Lipid nanoparticles comprising Venezuela equine encephalitis (VEE) replicons and uses thereof

Abstract

The present disclosure relates to lipid nanoparticles (LNPs) comprising a VEE replicon, wherein the LNPs can be used to selectively reduce and / or avoid expression of a payload within the liver. The present disclosure also relates to the use of such LNPs for the treatment of various diseases and disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 335,092, filed on April 26, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically via EFS-WEB

[0004] The contents of the sequence listing submitted electronically (name: 4597_016PC01_SequenceListing_ST26.XML; size: 410,263 bytes; creation date: April 24, 2023) and filed with the application are incorporated herein by reference in their entirety. Background Art

[0005] Treatment based on nucleic acid (e.g., mRNA vaccine) is becoming an increasingly important method for treating various diseases. When administered, the encoded protein can be produced to induce an immune response (e.g., tumor antigens induce anti-tumor immune response) or provide an important function to maintain a healthy state. However, the current delivery platform for delivering nucleic acid molecules is invalid or associated with undesirable side effects, thereby limiting the full potential of nucleic acid-based therapy. For example, in the case of mRNA, lipid nanoparticles (LNP) are generally used to encapsulate mRNA. And, in order to improve LNP delivery, many efforts are focused on identifying novel lipids or specific lipid compositions that can affect intracellular delivery and / or mRNA expression, such as in various types of mammalian tissues, organs and / or cells (e.g., mammalian hepatocytes). However, these existing methods are costly, time-consuming and unpredictable. In addition, when administered in vivo, LNP is also associated with hepatotoxicity. Therefore, there is a need for improved LNP in the art, which can provide safer and more effective means for delivering nucleic acid to a subject. Summary of the invention

[0006] Provided herein is a method for reducing or avoiding expression of a heterologous protein in the liver of a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the heterologous protein. In some aspects, reducing expression comprises (i) reducing the amount of the heterologous protein expressed in the liver, (ii) shortening the duration of expression of the heterologous protein in the liver, or (iii) both (i) and (ii), compared to a reference subject (e.g., a subject receiving the corresponding lipid nanoparticle but wherein the replicon is not a VEE replicon).

[0007] In some aspects, after administration, the amount of heterologous protein expressed in the liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference subject. In some aspects, after administration, the liver does not express the heterologous protein.

[0008] In some aspects, after administration, the duration of expression of the heterologous protein in the liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference subject.

[0009] In some aspects, after administration, the heterologous protein is expressed in the non-liver tissue of the subject, wherein the non-liver tissue is selected from spleen, lung, tumor or a combination thereof. In some aspects, after administration, the amount of the heterologous protein expressed in the non-liver tissue is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% or more compared to the amount observed in the non-liver tissue of the reference subject. In some aspects, the duration of heterologous protein expression in the non-liver tissue is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% or more compared to the duration observed in the non-liver tissue of the reference subject.

[0010] Also provided herein is a method for selectively expressing a heterologous protein in a non-liver tissue of a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the heterologous protein.

[0011] In some aspects, after administration, the amount of heterologous protein expressed in the non-hepatic tissue is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold the corresponding amount observed in the liver of the subject. In some aspects, after administration, the duration of expression of the heterologous protein in the non-hepatic tissue is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or longer than the corresponding duration observed in the liver of the subject. In some aspects, the non-hepatic tissue includes the spleen, lung, or both.

[0012] Disclosed herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the heterologous protein, and wherein upon administration, the heterologous protein is preferentially expressed in a non-liver tissue of the subject.

[0013] In some aspects, after administration, the amount of heterologous protein expressed in the non-hepatic tissue is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, or more, the corresponding amount observed in the liver of the subject. In some aspects, after administration, the duration of expression of the heterologous protein in the non-hepatic tissue is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or longer than the corresponding duration observed in the liver of the subject. In some aspects, the non-hepatic tissue includes the spleen, lung, or both.

[0014] In some aspects, the diseases or conditions treatable by the present disclosure include cancer, inflammatory conditions, monogenic conditions, neurological conditions, psychiatric conditions, or combinations thereof. In some aspects, cancer includes melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver tumors, stomach cancer, head and neck cancer, or combinations thereof.

[0015] The present disclosure further provides a method of increasing tolerability of a lipid nanoparticle-based therapy in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein.

[0016] In some aspects, the tolerability of the lipid nanoparticle-based therapy is increased in a subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold or more compared to the corresponding tolerability observed in a reference subject (e.g., a subject receiving the corresponding lipid nanoparticle but wherein the replicon is not a VEE replicon).

[0017] In some aspects, the tolerance increase of the therapy based on lipid nanoparticles is associated with reduced hepatotoxicity. In some aspects, after administration, compared with the corresponding hepatotoxicity observed in the reference subject, the subject shows reduced hepatotoxicity. In some aspects, compared with the hepatotoxicity of the reference subject, the hepatotoxicity in the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%.

[0018] In some aspects, following administration, the subject exhibits reduced expression of the heterologous protein in the liver as compared to corresponding expression observed in the liver of a reference subject.

[0019] In some aspects, after administration, the amount of the heterologous protein expressed in the liver of the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the amount observed in the liver of a reference subject. In some aspects, after administration, the duration of expression of the heterologous protein in the liver of the subject is shortened by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the duration observed in the liver of a reference subject. In some aspects, the amount of heterologous protein expressed in the non-hepatic tissue of the subject is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% or more compared to the amount observed in the non-hepatic tissue of a reference subject. In some aspects, the duration of expression of the heterologous protein in the non-hepatic tissue of the subject is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% or more compared to the amount observed in the non-hepatic tissue of a reference subject.

[0020] Provided herein is a method of producing a lipid nanoparticle-based therapy associated with reduced liver toxicity, the method comprising combining (i) lipid nanoparticles comprising one or more types of lipids and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein.

[0021] In any of the methods provided herein, in some aspects, the VEE replicon has a nucleotide sequence as shown in SEQ ID NO: 187. In some aspects, the heterologous protein comprises a cytokine, an antibody or antigen-binding fragment thereof, a chimeric antigen receptor, or a combination thereof. In some aspects, the cytokine comprises an interleukin (IL)-12 protein.

[0022] In some aspects, the nucleic acid sequence of the VEE replicon comprises: (i) encoding an IL-12 beta subunit and conjugated to SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. The sequence shown in NO:75 has a nucleotide sequence of at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity;(ii) Encoding IL-12 alpha subunit and associated with SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124 or SEQ ID The sequence shown in NO:125 has a nucleotide sequence that is at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical; or (iii) both of (i) and (ii). ;

[0023] In some aspects, the cytokine does not comprise an IL-12 protein.

[0024] In some aspects, one or more types of lipids include ionizable lipids, cationic lipids, lipids, phospholipids, sterols or their combinations. In some aspects, one or more types of lipids include TT3, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, C14-PEG2000 or their combinations. In some aspects, one or more types of lipids are TT3. In some aspects, C14-PEG2000 includes 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DMPE-PEG2000) or both. In some aspects, C14-PEG2000 is embedded in lipid nanoparticles. In some aspects, C14-PEG2000 is added after the separated polynucleotides have been encapsulated in lipid nanoparticles.

[0025] In some aspects, the lipid nanoparticle has a diameter of about 30-500nm. In some aspects, the lipid nanoparticle has a diameter of about 50-400nm. In some aspects, the lipid nanoparticle has a diameter of about 70-300nm. In some aspects, the lipid nanoparticle has a diameter of about 100-200nm. In some aspects, the lipid nanoparticle has a diameter of about 100-175nm. In some aspects, the lipid nanoparticle has a diameter of about 100-160nm.

[0026] In some aspects, the one or more types of lipids have a mass ratio to the VEE replicon of about 1:2 to about 15: 1. In some aspects, the one or more types of lipids have a mass ratio to the VEE replicon of 1:2, 1:1.5, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:1.

[0027] In some aspects, the VEE replicon comprises a 5' cap.

[0028] In some aspects, the VEE replicon further comprises a regulatory element. In some aspects, the regulatory element is selected from the group consisting of: at least one translation enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or its seed, a 3' tailing region connecting nucleosides, an AU-rich element (ARE), a post-transcriptional control regulator, a 5'UTR, a 3'UTR, and a combination thereof. In some aspects, the 3' tailing region connecting nucleosides comprises a poly-A tail, a polyA-G quadruplex, or a stem-loop sequence.

[0029] In some aspects, the VEE replicon comprises at least one modified nucleoside. In some aspects, at least one modified nucleoside is selected from the group consisting of: 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine , 8-oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrole-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine and combinations thereof.

[0030] In some aspects, the one or more types of lipids have a mass ratio of about 10:1 to the VEE replicon.

[0031] In some aspects, the lipid nanoparticles are administered to a subject via intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, transdermal, sublingual, submucosal, transdermal, or transmucosal administration.

[0032] In any method provided herein, in some aspects, the method further comprises administering at least one additional therapeutic agent to the subject. In some aspects, at least one additional therapeutic agent includes chemotherapeutics, targeted anticancer therapies, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, co-stimulatory molecule activators, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof. In some aspects, immune checkpoint inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-CTLA-4 antibodies, anti-GITR antibodies, anti-TIM3 antibodies, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A comparison of luminescence expression in mouse liver after intravenous administration of one of the following is provided: (1) RNA without any VEE replicon (RNA-no VEE), (2) RNA with VEE replicon (RNA-VEE), and (3) PBS. For comparison, luminescence expression in kidney, spleen and lung is also provided.

[0034] Figure 2A and 2BA comparison of td-Tomato protein fluorescence expression in mouse liver after a single intravenous administration of LNP1 or LNP2 is provided. Both LNP1 and LNP2 are loaded with mRNA that has a VEE replicon and encodes a Cre recombinase fused to a nuclear localization signal (Cre-NLS). Control animals received PBS. Figure 2A In , fluorescence expression is shown as percentage of radiant efficiency (normalized to control). Figure 2B In the figure, fluorescence expression is shown as the mean radiant efficiency. In both figures, each symbol represents one measured organ, and the error bars represent the standard error of the mean. The difference in fluorescence intensity between the LNP group and the control group was statistically analyzed using a two-tailed unpaired Student's t-test (N=3).

