin In some embodiments, the one or more helper lipids are selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the helper lipid of the LNP comprises, consists essentially of, or consists of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the LNP comprises DSPC. In some embodiments, the LNP comprises DOPC. In some embodiments, the LNP comprises DLPE. In some embodiments, the LNP comprises DOPE.

[0451] In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (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 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3(cis)PC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6(cis)PC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (PE(18:2 / 18:2), 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine (PE The glycerol-containing ester may be selected from the non-limiting group consisting of 18:3 (9Z, 12Z, 15Z), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE), 18:3 (9Z, 12Z, 15Z), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6 (cis)PE), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0452] In some embodiments, the helper lipid is distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylcholine (DPPG), palmitoyloleoyl ...ethanolamine (POPE), palmitoyloleoylphosphatidylcholine (PPP), palmitoyloleoylphosphatidylethanolamine (PPP), palmitoyloleoylphosphatidylcholine (DPPG), palmitoyloleoylphosphatidylethanolamine (PPP), palmitoyloleoylphos Dioleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethylphosphatidylethanolamine, 18-1-trans PE, l-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diercoylphosphatidylcholine (DEPC), palmitoyloleiophosphatidyl phatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine.

[0453] In some embodiments, the helper lipid of the present disclosure is DSPC.

[0454] In some embodiments, the LNPs comprise DSPC. In some embodiments, the LNPs comprise DOPE. In some embodiments, the LNPs comprise DMPE. In some embodiments, the LNPs comprise both DSPC and DOPE.

[0455] In some embodiments, the helper lipid is selected from the group consisting of DSPC, DMPE, and DOPC or a combination thereof.

[0456] In some embodiments of the present disclosure, the helper lipid is [ka] is.

[0457] In some embodiments, the phospholipid of the present disclosure comprises a modified tail. In some embodiments, the phospholipid is DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine) or its analog, comprising a modified tail. As described herein, "modified tail" can be a tail that comprises a shorter or longer aliphatic chain, a branched aliphatic chain, a substituted aliphatic chain, an aliphatic chain in which one or more methylenes are replaced with a cyclic group or a heteroatom group, or any combination thereof.

[0458] In some embodiments, the helper lipids of the present disclosure are alternative lipids that are not phospholipids.

[0459] In some embodiments, the phospholipids useful in the present disclosure comprise modified tails. In some embodiments, the phospholipids useful in the present disclosure are DSPC or its analogs, which comprise modified tails. As described herein, "modified tails" can be tails that comprise shorter or longer aliphatic chains, branched aliphatic chains, substituted aliphatic chains, aliphatic chains in which one or more methylenes are replaced with cyclic or heteroatom groups, or any combination thereof.

[0460] In some embodiments, phospholipids useful in the present disclosure comprise a modified phosphocholine moiety in which the alkyl chain connecting the quaternary amine to the phosphoryl group is not ethylene (eg, n is not 2).

[0461] In some embodiments, the LNPs of the present disclosure comprise oleic acid or an oleic acid analog as a helper lipid. In some embodiments, the oleic acid analog comprises a modified oleic acid tail, a modified carboxylic acid moiety, or both. In some embodiments, the oleic acid analog is a compound in which the carboxylic acid moiety of oleic acid is replaced with a different group.

[0462] In some embodiments, the LNPs of the present disclosure include a different zwitterionic group instead of a phospholipid as the helper lipid.

[0463] In some embodiments, the helper lipid of the present disclosure is a naturally occurring membrane lipid. In some embodiments, the helper lipid of the present disclosure is 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexadecanoyl-sn-glycero-3-phosphocholine (cycloPC), or a combination thereof. In some embodiments, the LNP of the present disclosure comprises a combination of helper lipids. In some embodiments, the combination of helper lipids does not include DSPC. In some embodiments, the combination of helper lipids includes DSPC. In some embodiments, LNPs comprising one or more naturally occurring membrane lipids (e.g., DGTS) exhibit improved hepatic transfection / delivery of target molecules encapsulated in the LNPs compared to LNPs comprising DSPC as the only helper lipid.