[0035] Figure 3A and 3B A comparison of td-Tomato protein fluorescence expression in mouse liver after a single intravenous administration of LNP3, LNP4, LNP5 or LNP6 is provided. Each of LNP3-LNP6 is loaded with mRNA that does not have any VEE replicon (RNA-without VEE) and encodes a Cre recombinase fused to a nuclear localization signal (Cre-NLS). Control animals receive PBS. Figure 3A In , fluorescence expression is shown as the mean percentage of radiant efficiency (normalized to control). Figure 3B In the figure, fluorescence expression is shown as mean radiant efficiency.

[0036] Figure 4A and 4B A comparison of td-Tomato protein fluorescence expression in mouse liver after a single intravenous administration of LNP7 or LNP3 is provided. Both LNP3 and LNP7 are loaded with mRNA that does not have any VEE replicon (RNA-no VEE) and encodes a Cre recombinase fused to a nuclear localization signal (Cre-NLS). Control animals receive PBS administration. Figure 4A In , fluorescence expression is shown as percentage of radiant efficiency (normalized to control). Figure 4B In the figure, fluorescence expression is shown as mean radiant efficiency.

[0037] Figure 5A and 5B A comparison of firefly luciferase expression in mouse livers following a single administration of LNP8, LNP9, or LNP2, each loaded with mRNA encoding firefly luciferase having a VEE replicon. LNP8 was administered intravenously (IV) or intratumorally (IT) to the animals. LNP9 and LNP2 were administered intravenously to the animals. Control animals received PBS. Figure 5ALuciferase expression is shown as percent radiance (normalized to control). Figure 5B Luciferase expression is shown as mean radiance. In both figures, each symbol represents one measured organ and the error bars represent the standard error of the mean.

[0038] Fig. 6A and 6B A comparison of firefly luciferase expression in mouse liver following a single intravenous administration of LNP8, LNP9 or LNP2, all loaded with mRNA that does not have any VEE replicon (RNA-VEE-free) and encodes firefly luciferase, is provided. Control animals received administration of PBS. Fig. 6A Luciferase expression is shown as percent radiance (normalized to control). Figure 6B In the Figures, luciferase expression is shown as mean radiance. In both figures, each symbol represents one measured organ, and the error bars represent the standard error of the mean. The difference in bioluminescence intensity between the LNP group and the control group was statistically analyzed using a two-tailed unpaired Student's t-test (N=5).

[0039] Fig. 7A and 7B Provided is a comparison of firefly luciferase expression in mouse liver after a single administration of (a) LNP8 loaded with mRNA without any VEE replicon (RNA-no VEE) and encoding firefly luciferase or (b) LNP8 loaded with mRNA with VEE replicon and encoding firefly luciferase. Fig. 7A Luciferase expression is shown as percent radiance (normalized to control). Figure 7B Luciferase expression is shown as mean radiance. In both figures, each symbol represents one measured organ, and the error bars represent the standard error of the mean. The difference in bioluminescence intensity between the LNP group and the control group was statistically analyzed using a two-tailed unpaired Student's t-test. DETAILED DESCRIPTION

[0040] The present disclosure relates to lipid nanoparticles (LNPs) and the use of such LNPs to selectively deliver a payload (e.g., a heterologous protein) in a subject. More specifically, the LNPs of the present disclosure comprise (i) one or more types of lipids, and (ii) a replicon ("VEE replicon") derived from Venezuelan equine encephalitis (VEE) virus, wherein the VEE replicon comprises a nucleic acid sequence encoding a payload. As demonstrated herein, in some aspects, when administered to a subject, the LNPs described herein can selectively avoid and / or prevent the expression of the encoded payload in the subject's liver. Therefore, in some aspects, compared to other delivery platforms available in the art, the LNPs of the present disclosure are associated with reduced hepatotoxicity. Additional aspects of the present disclosure are provided throughout the application.

[0041] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific compositions or method steps described, and therefore such compositions or method steps may of course vary. It will be apparent to those skilled in the art upon reading the present disclosure that each individual aspect described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any additional several aspects without departing from the scope or spirit of the present disclosure. Any enumerated method can be performed in the order of the events enumerated or in any other order that is logically possible.

[0042] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0043] I. Definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions) shall prevail. Unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0045] Throughout this disclosure, the term "a" or "an" entity refers to one or more of the entity; for example, "a polynucleotide" should be understood to mean one or more polynucleotides. Therefore, the terms "a" or ("a kind"), "one (kind) or more (kind)" and "at least one (kind)" are used interchangeably herein.

[0046] In addition, when used herein, "and / or" is considered to be a specific disclosure that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0047] It should be understood that wherever an aspect is described herein with the term "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0048] The term "about" is used herein to mean approximately, roughly, about, or around. When the term is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, unless otherwise indicated, the term "about" is used herein to modify a numerical value by a variation of 10% above and below (up or down) a value.

[0049] The term "at least" before a number or a series of numbers should be understood to include the numbers adjacent to the term "at least", as well as all subsequent numbers or integers that can be logically included, as can be clearly seen from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20 or 21 nucleotides have the specified properties. When at least appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the series or range. "At least" is also not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, 5.18%, without considering the number of significant figures).

[0050] As used herein, the term "derived from" refers to a component separated from a specific molecule or organism or prepared using a specific molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from a specific molecule or organism. For example, a nucleic acid sequence derived from a second nucleic acid sequence may include a nucleotide sequence identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of nucleotides or polypeptides, the source species may be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. The mutagenesis used to obtain nucleotides or polypeptides may be intentionally directed or intentionally random, or a mixture of the two. Mutagenesis of nucleotides or polypeptides to produce different nucleotides or polypeptides derived from the first nucleotide or polypeptide may be a random event (e.g., caused by polymerase distortion) and the source nucleotides or polypeptides may be identified by appropriate screening methods (e.g., as described herein). In some aspects, a nucleotide or amino acid sequence derived from a second nucleotide or amino acid sequence has at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about , at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity, wherein the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

[0051] "Nucleic acid," "nucleic acid molecule," "nucleotide sequence," "nucleic acid sequence," "polynucleotide," and grammatical variations thereof are used interchangeably and refer to a phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any of their phosphate analogs, such as phosphorothioates and thioesters, in single-stranded form or double-stranded helical form. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly a DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary form. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the normal convention of giving the sequence in the 5' to 3' direction only along the non-transcribed DNA strand (i.e., the strand with a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has been manipulated by molecular biology. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semisynthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein. As described herein, the polynucleotides of the present disclosure comprise DNA, RNA, or both. In some aspects, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, shRNA, siRNA, miRNA, and mRNA, whether spliced ​​or unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing an orthonucleotide backbone, such as polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that permits base pairing and base stacking, such as found in DNA and RNA.

[0052] As used herein, the term "replicon" refers to a polynucleotide comprising a replication origin that allows the polynucleotide to replicate in a cell of interest. As further described herein, in some aspects, a replicon is a self-replicating mRNA (i.e., capable of directing its own amplification or replication in a target cell) (also referred to herein as "repRNA" or "RNA-VEE"). In order to direct its own amplification, the RNA molecule should encode the enzymes necessary to catalyze RNA amplification (e.g., alphavirus nonstructural proteins nsP1, nsP2, nsP3, nsP4), and also contain cis RNA sequences required for replication, which are recognized and utilized by the encoded enzymes. The alphavirus RNA vector replicon should contain the following ordered elements: a 5' viral or cellular sequence required for nonstructural protein-mediated amplification (also referred to as a 5'CSE, or a 5' cis replication sequence, or a 5' viral sequence required for replication, or a 5' sequence capable of initiating alphavirus transcription); a sequence encoding a biologically active alphavirus nonstructural protein (e.g., nsP1, nsP2, nsP3, nsP4) when expressed; and a 3' viral or cellular sequence required for nonstructural protein-mediated amplification (also referred to as a 3'CSE, or a 3' viral sequence required for replication, or an alphavirus RNA polymerase recognition sequence). The alphavirus RNA vector replicon may contain a means for expressing one or more heterologous sequences, such as, for example, an IRES or a viral (e.g., alphavirus) subgenomic promoter (e.g., a linker region promoter), which in certain aspects may be modified to increase or decrease viral transcription of a subgenomic fragment, or to reduce homology to a defective accessory protein or structural protein expression cassette, as well as one or more heterologous sequences to be expressed. Replicons can also contain additional sequences, for example, one or more heterologous sequences encoding one or more polypeptides (e.g., protein-coding genes or 3' proximal genes) and / or polyadenylic acid tracts. Replicons should not contain sequences encoding all alphavirus structural proteins (capsid, E1, E2). Non-limiting examples of heterologous sequences that can be expressed by replicon vectors are described in, for example, U.S. Patent No. 6,015,686 (incorporated herein in its entirety by reference), and include, for example, antigens, lymphokines, cytokines, etc. As is apparent from the present disclosure, the VEE replicon provided herein is repRNA. Therefore, "VEE replicon", "RNA-VEE" and "repRNA" are used interchangeably in this application. In addition, the terms "mRNA without any VEE replicon", "RNA without any VEE replicon" and "RNA-free VEE" are used interchangeably with the term "modRNA" in this article.

[0053] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," and grammatical variations thereof encompass any agent approved by regulatory agencies of the U.S. federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, and any carrier or diluent that does not cause an undesirable physiological effect to such an extent that administration of the composition to a subject is prohibited and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that are useful in preparing pharmaceutical compositions and that are generally safe, nontoxic, and desirable are included.

[0054] As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, such as, for example, a polynucleotide of the present disclosure mixed or admixed or suspended therein with one or more other chemical components, such as pharmaceutically acceptable carriers and excipients.

[0055] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, for example, encoded by the polynucleotides described herein. The polymer may comprise modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function.

[0056] Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments of the foregoing and other equivalents, variants, and analogs.