[0464] In some embodiments, the helper lipid of the present disclosure is 5-heptadecylresorcinol or a derivative thereof.

[0465] structured lipids In some embodiments, the LNP of the present disclosure comprises one or more structured lipids. The incorporation of structured lipids into lipid nanoparticles can help reduce the aggregation of other lipids in the particle. The structured lipid can be, but is not limited to, a sterol or a lipid containing a sterol moiety.

[0466] In some embodiments, the structural lipid of the LNP is a sterol (e.g., a plant sterol or an animal sterol). In some embodiments, the sterol is cholesterol, or an analog or derivative thereof. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, alone or in combination, including analogs, salts, or esters thereof.

[0467] In some embodiments, the structural lipid of the LNP is cholesterol, a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0468] In some embodiments, the structural lipid of the LNP is a plant sterol, hi some embodiments, the plant sterol is sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, beta-sitosterol, stigmastanol, beta-sitostanol, ergosterol, lupeol, cycloartenol, A5-avenacerol, A7-avenacerol, or A7-stigmasterol, alone or in combination, including analogs, salts, or esters thereof.

[0469] In some embodiments, an LNP comprises one or more plant sterols. In some embodiments, the plant sterol component of an LNP is a single plant sterol. In some embodiments, the plant sterol component of an LNP of the present disclosure is a mixture of different plant sterols (e.g., 2, 3, 4, 5, or 6 different plant sterols). In some embodiments, the plant sterol component of an LNP of the present disclosure is a blend of one or more plant sterols and one or more animal sterols, for example, a blend of a plant sterol (e.g., a sitosterol, such as beta-sitosterol) and cholesterol.

[0470] In some embodiments of the present disclosure, the structural lipid of the LNP is cholesterol. [ka]

[0471] In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is DOTAP. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is DLin-MC3-DMA (MC3). In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is COATSOME® SS-EC. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is COATSOME® SS-LC. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is COATSOME® SS-OC. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is COATSOME® SS-OP. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the cationic lipid is L-319. In some embodiments, the LNPs further comprise a structured lipid. In some embodiments, the structural lipid is cholesterol.

[0472] In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the one or more helper lipids comprise DLPE. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the one or more helper lipids comprise DSPC. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the one or more helper lipids comprise DOPE. In some embodiments, the LNPs comprise a cationic lipid and one or more helper lipids, wherein the one or more helper lipids comprise DOPC. In some embodiments, the LNPs further comprise a structured lipid. In some embodiments, the structured lipid is cholesterol.

[0473] In some embodiments, the LNPs comprise a cationic lipid, a helper lipid, and a structural lipid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the cationic lipid is DOTAP and the helper lipid is DLPE. In some embodiments, the cationic lipid is MC3 and the helper lipid is DSPC. In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid is DSPC. In some embodiments, the LNPs comprise a cationic lipid, a structural lipid, and at least two helper lipids, wherein the cationic lipid is DOTAP and the at least two helper lipids comprise DLPE and DSPE. In some embodiments, the LNPs comprise a cationic lipid, a structural lipid, and at least two helper lipids, wherein the cationic lipid is MC3 and the at least two helper lipids comprise DSPC and DMG. In some embodiments, the at least two helper lipids comprise DOPE and DSPE. In some embodiments, the at least two helper lipids comprise DSPC and DMG. In some embodiments, the structural lipid is cholesterol. In some embodiments, the LNPs comprise DOTAP, cholesterol, and DLPE. In some embodiments, the LNPs comprise MC3, cholesterol, and DSPC. In some embodiments, the LNPs comprise DOTAP, cholesterol, and DOPE. In some embodiments, the LNPs comprise DOTAP, cholesterol, DLPE, and DSPE. In some embodiments, the LNPs comprise MC3, cholesterol, DSPC, and DMG. In some embodiments, the LNPs comprise DOTAP, cholesterol, DLPE, and DSPE-PEG. In some embodiments, the LNPs comprise MC3, cholesterol, DSPC, and DMG-PEG. In some embodiments, the LNPs comprise DOTAP, cholesterol, DOPE, and DSPE. In some embodiments, the LNPs comprise DOTAP, cholesterol, DOPE, and DSPE-PEG. In some embodiments, the LNPs comprise SS-OC, DSPC, cholesterol, and DPG-PEG (e.g., DPG-PEG2K).In some embodiments, the LNP comprises SS-OC, DSPC, cholesterol, and a PEG lipid of formula (I) (e.g., BRIJ™ S100).