[0057] Polypeptides can be single polypeptides or can be multimolecular complexes, such as dimers, trimers or tetramers. They can also comprise single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some aspects, a "peptide" can be less than or equal to about 50 amino acids long, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 amino acids long.

[0058] The term "coding sequence" or sequence "encoding" is used herein to refer to a region of DNA or RNA (transcribed region) that "encodes" a particular protein (e.g., such as IL-12). When placed under the control of a suitable regulatory region (such as a promoter), the coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide in vitro or in vivo. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxyl) terminus. The coding sequence may include, but is not limited to, cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences. The transcription termination sequence may be located 3' of the coding sequence.

[0059] Kozak consensus sequence, Kozak consensus or Kozak sequence, is referred to as a sequence that occurs on eukaryotic mRNA and has a consensus (gcc)gccRccAUGG (SEQ ID NO: 174), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another "G". In some aspects, the polynucleotide comprises a nucleic acid sequence that has at least about 95% or more, such as at least 99% sequence identity to the Kozak consensus sequence. In some aspects, the polynucleotide comprises a Kozak consensus sequence.

[0060] The term "RNA" is used herein to refer to a molecule comprising at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, recombinantly generated RNA (which differs from naturally occurring RNA by adding, deleting, replacing and / or changing one or more nucleotides). The term "mRNA" means "messenger-RNA", and refers to a "transcript" generated by using a DNA template and encoding a peptide or protein. Typically, mRNA comprises a 5'-UTR, a protein coding region and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In the context of the present disclosure, mRNA can be generated by in vitro transcription from a DNA template. In vitro transcription methods are known to technicians. For example, there are various commercially available in vitro transcription kits. In some aspects of the present disclosure, RNA, preferably mRNA, is modified with a 5'-cap structure.

[0061] As used herein, the term "identity" (e.g., sequence identity) refers to the overall monomer conservation between polymer molecules, such as between polynucleotide molecules. The term "identical" without any additional qualifiers, for example, polynucleotide A is identical to polynucleotide B, means that the polynucleotide sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity".

[0062] The calculation of percent identity between two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first polypeptide or polynucleotide sequence and the second polypeptide or polynucleotide sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some aspects, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then aligned, or bases in the case of polynucleotides are compared.

[0063] When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences varies with the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced for optimal alignment of the two sequences and the length of each space. Comparison of sequences between two sequences and determination of percent identity can be accomplished using a mathematical algorithm.

[0064] Suitable software programs that can be used for comparing different sequences (e.g., polynucleotide sequences) are available from various sources. A suitable program for determining percentage sequence identity is bl2seq, which is a part of the BLAST suite of programs available from the US government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are a part of the EMB OSS suite of, for example, Needle, Stretcher, Water or Matcher, bioinformatics programs, and can also be obtained from the European Bioinformatics Institute (EBI) at worldwideweb.ebi.ac.uk / Tools / psa.

[0065] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX or Clustal Omega), MUSCLE, and the like.

[0066] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It should be noted that the percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0067] In some aspects, the percent identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID=100x(Y / Z), wherein Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first sequence and the second sequence (such as alignment by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0068] It will be appreciated by those skilled in the art that the generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence-sequence comparisons driven only by primary sequence data. It should also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., the position of mutations), or phylogenetic data. An appropriate program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which can be obtained at www.tcoffee.org and can alternatively be obtained, for example, from EBI. It should also be appreciated that the final alignment for calculating percentage sequence identity can be verified automatically or manually.

[0069] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably and refer to a desired composition of the present disclosure, such as a prepared state of a polynucleotide of the present disclosure, that has been subjected to one or more purification processes. In some aspects, as used herein, separation or purification is a process of removing, partially removing (e.g., a portion) a composition of the present disclosure from a sample containing contaminants.

[0070] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide (e.g., a therapeutic protein, such as a coronavirus protein). It includes, but is not limited to, transcribing a polynucleotide into a microRNA binding site, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), or any other RNA product. It includes, but is not limited to, transcribing a polynucleotide into a messenger RNA (mRNA) and translating the mRNA into a polypeptide. Expression produces a "gene product". As used herein, a gene product may be, for example, a nucleic acid, such as an RNA produced by transcription of a gene. As used herein, a gene product may be a nucleic acid, an RNA, or a miRNA produced by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein also include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing; or polypeptides with post-translational modifications, such as phosphorylation, methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0071] "Heterologous" with respect to a polypeptide portion or a polynucleotide portion that is part of a larger polypeptide or polynucleotide, respectively, describes a polypeptide or polynucleotide that is derived from a polypeptide or polynucleotide that is different from the remainder of the polypeptide or polynucleotide molecule. The additional heterologous components of the polypeptide or polynucleotide can be derived from the same organism as the remainder of the polypeptide or polynucleotide, respectively, as described herein, or the additional components can be from a different organism. For example, a heterologous polypeptide can be synthetic, or derived from cells of the same or different types of species, different cell types of an individual, or different individuals. As described herein, a protein (or polypeptide) encoded by an ORF of a polynucleotide described herein is heterologous to the polynucleotide.

[0072] The term "half-life" refers to the time period required to eliminate half of the activity, amount or number of a molecule. In the context of the present disclosure, the half-life of an RNA indicates the stability of the RNA. The "emergence" or "progression" of a disease refers to the initial manifestation of the disease and / or the subsequent progression. The emergence of a disease can be detectable and can be assessed using standard clinical techniques as known in the art. However, emergence also refers to progression that may not be detectable. As used herein, emergence or progression refers to the biological process of a symptom. Emergence includes occurrence, recurrence and onset. As used herein, the onset or occurrence of a target disease or condition includes initial onset and / or recurrence.

[0073] II. Methods of the Disclosure

[0074] II.A. Methods for Selective Expression

[0075] The present disclosure generally relates to the delivery of payloads (also referred to herein as "bioactive molecules") to cells using lipid nanoparticles (LNPs). LNPs useful in the present disclosure have been modified or engineered so that the LNPs exhibit tissue-specific tropism. As used herein, "tissue-specific tropism" refers to the ability of LNPs to target specific tissues without targeting other tissues. For example, as demonstrated herein, the LNPs described herein are capable of inducing expression of payloads in non-hepatic tissues (e.g., spleen and lungs), but not inducing expression of payloads in the liver. Without being bound by any one theory, applicants have identified that modification or engineering of LNPs to include a replicon ("VEE replicon") derived from Venezuelan equine encephalitis (VEE) virus allows for selective delivery of payloads such that expression of the payload in the liver is avoided or reduced.

[0076] Thus, in some aspects, provided herein is a method for reducing or avoiding expression of a payload (e.g., a heterologous protein) in the liver of a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the payload.

[0077] In some embodiments, the expression of the payload is completely avoided (i.e., the payload is not expressed in the liver after administration). In some aspects, the expression of the payload in the liver is reduced compared to non-liver tissues (e.g., spleen and lungs) in the subject. For example, in some aspects, after administration, compared to the corresponding expression in non-liver tissues, the expression of the payload in the liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%. In some aspects, compared to the expression of the payload in the liver of a reference subject (e.g., a subject receiving a corresponding lipid nanoparticle that does not include a VEE replicon), the expression of the payload in the liver is reduced. In some aspects, after administration, expression of the payload in the liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the corresponding expression in the liver of a reference subject.

[0078] As used herein, "reduced expression" means (i) a decrease in the total amount of the payload expressed in a tissue (e.g., liver); (ii) a decrease in the time (i.e., duration) that the payload is expressed in a tissue (e.g., liver); or (iii) both (i) and (ii).

[0079] Thus, in some aspects, after administering LNPs described herein (e.g., comprising a VEE replicon encoding a payload), the total amount of payload expressed in the liver is reduced compared to non-liver tissues (e.g., spleen and lungs). In some aspects, the total amount of payload expressed in the liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the corresponding amount in non-liver tissue. In some aspects, the total amount of payload expressed in the liver is reduced compared to the total amount of payload expressed in the liver of a reference subject (e.g., a subject receiving a corresponding lipid nanoparticle that does not comprise a VEE replicon). In some aspects, following administration of an LNP described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload), the total amount of payload expressed in the liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the corresponding amount in the liver of a reference subject.

[0080] As described herein, in some aspects, after administration of the LNPs described herein (e.g., comprising a VEE replicon containing a nucleic acid sequence encoding a payload), the payload is only transiently expressed in the liver, e.g., compared to corresponding expression in non-hepatic tissues and / or compared to corresponding expression in the liver of a reference subject (e.g., a subject receiving a corresponding lipid nanoparticle that does not comprise a VEE replicon). Thus, in some aspects, administration of the LNPs disclosed herein results in a shortened duration of payload expression in the liver compared to non-hepatic tissues (e.g., spleen and lungs). In some aspects, the duration of payload expression in the liver is shortened by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, compared to the corresponding duration in non-hepatic tissues. In some aspects, after administering an LNP as described herein (e.g., comprising a VEE replicon containing a nucleic acid sequence encoding a payload) to a subject in need, the duration of payload expression in the subject's liver is shortened compared to the duration of payload expression in the liver of a reference subject (e.g., a subject receiving a corresponding lipid nanoparticle that does not comprise a VEE replicon). In some aspects, the duration of payload expression in the subject's liver is shortened by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, compared to the corresponding duration in the liver of a reference subject.

[0081] In some aspects, after administration of LNPs described herein (e.g., comprising a VEE replicon containing a nucleic acid sequence encoding a payload), the total amount of (i) expressed payload and the duration of (ii) payload expression in the liver are reduced compared to non-hepatic tissues (e.g., spleen and lung). In some aspects, the total amount of (i) expressed payload and the duration of (ii) payload expression are reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to corresponding values ​​in non-hepatic tissues. In some aspects, after administration of LNPs described herein (e.g., comprising a VEE replicon containing a nucleic acid sequence encoding a payload), the total amount of (i) expressed payload and the duration of (ii) payload expression in the liver are reduced compared to corresponding values ​​in the liver of a reference subject (e.g., a subject receiving a corresponding lipid nanoparticle that does not comprise a VEE replicon). In some aspects, (i) the total amount of payload expressed and (ii) the duration of payload expression are both reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to corresponding values ​​in the liver of a reference subject.

[0082] As will be apparent from this disclosure, in some aspects, the LNPs described herein have minimal effect on expression of the payload in non-lymphoid tissues compared to corresponding expression in a reference subject (e.g., a subject receiving a corresponding lipid nanoparticle that does not comprise a VEE replicon). Thus, in some aspects, after administration, the total amount of payload expressed in the non-hepatic tissue of the subject is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% or more compared to the total amount observed in the non-hepatic tissue of the reference subject. In some aspects, following administration of an LNP described herein (e.g., to a subject receiving a corresponding lipid nanoparticle that does not comprise a VEE replicon), the duration of payload expression in a non-hepatic tissue is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% as compared to the duration observed in a non-hepatic tissue of a reference subject.

[0083] As at least apparent from the above disclosure, some aspects of the present disclosure relate to methods for selectively expressing a payload (e.g., a heterologous protein) in a non-hepatic tissue of a subject in need. In some aspects, such methods include administering a lipid nanoparticle to a subject in need, the lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon ("VEE replicon") derived from a Venezuelan equine encephalitis (VEE) virus, wherein the VEE replicon comprises a nucleic acid sequence encoding a payload, and wherein after administration, compared to the corresponding expression in non-hepatic tissue, the payload expression in the liver is reduced. As further described herein, non-hepatic tissue may include any suitable tissue other than the liver in a subject. Non-limiting examples of such non-hepatic tissues include spleen, lung, or both. As used herein, "selective expression" or "preferential expression" or grammatical variants thereof refer to an increase in expression of (e.g., payload) in a first tissue (e.g., non-hepatic tissue) compared to a second tissue (e.g., liver). In some aspects, the payload is selectively or preferentially expressed in non-liver tissue, wherein the expression in the non-liver tissue (e.g., the total amount of payload and / or the duration of payload expression) increases compared to the corresponding expression in the liver. In some embodiments, the expression of the payload in the non-liver tissue is identical to the corresponding expression in other non-liver tissues of the subject. In some aspects, the expression of the payload in the non-liver tissue increases compared to the corresponding expression in other non-liver tissues of the subject. In some aspects, the expression of the payload in the non-liver tissue decreases compared to other non-liver tissues in the subject, but increases compared to the corresponding expression in the liver.

[0084] As demonstrated herein, in some aspects, LNPs of the present disclosure can be used to induce selective (or preferential) expression of a payload in the spleen compared to the liver. Thus, in some aspects, the total amount of payload expressed in the spleen increases by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, or at least about 100 times or more compared to the corresponding amount observed in the liver. In some aspects, the duration of payload expression in the spleen is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, or at least about 100 fold or more compared to the corresponding duration observed in the liver.

[0085] As also demonstrated herein, in some aspects, LNPs of the present disclosure can be used to induce selective expression of a payload in the lungs compared to the liver. Thus, in some aspects, the total amount of payload expressed in the lungs is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, or at least about 100 fold or more compared to the corresponding amount observed in the liver. In some aspects, the duration of payload expression in the lung is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 75 fold, or at least about 100 fold or more compared to the corresponding duration observed in the liver.

[0086] II.B. Treatment Methods

[0087] As is apparent from the present disclosure, the LNPs described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload) can be used to treat a variety of diseases or conditions. Thus, in some aspects, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from a Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the payload, and wherein upon administration, the payload is preferentially expressed in non-liver tissue of the subject. In some aspects, following administration of the LNP, the amount of payload expressed in non-hepatic tissue is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, or at least about 100-fold or more of the corresponding amount observed in the liver of a subject. In some aspects, after administration of the LNP, the duration of payload expression in non-hepatic tissue is at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, or at least about 100 times or longer than the corresponding duration observed in the liver of a subject.

[0088] The LNPs described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload) can be used to treat any suitable disease or condition known in the art. Non-limiting examples of treatable diseases and conditions include cancer, inflammatory conditions, monogenic conditions, neurological conditions, psychiatric conditions, or combinations thereof. As will be apparent from this disclosure, in some aspects, the LNPs described herein can be used to modulate (e.g., increase or decrease) an immune response. In some aspects, the LNPs described herein can be used as vaccines. In some aspects, a disease or condition includes cancer. Non-limiting examples of cancer include: melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver tumors, gastric cancer, head and neck cancer, or combinations thereof.

[0089] As further described elsewhere in the present disclosure, in some aspects, compared with other delivery platforms available in the art, the LNP described herein allows increased safety while reducing undesirable side effects. For example, for many past LNP-based intratumoral treatment methods, hepatotoxicity has always been a major problem (e.g., due to the accumulation of LNP in the liver). See, for example, Jain et al., Nucleic Acid Ther 28 (5): 285-296 (Oct. 2018), which is incorporated herein by reference in its entirety. Not bound by any one theory, in some aspects, by avoiding or reducing the expression of the payload in the liver, the LNP described herein (e.g., comprising a VEE replicon, the VEE replicon comprising a nucleic acid sequence encoding a payload) is associated with reduced hepatotoxicity. For example, in some aspects, after administering the LNP provided herein, compared with a reference subject (e.g., a subject receiving the corresponding LNP but without a VEE replicon), the hepatotoxicity in the subject is reduced. In some aspects, compared with the hepatotoxicity of a reference subject, the hepatotoxicity in the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%. Hepatotoxicity can be measured using any suitable method available in the art. In some aspects, hepatotoxicity can be assessed by performing a general toxicity assessment (e.g., clinical tolerance and general health) on the subject. Non-limiting criteria that can be used for such assessments include: the activity of the subject, food consumption, body score index, hydration status, and combinations thereof. In some aspects, biological samples (e.g., blood or serum) can be obtained from the subject, and hematology and / or clinical chemistry can be performed. For example, in some aspects, the level of liver transaminases (ALT / AST), total bilirubin, serum albumin, or a combination thereof can be measured. Such measured values ​​can be compared with reference values ​​(e.g., corresponding amounts in healthy subjects) to assess hepatotoxicity. In some aspects, more invasive methods (e.g., microscopic assessment of the liver) can also be used.

[0090] As will be apparent to one skilled in the art, in some aspects, reduced hepatotoxicity can allow for greater tolerability of the administered LNPs. Thus, in some aspects, provided herein is a method of increasing tolerability of an LNP-based therapy in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from a Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a payload (e.g., a heterologous protein). In some aspects, the tolerability of the lipid nanoparticle-based therapy is increased in a subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold or more compared to the corresponding tolerability observed in a reference subject (e.g., a subject receiving the corresponding lipid nanoparticle but wherein the replicon is not a VEE replicon).

[0091] In some aspects of the present disclosure, the polynucleotides, vectors, lipid nanoparticles and / or pharmaceutical compositions described herein (also collectively referred to herein as "compositions") are used to treat a disease or condition. In some aspects, the disease or condition includes cancer. Non-limiting examples of treatable cancers are provided elsewhere in the present disclosure.

[0092] In some aspects, an effective amount of any LNP described herein or a composition comprising such LNP (also collectively referred to herein as "composition") is administered to a subject in need via a suitable route (such as intratumoral administration, intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerebrospinal fluid, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhaled, or topical route). Commercially available nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers can be used for administration. Liquid formulations can be atomized, and lyophilized powders can be atomized after reconstitution. In some aspects, pharmaceutical compositions described herein are atomized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and ground powders. In some aspects, pharmaceutical compositions described herein are formulated for intratumoral injection. In some aspects, pharmaceutical compositions described herein are administered to a subject via a topical route, for example, by injection into a local site, such as a tumor site or an infection site. In some aspects, the subject is a human.

[0093] As will be apparent from the present disclosure, in some respects, compositions as herein described are administered to a subject in effective amount alone or in combination with one or more other activating agents, to confer therapeutic effect. In some respects, compositions are administered to a subject suffering from cancer, and therapeutic effect includes tumor load reduction, cancer cell reduction, increased immunocompetence, or a combination thereof. Any suitable method known in the art (for example, measuring tumor volume and / or T cell activity) can be used to determine whether the compositions (for example, lipid nanoparticles) administered achieves therapeutic effect. As recognized by those skilled in the art, effective amount is different, depending on the specific condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, sex, and body weight, treatment duration, the nature (if any) of simultaneous therapy, specific route of administration, and similar factors within the professional skills knowledge of health practitioners.

[0094] Empirical considerations such as half-life will generally help determine dosage. Frequency of administration can be determined and adjusted during treatment, and is generally but not necessarily based on the treatment and / or inhibition and / or improvement and / or delay of the target disease / disorder. Alternatively, sustained release formulations of compositions described herein (e.g., lipid nanoparticles) may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0095] In some aspects of the disclosure, the treatment is a single injection of a composition disclosed herein. In some aspects, a single injection is administered intratumorally to a subject in need thereof.

[0096] In some aspects of the present disclosure, the dosage of the compositions described herein can be determined empirically in individuals who have been given one or more administrations of the compositions (e.g., lipid nanoparticles described herein). In some aspects, the compositions described herein of individual incremental doses are given. In order to assess the efficacy of the compositions herein, the index of the disease / illness can be tracked. For repeated administration over several days or longer, depending on the condition, in some aspects, continued treatment occurs until the desired symptom suppression or until a treatment level sufficient to alleviate the target disease or illness or its symptoms is reached.

[0097] In some aspects of the present disclosure, the administration frequency is about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, or about once every 10 weeks; or about once a month, about once every 2 months, or about once every 3 months or longer. The dosage regimen (e.g., dosage and / or administration frequency) of the compositions described herein (e.g., lipid nanoparticles) used can change over time.

[0098] In some aspects of the disclosure, the methods comprise administering to a subject in need thereof one or more doses of a composition described herein.