[0474] Lipid molar ratio in LNP composition In some embodiments, LNPs of the present disclosure comprise 40 mol% to 70 mol% cationic lipids, up to 50 mol% helper lipids, 10 mol% to 50 mol% structural lipids, and 0.001 mol% to 5 mol% PEG lipids (inclusive). In some embodiments, the total mol% of cationic lipids, helper lipids, structural lipids, and PEG lipids is 100%.

[0475] In some embodiments, the mol% of cationic lipid in the LNP is 40-70 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 44-54 mol%, 45-60 mol%, 45-55 mol%, 45-50 mol%, 50-60 mol%, 49-64 mol%, 50-55 mol%, or 55-60 mol%. In some embodiments, the mol% of cationic lipid in the LNP is 44-54 mol%. In some embodiments, the mol% of cationic lipid in the LNP is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%. In some embodiments, the mol% of cationic lipid in the LNP is about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 mol%. All values ​​include all endpoints.

[0476] In some embodiments, the mol% of structured lipids in the LNP is 10-60 mol%, 10-30 mol%, 15-35 mol%, 20-40 mol%, 20-45 mol%, 25-33 mol%, 24-32 mol%, 25-45 mol%, 30-50 mol%, 35-43 mol%, 35-55 mol%, or 40-60 mol%. In some embodiments, the mol% of structured lipids in the LNP is 20-45 mol%. In some embodiments, the mol% of structured lipids in the LNP is 24-32 mol%. In some embodiments, the mol% of structured lipids in the LNP is 25-33 mol%. In some embodiments, the mol% of structured lipids in the LNP is 22-28 mol%. In some embodiments, the mol% of structured lipids in the LNP is 35-45 mol%. In some embodiments, the mol% of structured lipids in the LNP is 35-43 mol%. In some embodiments, the mol% of structural lipids in the LNP is 10-60 mol%, hi some embodiments, the mol% of structural lipids in the LNP is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%. In some embodiments, the mol% of the structural lipid in the LNP is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 mol%. In some embodiments, the structural lipid is cholesterol. All values ​​include all endpoints.

[0477] In some embodiments, the mol% of helper lipid in the LNP is 1-50 mol%. In some embodiments, the mol% of helper lipid in the LNP is up to 29 mol%. In some embodiments, the mol% of helper lipid in the LNP is 1-10 mol%, 5-9 mol%, 5-15 mol%, 8-14 mol%, 18-22%, 19-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 20-30 mol%, 25-35 mol%, 30-40 mol%, or 35-50 mol%. In some embodiments, the mol% of helper lipid in the LNP is 10-25 mol%. In some embodiments, the mol% of helper lipid in the LNP is 5-9 mol%. In some embodiments, the mol% of helper lipid in the LNP is 8-14 mol%. In some embodiments, the mol% of helper lipid in the LNP is 18-22 mol%. In some embodiments, the mol% of helper lipid in the LNP is 19-25 mol%, hi some embodiments, the mol% of helper lipid in the LNP is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mol%. In some embodiments, the mol% of the helper lipid in the LNP is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 mol%. In some embodiments, the helper lipid is DSPC. All values ​​include all endpoints.

[0478] In some embodiments, the mol% of PEG-lipid in the LNP is greater than 0 mol% and up to 5 mol% of the total lipid present in the LNP. In some embodiments, the mol% of PEG-lipid is 0.1 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, or 2.0 mol% of the total lipid present in the LNP. mol%, 1.8mol%, 1.9mol%, 2.0mol%, 2.1mol%, 2.2mol%, 2.3mol%, 2.4mol%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8 mol%, 2.9mol%, 3.0mol%, 3.1mol%, 3.2mol%, 3.3mol%, 3.4mol%, 3.5mol%, 4.0mol%, 4.5mol%, or 5mol%. In some embodiments, the mol% of PEG lipid is about 0.1 mol%, about 0.2 mol%, about 0.25 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol% of the total lipid present in the LNP. ol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 4.0 mol%, about 4.5 mol%, or about 5 mol%.In some embodiments, the mol% of PEG lipids is at least 0.1 mol%, at least 0.2 mol%, at least 0.25 mol%, at least 0.3 mol%, at least 0.4 mol%, at least 0.5 mol%, at least 0.6 mol%, at least 0.7 mol%, at least 0.8 mol%, at least 0.9 mol%, at least 1.0 mol%, at least 1.1 mol%, at least 1.2 mol%, at least 1.3 mol%, at least 1.4 mol%, at least 1.5 mol%, at least 1.6 mol%, at least 1.7 mol%, or less of the total lipids present in the LNP. at least 1.8 mol%, at least 1.9 mol%, at least 2.0 mol%, at least 2.1 mol%, at least 2.2 mol%, at least 2.3 mol%, at least 2.4 mol%, at least 2.5 mol%, at least 2.6 mol%, at least 2.7 mol%, at least 2.8 mol%, at least 2.9 mol%, at least 3.0 mol%, at least 3.1 mol%, at least 3.2 mol%, at least 3.3 mol%, at least 3.4 mol%, at least 3.5 mol%, at least 4.0 mol%, at least 4.5 mol%, or at least 5 mol%. In some embodiments, the mol% of PEG lipid is up to 0.1 mol%, up to 0.2 mol%, up to 0.25 mol%, up to 0.3 mol%, up to 0.4 mol%, up to 0.5 mol%, up to 0.6 mol%, up to 0.7 mol%, up to 0.8 mol%, up to 0.9 mol%, up to 1.0 mol%, up to 1.1 mol%, up to 1.2 mol%, up to 1.3 mol%, up to 1.4 mol%, up to 1.5 mol%, up to 1.6 mol%, up to 1.7 mol %, maximum 1.8mol%, maximum 1.9mol%, maximum 2.0mol%, maximum 2.1mol%, maximum 2.2mol%, maximum 2.3mol%, maximum 2.4mol%, maximum 2.5mol%, maximum 2.6mol%, maximum 2.7mol%, maximum 2.8m ol%, up to 2.9 mol%, up to 3.0 mol%, up to 3.1 mol%, up to 3.2 mol%, up to 3.3 mol%, up to 3.4 mol%, up to 3.5 mol%, up to 4.0 mol%, up to 4.5 mol%, or up to 5 mol%.In some embodiments, the mol% of PEG lipids is 0.1-4 mol% of the total lipids present in the LNP. In some embodiments, the mol% of PEG lipids is 0.1-2 mol% of the total lipids present in the LNP. In some embodiments, the mol% of PEG lipids is 0.2-0.8 mol%, 0.4-0.6 mol%, 0.7-1.3 mol%, 1.2-1.8 mol%, or 1-3.5 mol% of the total lipids present in the LNP. In some embodiments, the mol% of PEG lipids is 0.1-0.7 mol%, 0.2-0.8 mol%, 0.3-0.9 mol%, 0.4-0.8 mol%, 0.4-0.6 mol%, 0.4-1 mol%, 0.5-1.1 mol%, 0.6-1.2 mol%, 0.7-1.3 mol%, 0.8-1.4 mol%, or 0.9-1.5 mol% of the total lipids present in the LNP. , 1-3.5 mol%, 1-1.6 mol%, 1.1-1.7 mol%, 1.2-1.8 mol%, 1.3-1.9 mol%, 1.4-2 mol%, 1.5-2.1 mol%, 1.6-2.2 mol%, 1.7-2.3 mol%, 1.8-2.4 mol%, 1.9-2.5 mol%, 2-2.6 mol%, 2.4-3.8 mol%, or 2.6-3.4 mol%. All values ​​are inclusive.