[0099] The appropriate dosage of the composition (e.g., lipid nanoparticles as described herein) will depend on the specific composition (e.g., lipid nanoparticles), the type and severity of the disease / disorder (e.g., cancer), whether the composition (e.g., lipid nanoparticles) is administered for prevention or treatment purposes, previous therapy, the clinical history of the subject and the response to the composition (e.g., lipid nanoparticles) and the judgment of the attending physician. In some aspects, the clinician can administer the composition disclosed herein until the dosage for achieving the desired result is reached. In some aspects, the desired result is a reduction in tumor load, a reduction in cancer cells, or an increase in immune activity. The administration of one or more compositions as described herein can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to skilled practitioners. The administration of the composition as described herein can be substantially continuous over a preselected time period, or a series of interval doses can be used, for example, before, during, or after the occurrence of a target disease or disorder.

[0100] As used herein, alleviating a target disease / illness includes delaying the appearance or progression of a disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a cure. As used herein, "delaying" the appearance of a target disease or illness means postponing, hindering, slowing down, slowing down, stabilizing and / or delaying the progression of a disease. This delay can have different lengths of time, depending on the disease and / or the medical history of the subject being treated. Delaying or alleviating the appearance of a disease or delaying the onset of a disease is a method of reducing the possibility of one or more symptoms of a disease occurring within a given time frame and / or reducing the degree of symptoms within a given time frame compared to not using the method. This comparison is usually based on clinical studies, using many subjects that are sufficient to give statistically significant results.

[0101] In some aspects, the compositions described herein are administered to a subject in need thereof in an amount sufficient to reduce tumor burden or cancer cell growth in vivo by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more. In some aspects, the compositions described herein are administered in an effective amount to increase immune activity by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more.

[0102] In some aspects, administering compositions to a subject can enhance the immunocompetence of the subject, such as T cell activity. In some aspects, compared with the immunocompetence of a reference subject (e.g., a subject before administering the compositions or a corresponding subject without responsiveness to administering the compositions), the immunocompetence is enhanced or increased by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 50 times or more.

[0103] In some respects, the subject is a person suffering from cancer, suspected of suffering from cancer or being in a risk of cancer. In some respects, cancer is selected from the group consisting of: melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer and various types of head and neck cancer, including squamous cell head and neck cancer. In some respects, cancer can be melanoma, lung cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma or Hodgkin lymphoma.

[0104] Subjects with target diseases or conditions can be identified by routine medical examinations (e.g., laboratory tests, organ function tests, CT scans, or ultrasound). Subjects suspected of having target diseases or conditions may show one or more symptoms of the disease or condition. Subjects at risk of a disease or condition can be subjects with one or more risk factors associated with the disease or condition. Subjects at risk of a disease or condition can also be identified by routine medical practice.

[0105] In some aspects, the compositions described herein are co-administered with at least one additional suitable therapeutic agent. In some aspects, at least one additional suitable therapeutic agent includes anticancer agents, antiviral agents, antibacterial agents or other agents for enhancing and / or supplementing the immunostimulatory effects of compositions described herein (e.g., lipid nanoparticles). Other examples of additional therapeutic agents that can be used in combination with compositions described herein include: chemotherapeutics, targeted anticancer therapies, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy or any combination thereof. In some aspects, compositions described herein and at least one additional therapeutic agent are administered to a subject in a sequential manner, that is, each therapeutic agent is administered at different times. In some aspects, compositions described herein and at least one additional therapeutic agent are administered to a subject in a substantially simultaneous manner.

[0106] Those skilled in the art will appreciate that any combination of the compositions described herein and another anticancer agent (e.g., chemotherapeutic agent) can be used in any order to treat cancer. The combinations described herein can be selected based on many factors, including but not limited to the effectiveness of reducing tumor formation or tumor growth, reducing cancer cells, increasing immune activity and / or alleviating at least one symptom associated with cancer, or alleviating the effectiveness of the side effects of another agent in the combination. For example, the combination therapy described herein can reduce any side effects associated with each individual member of the combination, for example, side effects associated with an anticancer agent.

[0107] In some aspects, other anticancer therapeutic agents are chemotherapy, radiotherapy, surgical therapy, immunotherapy or their combination. In some aspects, chemotherapeutic agents are carboplatin (carboplatin), cisplatin (cisplatin), docetaxel (docetaxel), gemcitabine (gemcitabine), albumin-bound paclitaxel (nab-paclitaxel), pemetrexed (pemetrexed), vinorelbine (vinorelbine) or their combination. In some aspects, radiotherapy is ionizing radiation, gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioisotope, radiosensitizer or their combination. In some aspects, surgical therapy is curative surgery (for example, tumor removal surgery), preventive surgery, laparoscopic surgery, laser surgery or their combination. In some aspects, immunotherapy is adoptive cell transfer, therapeutic cancer vaccine or their combination.

[0108] In some aspects, the chemotherapeutic agent is a platinum-containing agent such as Carboplatin, Oxaliplatin, Cisplatin, Nedaplatin, Satraplatin, Lobaplatin, Triplatin, Tetranitrate, Picoplatin, Prolindac, Aroplatin and other derivatives; a topoisomerase I inhibitor such as Camptothecin, Topotecan, Irinotecan, otecan / SN38, rubitecan, Belotecan and other derivatives; topoisomerase II inhibitors such as etoposide (VP-16), daunorubicin, doxorubicin agents (e.g., doxorubicin in liposomes, doxorubicin HCl, doxorubicin analogs or doxorubicin and its salts or analogs), mitoxantrone, aclarubicin, epirubicin, irritin Idarubicin, Amrubicin, Amsacrine, Pirarubicin, Valrubicin, Zorubicin, Teniposide and other derivatives; Antimetabolites, such as the folate family (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin and relatives); Purine antagonists (Thioguanine, Fluorine, and Cyclopentane); Fludarabine, Cladribine, 6-mercaptopurine, Pentostatin, clofarabine and relatives) and pyrimidine antagonists (Cytarabine, Floxuridine, Azacitidine, Tegafur, Carmofur, Capacitabine, Gemcitabine, hydroxyurea, 5-fluorouracil (5FU) and relatives);Alkylating agents such as nitrogen mustards (e.g., Cyclophosphamide, Melphalan, Chlorambucil, mechlorethamine, Ifosfamide, Trofosfamide, Prednimustine, Bendamustine, Uramustine, Estramustine and relatives); Nitrosoureas (e.g., Carmustine, Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin and relatives); Triazenes (e.g., Dacarbazine (Dacarbazine, Altretamine, Temozolomide and relatives); Alkyl sulfonates (e.g., Busulfan, Mannosulfan, Treosulfan and relatives); Procarbazine; Mitobronitol and aziridines (e.g., Carboquone, Triaziquone, ThioTEPA, triethylenemalamine and relatives); Antibiotics such as Hydroxyurea, Anthracyclines (e.g., Doxorubicin, Daunorubicin, Epirubicin and other derivatives); Anthracenediones (e.g., Mitoxantrone and relatives); Streptomyces family (e.g., Bleomycin, Mitomycin C, C), actinomycin, plicamycin); ultraviolet light; and combinations thereof. ;

[0109] In some aspects, other anticancer therapeutic agents are antibodies. Antibodies (preferably monoclonal antibodies) achieve their therapeutic effects for cancer cells through various mechanisms. They can have a direct effect in producing apoptosis or programmed cell death. They can block components of signal transduction pathways, such as growth factor receptors, thereby effectively preventing the proliferation of tumor cells. In cells expressing monoclonal antibodies, they can cause the formation of anti-idiotypic antibodies. Indirect effects include raising cells with cytotoxicity, such as monocytes and macrophages. This type of antibody-mediated cell killing is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind complement, thereby causing direct cytotoxicity, which is referred to as complement-dependent cytotoxicity (CDC). Combining surgical methods with immunotherapy drugs or methods is a successful method, as demonstrated in, for example, Gadri et al. 2009:Synergistic effect of dendritic cell vaccination andanti-CD20 antibody treatment in the therapy of murine lymphoma.JImmunother.32 (4):333-40. The following list provides some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present disclosure: Abagovomab (CA-125), Abciximab (CD41), Adecatumumab (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Altumomab pentetate (CEA), Amatuximab (MORAb-009), Anatumomab (CNS), mafenatox (TAG-72), Apolizumab (HLA-DR), Arcitumomab (CEA), Bavituximab (phosphatidylserine), Bectumomab (CD22), Belimumab (BAFF), Bevacizumab (VEGF-A), Bivatuzumab mertansine (CD44 v6), Blinatumomab (CD19), Brentuximab vedotin (CD30 TNFRSF8), Cantuzumabmertansin)(mucin CanAg), Cantuzumab ravtansine(MUC1), Capromab pendetide(prostate cancer cells), Carlumab(CNT0888), Catumaxomab(Ep CAM)(CD3), Cetuximab(EGFR), Citatuzu mab bogatox(EpCAM), Cixutumumab(IGF-1 receptor), Claudiximab(Claudin), Clivatuzumab tetraxe tan(MUC1), Conatumumab(TRAIL-R2), Dacillinomab(Da cetuzumab (CD40), Dalotuzumab (insulin-like growth factor I receptor), Denosumab (RANKL), Detumomab (B lymphoma cells), Drozitumab (DR5), Ecromeximab (GD3 ganglioside), Edrecolomab (EpCAM), Elotuzumab (SLAMF7), Enavatuzumab (PDL192), Ensit uximab)(NPC-1C), Epratuzumab(CD22), Ertumaxomab(HER2 / neu)(CD3), Etaracizumab(integrin αvβ3), Farletuzumab(folate receptor 1), FBTA05(CD20), Ficlatuzumab(SCH900105), Figitumumab(IGF-1 receptor), Flanvotumab(glycoprotein 75), Fresol imumab(TGF-β), Galiximab(CD80), Ganitu mab(IGF-I), Gemtuzumabozogamicin)(CD33), Gevokizumab(IL-1β), Girentuximab(Carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin(GPNMB), Ibritumomab tiuxetan(CD20), Icrucumab(VE GFR-1), Igovoma(CA-125), Indatuximab ra vtansine(SDC1), Intetumumab(CD51), Inotuzumab ozogamicin)(CD22), Ipilimumab(CD152), Iratumumab(CD30), Labetuzumab(CEA), Lexatumumab(TRAIL-R2), Libivirumab(HBsAg), Lintuzumab(CD33), Lorvotuzumab mertansine(CD56), Lucatumumab(CD40), Lumiliximab(CD23), Mapatumumab(TRAIL-R1), Matuzumab(EGFR), Mepol izumab(IL-5), Milatuzumab(CD74), Mituzumab(Mituzumab) umomab)(GD3 ganglioside), Mogamulizumab(CCR 4), Moxetumomab pasudotox(CD22), Nacolomab tafenatox(C242 antigen), Naptumomab estafe natox(5T4), Narnatumab(RON), Necit umumab(EGFR), Nimotuzumab(EGFR), Nivolumab(IgG4), Ofatumumab(CD20), Olaratumab(OLAratumab)(PDGF-Rα), Onartuzumab(Human scatter factor receptor kinase), Oportuzumab monatox(EpCAM), Oregovomab(CA-125), Oxelumab(OX-40), Panitumumab(EGFR), Patritumab(HER3), Pemtumoma(MUC1), Pertuzumab(HER2 / neu), Pintumomab(Pint umomab (adenocarcinoma antigen), Pritumumab (vimentin), Racotumomab (N-glycolylneuraminic acid), Radretumab (fibronectin extra domain-B), Rafivirumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Sibrot uzumab (FAP), Siltuximab (IL-6), Tabalumab (BAFF), Tacatuzumab tetraxetan (alpha-fetoprotein), Tapitumomab paptox (CD19), tenatumomab (tenasin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimuma b (CTLA-4), tucotuzumabcelmoleukin)(EpCAM), Ublituximab(MS4A1), Urelumab(4-1BB), Volociximab(integrin α5β1), Votumumab(tumor antigen CTAA16.88), Zalutumumab(EGFR), Zanolimumab(CD4).

[0110] In some aspects, other anticancer therapeutic agents are cytokines, chemokines, costimulatory molecules, fusion proteins or combinations thereof. The example of chemokines includes but is not limited to CCR7 and its ligands CCL19 and CCL21, in addition to CCL2, CCL3, CCL5 and CCL16. Other examples are CXCR4, CXCR7 and CXCL12. In addition, costimulatory or regulatory molecules such as, for example, B7 ligands (B7.1 and B7.2) are useful. Other cytokines are also useful, such as, for example, interleukins, especially (e.g., IL-1 to IL17), interferons (e.g., IFNalpha1 to IFNalpha8, IFNalpha10, IFNalpha13, IFNalpha14, IFNalpha16, IFNalpha17, IFNalpha21, IFNbeta1, IFNW, IFNE1, and IFNK), hematopoietic factors, TGFs (e.g., TGF-α, TGF-β, and other members of the TGF family), and finally members of the tumor necrosis factor receptor family and their ligands, as well as other stimulatory molecules, including, but not limited to, 41BB, 41BB-L, CD137, CD137-1, and CD137-2. L, CTLA-4GITR, GITRL, Fas, Fas-L, TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, LT.beta.R, RANK, EDAR1, XEDAR, Fn114, Troy / Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT, and CD95 (Fas / APO-1), glucocorticoid-induced TNFR-related protein, TNF receptor-related apoptosis-mediating protein (TRAMP) and death receptor-6 (DR6). In particular, CD40 / CD40L and OX40 / OX40L become important targets for combined immunotherapy due to their direct effects on T cell survival and proliferation. For review, see Lechner et al. 2011: Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors. Immunotherapy 3(11), 1317-1340.

[0111] In some aspects, other anticancer therapeutic agents are bacterial therapies. Researchers have been using anaerobic bacteria, such as Clostridium novyi, to consume the interior of hypoxic tumors. When these bacteria come into contact with the oxygenated side of the tumor, they should die, which means they are harmless to the rest of the body. Another strategy is to use anaerobic bacteria transformed by enzymes that can convert non-toxic prodrugs into toxic drugs. As the tumor necroses and the bacteria proliferate in the hypoxic zone, the enzyme is only expressed in the tumor. Therefore, the prodrug applied systemically is metabolized to toxic drugs only in the tumor. This has been shown to be effective for the non-pathogenic anaerobic Clostridium sporogenes.

[0112] In some aspects, other anticancer therapeutic agents are kinase inhibitors. The growth and survival of cancer cells are closely related to the disorder of kinase activity. In order to restore normal kinase activity and thus reduce tumor growth, a wide range of inhibitors are used. The targeted kinase group includes receptor tyrosine kinases (e.g., BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-α, PDGFR-β, c-Kit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, ALK), cytoplasmic tyrosine kinases (e.g., c-SRC, c-YES, Abl, JAK-2), serine / threonine kinases (e.g., ATM, Aurora A&B, CDKs, mTOR, PKCi, PLKs, b-Raf, S6K, STK11 / LKB1) and lipid kinases (e.g., PI3K, SK1). Small molecule kinase inhibitors are, for example, PHA-739358, Nilotinib, Dasatinib and PD166326, NSC743411, Lapatinib (GW-572016), Canertinib (CI-1033), Semaxinib (SU5416), Vatalanib (PTK787 / ZK222584), Sunitinib (Sutent) (SU11248), Sorafenib (BAY 43-9006) and Leflunomide (SU101). For more information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

[0113] In some aspects, other anticancer therapeutic agents are toll-like receptors. Members of the Toll-like receptor (TLR) family are important links between innate immunity and adaptive immunity, and the effects of many adjuvants depend on the activation of TLRs. A large number of established anticancer vaccines incorporate TLR ligands to enhance vaccine response. In addition to TLR2, TLR3, TLR4, especially TLR7 and TLR8 have been used for cancer therapy in passive immunotherapy methods. Closely related TLR7 and TLR8 contribute to antitumor response by affecting immune cells, tumor cells and tumor microenvironment, and can be activated by nucleoside analog structures. All TLRs have been used as independent immunotherapeutic agents or cancer vaccine adjuvants, and can be combined synergistically with the formulations and methods disclosed herein. For more information, see van Duin et al. 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27 (1): 49-55.

[0114] In some aspects, other anti-cancer therapeutic agents are angiogenesis inhibitors. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) required for tumor survival. For example, the angiogenesis promoted by tumor cells to meet their growing nutritional and oxygen needs can be blocked by targeting different molecules. Non-limiting examples of angiogenesis mediating molecules or angiogenesis inhibitors that can be combined with the present disclosure are soluble VEGF (VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors neuropilin-1 and neuropilin-2) 1 and NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2, IFN-α, -β and -γ, CXCL10, IL-4, -12 and -18, prothrombin (kringle domain-2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferation protein related Proteins, restin and drugs such as bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-α, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids + heparin, cartilage-derived angiogenesis inhibitors, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin Vβ3 inhibitors, linomide, tasquinimod. For a review, see Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9): 976-81.

[0115] In some aspects, other anticancer therapeutic agents are virus-based vaccines. There are many available or developing virus-based cancer vaccines that can be used in combination therapy methods with the formulations disclosed herein. An advantage of using such viral vectors is their inherent ability to elicit immune responses, in which inflammatory responses occur due to viral infection, thereby generating danger signals required for immune activation. Ideal viral vectors should be safe, and anti-vector immune responses should not be introduced to allow enhanced anti-tumor specific responses. Recombinant viruses (such as vaccine viruses, herpes simplex viruses, adenoviruses, adeno-associated viruses, retroviruses, and fowl pox viruses) have been used in animal tumor models, and based on their encouraging results, human clinical trials have been initiated. Particularly important virus-based vaccines are virus-like particles (VLPs), i.e., small particles containing certain proteins from viral shells. Virus-like particles do not contain any genetic material of the virus and cannot cause infection, but they can be constructed to present tumor antigens on their shells. VLPs can be derived from various viruses, such as, for example, hepatitis B virus or other virus families, including Parvoviridae (e.g., adeno-associated virus), Retroviridae (e.g., HIV), and Flaviviridae (e.g., hepatitis C virus). For a general review, see Sorensen and Thompson 2007: "Virus-based immunotherapy of cancer: what do we know and where are we going?" APMIS 115(11): 1177-93; virus-like particles against cancer are reviewed in the following literature: Buonaguro et al. 2011: Developments in virus-like particle-based vaccines for infectious diseases and cancer. Expert Rev Vaccines 10(11): 1569-83; and Guillén et al. 2010: Virus-like particles as vaccine antigens and adjuvants: application to chronic disease, cancer immunotherapy and infectious disease preventive strategies. Procedia in Vaccinology 2(2), 128-133.

[0116] In some aspects, other anticancer therapeutic agents are peptide-based targeted therapies. Peptides can bind to cell surface receptors or the extracellular matrix affected around tumors. If the nuclide decays near the cell, the radionuclides attached to these peptides (e.g., RGD) will eventually kill cancer cells. Oligomers or polymers of these binding motifs are particularly interesting because this can lead to enhanced tumor specificity and avidity. For non-limiting examples, see Yamada 2011: Peptide-based cancer vaccine therapy for prostate cancer, bladder cancer, and malignant glioma. Nihon Rinsho 69 (9): 1657-61.

[0117] III. Lipid Nanoparticles

[0118] As described herein, LNP of the present disclosure exhibits tissue-specific tropism, so that LNP can allow payload (e.g., heterologous protein) to be selectively delivered to cells (e.g., cells in non-hepatic tissue). In some aspects, delivery can occur in vivo (e.g., by administering LNP as described herein to a subject) or in vitro (e.g., by culturing LNP as described herein with cells in vitro). As used herein, "lipid nanoparticle" (LNP) refers to a vesicle with a continuous lipid bilayer, such as a spherical vesicle. Lipid nanoparticles can be used in methods for delivering drug therapy to a targeted location. Non-limiting examples of LNP include liposomes, biphasic amphiphilic molecules (bolaamphihiles), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), monolayer membrane structures (e.g., archaeal liposomes (archaeosomes) and micelles), lipid nanoparticles (LLN), polymer nanoparticles (PNP), lipid polymer hybrid nanoparticles (LPN) or combinations thereof.