[0479] In some embodiments, LNPs of the present disclosure comprise 44-60 mol% cationic lipid, 19-25 mol% helper lipid, 25-33 mol% structural lipid, and 0.2-0.8 mol% PEG lipid (inclusive). In some embodiments, LNPs of the present disclosure comprise 44-54 mol% cationic lipid, 19-25 mol% helper lipid, 24-32 mol% structural lipid, and 1.2-1.8 mol% PEG lipid (inclusive). In some embodiments, LNPs of the present disclosure comprise 44-54 mol% cationic lipid, 8-14 mol% helper lipid, 35-43 mol% structural lipid, and 1.2-1.8 mol% PEG lipid (inclusive). In some embodiments, LNPs of the present disclosure comprise 45-55 mol% cationic lipid, 5-9 mol% helper lipid, 36-44 mol% structural lipid, and 2.5-3.5 mol% PEG lipid (endpoints included).

[0480] In some embodiments, the LNPs of this disclosure comprise one or more cationic lipids of this disclosure, one or more helper lipids of this disclosure, one or more structural lipids of this disclosure, and one or more PEG lipids of this disclosure, in mol% of total lipids (or mol% ranges of total lipids) in the LNP according to Table 6 below. In some embodime...

Claims

1. A lipid nanoparticle (LNP), a. a synthetic RNA viral genome encoding an oncolytic virus; and b. A lipid composition comprising a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid, the PEG lipid being a compound of formula (A″): 【Chemistry 108】 or a pharma- ceutically acceptable salt thereof, n is an integer from 10 to 200, inclusive; L Ｐ１” is a bond, -[(CH ２ ) ０－３ -C(O)O] １－３ -, -(CH ２ ) ０－３ -C(O)O-(CH ２ ) １－３ -OC(O)-, or -C(O)N(H)-, R Ｐ１” is C ５ -C ２５ Alkyl or C ５ -C ２５ alkenyl, R Ｐ２” is hydrogen or -CH ３ and the LNPs have a molar ratio of PEG lipids of about 0.001% to about 5%; The lipid nanoparticle (LNP).

2. L Ｐ１” The LNP of claim 1 , wherein is a bond.

3. R Ｐ２” The LNP of claim 1 or 2, wherein is hydrogen.

4. The PEG lipid may be a compound of formula (A"-f1), (A"-f2), or (A"-f3): 【Chemistry 109】 or a pharma- ceutically acceptable salt thereof.

5. A lipid nanoparticle (LNP), a. a synthetic RNA viral genome encoding an oncolytic virus; and b. A cationic lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is Compound of formula (B): 【Chemistry 111】 or a pharma- ceutically acceptable salt thereof, n is an integer from 10 to 200, inclusive; R Ｂ１ is C ５ -C ２５ Alkyl or C ５ -C ２５ alkenyl, the LNPs have a molar ratio of PEG lipids of about 0.001% to about 5%; The lipid nanoparticle (LNP).

6. The LNP of any one of claims 1 to 5, wherein the LNP induces a reduced immune response in vivo compared to a control LNP lacking the PEG lipid of Formula (A") or Formula (B), and optionally, the PEG lipid in the control LNP is PEG2K-DPG or PEG2K-DMG.