[0119] In some aspects, lipid nanoparticles include one or more types of lipids. As used herein, "lipid" refers to a group of organic compounds, which includes but is not limited to esters of fatty acids, and in some aspects, is characterized in that it is insoluble in water, but soluble in many organic solvents. Therefore, unless otherwise indicated, lipids include natural lipids and lipidoid materials (e.g., lipids). They are generally divided into at least three categories: (1) "simple lipids", which include fats and oils and waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids. Non-limiting examples of lipids include triglycerides (e.g., tristearin), diglycerides (e.g., behenic acid glyceryl), monoglycerides (e.g., monostearin), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol) and waxes (e.g., cetyl palmitate). In some aspects, lipids that can be used in the present disclosure include ionizable lipids, cationic lipids, lipids, phospholipids, sterols or combinations thereof.

[0120] In some aspects, one or more types of lipids in the LNP comprise ionizable lipids.Thus, in some aspects, LNPs useful in the present disclosure comprise (i) ionizable lipids and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload.

[0121] Non-limiting examples of ionizable lipids include: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-((2-hydroxyethyl)(6-oxo-6-(undecyoxy)hexyl)amino)octanoic acid heptadecan-9-yl ester (SM-102), 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid heptadecan-9-yl ester (lipid 5), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3- (Docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), Nl-[2(docosylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraaza-triacontane (KL25), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptahexanoic acid-6, 9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDM A), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), or any combination thereof.

[0122] In some aspects, one or more types of lipids in the LNP include cationic lipids. In some aspects, LNPs useful for the present disclosure include (i) cationic lipids and (ii) VEE replicons, wherein the VEE replicons include nucleic acid sequences encoding payloads. In some aspects, the LNPs (i.e., including cationic lipids and VEE replicons) further include ionizable lipids.

[0123] As used herein, "cationic lipid" refers to any of a number of lipid species having a net positive charge at a selected pH, such as physiological pH. Non-limiting examples of cationic lipids include: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N -Dimethyl-l-trifluoroacetic acid propylamine (DOSPA), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxy-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(l,2-dioleoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), l,2-dilauroyl-s n-glycero-3-ethylphosphocholine (DLePC), l,2-distearoyl-3-trimethylammonium-propane (DSTAP), l,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), l,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), l,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC), l,2-dipalmitoyl-sn -glycero-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC), 1,2-dioleoyl sn-glycero-3-ethylphosphocholine (DOePC), 1,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1EPC), or any combination thereof. Additional examples are provided throughout this disclosure.

[0124] In some aspects, one or more types of lipids in the LNP include lipids. Therefore, in some aspects, the LNP described herein includes: (i) lipids and (ii) VEE replicon, wherein the VEE replicon includes a nucleic acid sequence encoding a payload. In some aspects, the LNP (i.e., including lipids and VEE replicon) further includes ionizable lipids, cationic lipids, or both.

[0125] As used herein, the term "lipid" refers to a molecule having one or more properties of a lipid (e.g., a synthetic cationic lipid). In some aspects, the lipid can have a series of secondary and tertiary amines, which increases the net positive charge of the LNP. Non-limiting examples of lipid materials and / or lipids include: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine 2,5-dione (cKK-E12), tetra(8-methylnonyl) 3,3',3″, 3″′-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecadien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(ethane -2,1-diyl)(9Z,9'Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetra(octadec-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))tetra(azanetriyl))tetra(butane-4,1-diyl)(9Z,9′Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetra(decadien-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))tetra(azanetriyl))tetra(butane-4,1-diyl)(9Z,9′Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetra(decadien-9,12-dienoate) Octa-9,12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tri(3-(docosylamino)propyl)benzene 1,3,5-tricarboxamide (TT3), hexa(oct-3-yl) 9,9′,9″,9″′,9″″,9″′″-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))nonanoic acid hexyl ester (FTT5), PL-1 [published in Nature Communications, 12-7264 (2021), which is hereby incorporated by reference], 98N12-5 [published in Molecular Therapy Vol. 17 No. 5 May 2009, which is hereby incorporated by reference], ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18(A2)) and A12-Iso5-2DC18(A12), or any combination thereof.

[0126] In some aspects, the lipid comprises a sterol. In some aspects, LNPs useful in the present disclosure comprise (i) a sterol and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some aspects, the LNP (i.e., comprising a sterol and a VEE replicon) further comprises an ionizable lipid, a cationic lipid, a lipid, or any combination thereof.

[0127] As used herein, "sterol" refers to cholesterol or cholesterol analogs that can be used to fill lipid membrane packing defects and provide structural integrity. Non-limiting examples of sterols include: cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, alpha-tocopherol and combinations thereof. In some aspects, the sterol is cholesterol.

[0128] In some aspects, the lipid comprises a phospholipid. In some aspects, LNPs useful in the present disclosure comprise (i) a phospholipid and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some aspects, the LNP (i.e., comprising a phospholipid and a VEE replicon) further comprises an ionizable lipid, a cationic lipid, a lipid, a sterol, or any combination thereof.

[0129] Non-limiting examples of phospholipids include: 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-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2 -Henicosanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidyl-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and any combination thereof. In some aspects, the phospholipid is selected from the group consisting of: 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0PC, MSPC), 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0PC), 1-palmitoyl-2-myristoyl- sn-glycero-3-phosphocholine (16:0-14:0PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0PC, PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2PC,PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1PC , SOPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0PC, OPPC ), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0PC, OSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE, POPE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2PE), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (16:0-20:4PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1PE), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:2-PE), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (18:0-20:4-PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0-22:6PE), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), and any combination thereof. ,

[0130] Thus, in some aspects, LNPs useful for the present disclosure comprise (i) one or more natural lipids (e.g., natural cationic lipids) and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some aspects, LNPs useful for the present disclosure comprise (i) one or more synthetic lipids (e.g., lipids) and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some aspects, LNPs useful for the present disclosure comprise (i) both natural lipids (e.g., cationic lipids) and synthetic lipids (e.g., lipids), and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload.

[0131] Examples of lipids that can be used with the LNPs of the present disclosure include, but are not limited to, N1,N3,N5-tris(3-(docosylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); lipofectamine; 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLenDMA); dioctadecyldimethylammonium (DO ammonium bromide (DMRIE); N-(1,2-dimyristyloxypropyl)-N,N-dimethyl-N-hydroxyethyl-N-(1,2-dioctyloxypropyl)-N,N-dimethyl ...

[0132] In some aspects, the lipid is represented by Formula I:

[0133]

[0134] and its salts; wherein each R 1 is independently an unsubstituted alkyl group; each R 2 is independently an unsubstituted alkyl group; each R 3 is independently hydrogen or substituted or unsubstituted alkyl; and each m is independently 3, 4, 5, 6, 7 or 8. In some aspects, each R 1 is independently an unsubstituted alkyl group; each R 2 are independently unsubstituted alkyl; R 3 is hydrogen; and each m is 3. In some aspects, at least one R 1is an unsubstituted C1-24 alkyl. In some aspects, at least one R 1 For unsubstituted C 1-18 In some aspects, at least one R 1 For unsubstituted C 1-12 In some aspects, at least one R 1 For unsubstituted C 6-18 In some aspects, at least one R 1 For unsubstituted C 6-12 In some aspects, at least one R 1 For unsubstituted C 8-12 In some aspects, at least one R 1 For unsubstituted C 10-12 In some aspects, at least one R 1 For unsubstituted C 11 alkyl.

[0135] In some aspects, at least one R 2 For unsubstituted C 1-24 In some aspects, at least one R 2 For unsubstituted C 1-18 In some aspects, at least one R 2 For unsubstituted C 1-12 In some aspects, at least one R 2 For unsubstituted C 6-18 In some aspects, at least one R 2 For unsubstituted C 6-12 In some aspects, at least one R 2 For unsubstituted C 8-12 In some aspects, at least one R 2 For unsubstituted C 10-12 In some aspects, at least one R 2 For unsubstituted C 11 alkyl.

[0136] In some aspects, at least two R 1 For unsubstituted C 1-24 In some aspects, at least two R 1 For unsubstituted C 1-18 In some aspects, at least two R 1 For unsubstituted C 1-12 In some aspects, at least two R 1 For unsubstituted C 6-18 In some aspects, at least two R 1 For unsubstituted C 6-12 In some aspects, at least two R1 For unsubstituted C 8-12 In some aspects, at least two R 1 For unsubstituted C 10-12 In some aspects, at least two R 1 For unsubstituted C 11 alkyl.

[0137] In some aspects, at least two R 2 For unsubstituted C 1-24 In some aspects, at least two R 2 For unsubstituted C 1-18 In some aspects, at least two R 2 For unsubstituted C 1-12 In some aspects, at least two R 2 For unsubstituted C 6-18 In some aspects, at least two R 2 For unsubstituted C 6-12 In some aspects, at least two R 2 For unsubstituted C 8-12 In some aspects, at least two R 2 For unsubstituted C 10-12 In some aspects, at least two R 2 For unsubstituted C 11 alkyl.

[0138] In some respects, R 1 All instances of unsubstituted C 1-24 In some aspects, R 1 All instances of unsubstituted C 1-18 In some aspects, R 1 All instances of unsubstituted C 1-12 In some aspects, R 1 All instances of unsubstituted C 6-18 In some aspects, R 1 All instances of unsubstituted C 6-12 In some aspects, R 1 All instances of unsubstituted C 8-12 In some aspects, R 1 All instances of unsubstituted C 10-12 In some aspects, R 1 All instances of unsubstituted C 11 alkyl.

[0139] In some respects, R 2 All instances of unsubstituted C 1-24In some aspects, R 2 All instances of unsubstituted C 1-18 In some aspects, R 2 All instances of unsubstituted C 1-12 In some aspects, R 2 All instances of unsubstituted C 6-18 In some aspects, R 2 All instances of unsubstituted C 6-12 In some aspects, R 2 All instances of unsubstituted C 8-12 In some aspects, R 2 All instances of unsubstituted C 10-12 In some aspects, R 2 All instances of unsubstituted C 11 alkyl.