7. The LNP of claim 6 , wherein the immune response is accelerated blood clearance (ABC) of the LNP and / or an anti-PEG IgM response.

8. The LNP of any one of claims 1 to 7, wherein the cationic lipid is COATSOME (registered trademark) SS-OC, the structural lipid is cholesterol, the helper lipid is DSPC, and the PEG lipid is BRIJ (trademark) S100.

9. The LNP described in any one of claims 1 to 8, wherein the oncolytic virus is a Coxsackievirus or a Seneca Valley virus (SVV).

10. The LNP of claim 9, wherein the oncolytic virus is a Coxsackievirus, the synthetic RNA viral genome comprises a polynucleotide sequence having at least 95% or 100% sequence identity to SEQ ID NO:5, and the LNP comprises COATSOME® SS-OC (SS-OC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and BRIJ™ S100, wherein the ratio of SS-OC:DSPC:Chol:BRIJ™ S100 (as a percentage of total lipid content) is A:B:C:D, where A+B+C+D=100%, with A=40%-60%, B=10%-25%, C=20%-30%, and D=0.01%-3%.

11. The LNP of claim 9, wherein the oncolytic virus is the Seneca Valley Virus (SVV), the synthetic RNA viral genome comprises a polynucleotide sequence having at least 95% or 100% sequence identity to SEQ ID NO:68, and the LNP comprises COATSOME® SS-OC (SS-OC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and BRIJ™ S100, wherein the ratio of SS-OC:DSPC:Chol:BRIJ™ S100 (as a percentage of total lipid content) is A:B:C:D, where A+B+C+D=100%, with A=40%-60%, B=10%-25%, C=20%-30%, and D=0.01%-3%.

12. A lipid nanoparticle (LNP) comprising a synthetic RNA viral genome encoding an oncolytic Coxsackievirus, wherein the Coxsackievirus is a CVA21 strain selected from the group consisting of EF and KY strains.

13. 13. The LNP of claim 12, wherein the Coxsackievirus is a CVA21-KY strain, and the CVA21-KY strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:

5.

14. 13. The LNP of claim 12, wherein the Coxsackievirus is a CVA21-EF strain, and the CVA21-EF strain comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:

9.

15. The LNP according to any one of claims 12 to 14, comprising a cationic lipid, a helper lipid, a structural lipid, and a PEG lipid.

16. The cationic lipid is a compound of formula (I): 【Chemistry 101】 or a pharma- ceutically acceptable salt or solvate thereof, wherein A is -N(CH ２ R Ｎ１ ) (CH ２ R Ｎ２ ), or a 4- to 7-membered heterocyclyl ring containing at least one N, said 4- to 7-membered heterocyclyl ring being selected from 0 to 6 R ３ and optionally substituted with Each X is independently -O-, -N(R １ )- or -N(R ２ ) - and R １ is an optionally substituted C １ -C ３１ Selected from the group consisting of aliphatic and steroidal; R ２ is an optionally substituted C １ -C ３１ Selected from the group consisting of aliphatic and steroidal; R ３ is an optionally substituted C １ -C ６ It is aliphatic, R Ｎ１ and R Ｎ２ are each independently hydrogen, hydroxy-C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, or C ３ -C ７ is cycloalkyl, L １ is an optionally substituted C １ -C ２０ An alkylene chain and a divalent optionally substituted C ２ -C ２０ alkenylene chains, L ２ is an optionally substituted C １ -C ２０ An alkylene chain and a divalent optionally substituted C ２ -C ２０ alkenylene chains, L ３ is a bond, optionally substituted C １ -C ６ Alkylene chain or divalent optionally substituted C ３ -C ７ Although it is a cycloalkylene, However, A is -N(CH ３ ) (CH ３ ) and when X is O, L ３ is C １ -C ６ provided that it is not an alkylene chain. The LNP of claim 15.

17. The PEG lipid is a compound of formula (A″): 【Chemistry 104】 or a pharma- ceutically acceptable salt thereof, n is an integer from 10 to 200, inclusive; L Ｐ１” is a bond, -[(CH ２ ) ０－３ -C(O)O] １－３ -, -(CH ２ ) ０－３ -C(O)O-(CH ２ ) １－３ -OC(O)-, or -C(O)N(H)-, R Ｐ１” is C ５ -C ２５ Alkyl or C ５ -C ２５ alkenyl, R Ｐ２” is hydrogen or -CH ３ and the LNPs have a molar ratio of PEG lipids of about 0.001% to about 5%; The LNP of claim 15 or 16.

18. L Ｐ１” The LNP of claim 17, wherein is a bond.

19. R Ｐ２” The LNP of claim 17 or 18, wherein is hydrogen.

20. The PEG lipid is a compound of formula (B): 【Chemistry 105】 or a pharma- ceutically acceptable salt thereof, n is an integer from 10 to 200, inclusive; R Ｂ１ is C ５ -C ２５ Alkyl or C ５ -C ２５ alkenyl, the LNPs have a molar ratio of PEG lipids of about 0.001% to about 5%; The LNP of claim 15 or 16.

21. The cationic lipid comprises COATSOME® SS-OC, the helper lipid comprises DSPC, the structural lipid comprises cholesterol (Chol), and the PEG lipid comprises a compound of formula (A″): 【Chemistry 106】 or a pharma- ceutically acceptable salt thereof, n is an integer from 10 to 200, inclusive; L Ｐ１” is a bond, R Ｐ１” is C ５ -C ２５ Alkyl or C ５ -C ２５ alkenyl, R Ｐ２” is hydrogen, the LNPs have a molar ratio of PEG lipids of about 0.001% to about 5%; The LNP of claim 15.

22. The LNP of any one of claims 15 to 19 and 21, wherein the PEG-lipid is selected from the group consisting of BRIJ™ S100, BRIJ™ S20, BRIJ™ O20, and BRIJ™ C20.

23. The LNP of claim 22, wherein the PEG lipid is BRIJ™ S100.

24. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to any one of claims 1 to 23.

25. 25. The pharmaceutical composition of claim 24, wherein the plurality of LNPs have an average diameter of about 50 nm to about 120 nm.

26. The pharmaceutical composition of any one of claims 24 to 25, wherein the composition has a longer duration of in vivo therapeutic effect than that of a composition lacking the PEG lipid of formula (A") and / or the ionizable lipid of formula (I).

27. 27. The pharmaceutical composition of claim 26, wherein the composition is formulated for multiple administration.

28. 28. An in vitro method of killing a cancerous cell, comprising exposing said cancerous cell to the LNP of any one of claims 1-23, or the pharmaceutical composition of any one of claims 24-27, under conditions sufficient to intracellularly deliver said particle to said cancerous cell, wherein said replication-competent virus produced by an encapsulated polynucleotide results in killing of said cancerous cell.

29. A composition comprising the LNP described in any one of claims 1 to 23, or a pharmaceutical composition described in any one of claims 24 to 27, for treating cancer in a subject suffering from cancer.

30. 30. The composition or pharmaceutical composition of claim 29, wherein the composition is administered repeatedly to the subject.

31. The composition or pharmaceutical composition of any of claims 29 to 30, wherein the cancer is lung cancer, breast cancer, colon cancer, or pancreatic cancer, and the synthetic RNA virus genome comprises a polynucleotide sequence derived from the KY strain.

32. The composition or pharmaceutical composition of any of claims 29 to 30, wherein the cancer is bladder cancer, renal cell carcinoma, ovarian cancer, gastric cancer or liver cancer, and the synthetic RNA virus genome comprises a polynucleotide sequence derived from the EF strain.

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