[0140] In some aspects, at least one R 3 is hydrogen. In some aspects, at least one R 3 is a substituted or unsubstituted alkyl group. In some aspects, at least one R 3 is substituted or unsubstituted C 1-18 In some aspects, at least one R 3 is substituted or unsubstituted C 1-12 In some aspects, at least one R 3 is substituted or unsubstituted C 1-6 In some aspects, at least one R 3 is substituted or unsubstituted C 1-4 In some aspects, at least one R 3 is substituted or unsubstituted C 2-4 In some aspects, at least one R 3 is a substituted or unsubstituted methyl group.

[0141] In some aspects, at least one R 3 is a substituted alkyl group, wherein the substituted alkyl group is substituted with halogen. 3 is a substituted alkyl group, wherein the substituted alkyl group is substituted with fluorine. 3 is a substituted alkyl group, wherein the substituted alkyl group is substituted by a haloalkyl group.

[0142] In some aspects, at least two R 3 In some aspects, at least two R 3 is a substituted or unsubstituted alkyl group. In some aspects, at least two R 3 is substituted or unsubstituted C1-18 In some aspects, at least two R 3 is substituted or unsubstituted C 1-12 In some aspects, at least two R 3 is substituted or unsubstituted C 1-6 In some aspects, at least two R 3 is substituted or unsubstituted C 1-4 In some aspects, at least two R 3 is substituted or unsubstituted C 2-4 In some aspects, at least two R 3 is a substituted or unsubstituted methyl group.

[0143] In some aspects, at least two R3 are substituted alkyl, wherein the substituted alkyl is substituted by halogen. In some aspects, at least two R3 are substituted alkyl, wherein the substituted alkyl is substituted by fluorine. In some aspects, at least two R3 are substituted alkyl, wherein the substituted alkyl is substituted by haloalkyl.

[0144] In some respects, R 3 All instances of are hydrogen. In some aspects, R 3 All examples of are substituted or unsubstituted alkyl. In some aspects, R 3 All instances of are substituted or unsubstituted C 1-18 In some aspects, R 3 All instances of are substituted or unsubstituted C 1-12 In some aspects, R 3 All instances of are substituted or unsubstituted C 1-6 In some aspects, R 3 All instances of are substituted or unsubstituted C 1-4 In some aspects, R 3 All instances of are substituted or unsubstituted C 2-4 In some aspects, R 3 All examples of are substituted or unsubstituted methyl.

[0145] In some respects, R 3 All examples of are substituted alkyl, wherein the substituted alkyl is substituted with halogen. In some aspects, R 3 All examples of are substituted alkyl, wherein the substituted alkyl is substituted with fluorine. 3 All examples of are substituted alkyl groups, wherein the substituted alkyl groups are substituted with haloalkyl groups.

[0146] In some aspects, at least one m is 3. In some aspects, at least one m is 4. In some aspects, at least one m is 5. In some aspects, at least one m is 6. In some aspects, at least one m is 7. In some aspects, at least one m is 8. In some aspects, at least two m are 3. In some aspects, at least two m are 4. In some aspects, at least two m are 5. In some aspects, at least two m are 6. In some aspects, at least two m are 7. In some aspects, at least two m are 8.

[0147] In some aspects, all instances of m are 3. In some aspects, all instances of m are 4. In some aspects, all instances of m are 5. In some aspects, all instances of m are 6. In some aspects, all instances of m are 7. In some aspects, all instances of m are 8.

[0148] In some aspects of the disclosure, the lipid is TT3, which is represented by the formula:

[0149]

[0150] wherein all instances of m = 3. The compositions, synthesis and uses of Formula I and TT3 are described in WO2016187531A1, which is incorporated herein by reference in its entirety.

[0151] Therefore, in some aspects, LNP described herein comprises (i) TT3 and (ii) VEE replicon, and the VEE replicon comprises a nucleic acid sequence encoding a payload. As used herein, "TT3" can form lipid nanoparticles for delivering various bioactive agents (e.g., payloads described herein) to cells. In addition, the disclosure also demonstrates that unloaded TT3-LNP can induce immunogenic cell death (ICD) in cancer cells in vivo and in vitro. As described herein, immunogenic cell death refers to a form of cell death that can induce an effective immune response by activating dendritic cells (DC) and subsequently activating specific T cell responses. In some aspects, the cells experiencing immunogenic cell death are tumor cells.

[0152] In some aspects, the lipid is DOTAP. Therefore, in some aspects, the LNP of the present disclosure comprises (i) DOTAP and (ii) VEE replicon, and the VEE replicon comprises a nucleic acid sequence encoding a payload. As used herein, "DOTAP" can also form lipid nanoparticles. DOTAP can be used for efficient transfection of DNA including yeast artificial chromosomes (YAC) into eukaryotic cells for transient or stable gene expression, and is also suitable for effectively transferring other negatively charged molecules (such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes and proteins) into research samples of mammalian cells.

[0153] In some aspects of the present disclosure, the lipid is lipofectamine. In some aspects, the LNP described herein comprises (i) lipofectamine and (ii) VEE replicon, and the VEE replicon comprises a nucleic acid sequence encoding a payload. As used herein, "lipofectamine" is a common transfection reagent for molecular and cell biology produced and sold by Invitrogen. It is used to improve the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell culture by lipid transfection. Lipofectamine contains lipid subunits, which can form liposomes or lipid nanoparticles in an aqueous environment, and the liposomes or lipid nanoparticles carry a transfection payload. Since neutral co-lipids mediate the fusion of liposomes with cell membranes, RNA-containing liposomes (positively charged on their surfaces) can fuse with negatively charged plasma membranes of living cells, thereby allowing nucleic acid cargo molecules to pass through the cytoplasm for replication or expression.

[0154] In some aspects, LNP described herein comprises a single type of lipid (e.g., all lipids, e.g., all TT3). In some aspects, LNP of the present disclosure comprises multiple types of lipids. For example, in some aspects, LNP comprises lipids (e.g., TT3) and other lipid components. These generally include other lipid molecules belonging to, but not limited to, phosphatidylcholine (PC) (e.g., 1,s-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sterols (e.g., cholesterol), and polyethylene glycol (PEG)-lipid conjugates (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000 (DSPE-PEG2000) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (C14-PEG2000)). Table 1 shows the formulations of exemplary LNPs, TT3-LNPs, and DOTAP-LNPs.

[0155] Table 1.

[0156]

[0157] In some aspects, LNP comprises C14-PEG2000. Therefore, in some aspects, LNP that can be used for the present disclosure comprises (i) C14-PEG2000 and (ii) VEE replicon, and the VEE replicon comprises the nucleic acid sequence encoding the payload. In some aspects, C14-PEG2000 comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000] (DMPE-PEG2000) or both. In some aspects, C14-PEG2000 (or other lipid components disclosed herein) can be embedded in LNP before encapsulating VEE replicon. In some aspects, C14-PEG2000 (or other lipid components disclosed herein) can be added to LNP after encapsulating VEE replicon. For example, in some aspects, the VEE replicon is encapsulated in LNPs and then C14-PEG2000 (other lipid components disclosed herein) is attached to the LNPs using, for example, micelles.

[0158] In some aspects, LNPs useful for the present disclosure include polymeric materials. Therefore, in some aspects, LNPs provided herein include (i) polymeric materials and (ii) VEE replicons, wherein the VEE replicons include nucleic acid sequences encoding payloads. In some aspects, LNPs include (i) one or more types of lipids, (ii) polymeric materials, and (iii) VEE replicons, wherein the VEE replicons include nucleic acid sequences encoding payloads. In some aspects, one or more types of lipids include ionizable lipids, cationic lipids, lipids, phospholipids, sterols, or combinations thereof.

[0159] In some aspects, the polymer material includes a cationic polymer or a non-cationic polymer, such as those described herein. Non-limiting examples of polymer materials are disclosed in Jiang et al. Reference Module in Materials Science and Materials Engineering. (2021) and Byun et al. BioChip J (2022), both of which are hereby incorporated by reference. Non-limiting examples of polymer materials include: polyethyleneimine (PEI), poly (amidoamine) (PAMAM), poly (β-amino ester) (PBAE), poly (methacrylate 2-N, N-dimethylaminoethyl ester) (PDMAEMA), poly (amino acid) (PAA), chitosan dextran (Raemdonck et al., 2009), cyclodextrin (Singh et al., 2019), cellulose (Kim et al., 2020), hyaluronic acid, polylactic acid (PLA), poly (lactic acid-co-glycolic acid) (PLGA) and polycaprolactone (PCL).

[0160] At least from the above disclosure, it is apparent that in some aspects, LNPs useful in the present disclosure comprise a VEE replicon comprising a nucleic acid sequence encoding a payload, and one or more of the following components: (i) an ionizable lipid; (ii) a cationic lipid; (iii) a lipidoid material (e.g., a lipid); (iv) a phospholipid; (v) a sterol; (vi) a pegylated lipid; and (vii) a polymeric material. In some aspects, LNPs described herein comprise a VEE replicon comprising a nucleic acid sequence encoding a payload, and one or more of the following: (i) an ionizable lipid; (ii) a cationic lipid; (iii) a phospholipid; (iv) a sterol; and (v) a pegylated lipid. In some aspects, LNPs described herein include VEE replicons, comprising nucleic acid sequences encoding payloads, and one or more of: (i) lipidoid materials (e.g., lipids); (ii) phospholipids; (iii) sterols; and (iv) pegylated lipids. In some aspects, LNPs described herein include VEE replicons, comprising nucleic acid sequences encoding payloads, and one or more of: (i) polymeric materials; (ii) phospholipids; (iii) sterols; and (iv) pegylated lipids. In some aspects, LNPs described herein include VEE replicons, comprising nucleic acid sequences encoding payloads, and one or more of: (i) polymeric materials; (ii) lipids; (iii) phospholipids; (iv) sterols; and (v) pegylated lipids.

[0161] The particle size of lipid nanoparticles can affect drug release rate, biodistribution, mucosal adhesion, cell to the uptake of water and the buffer exchange and protein diffusion inside the nanoparticle. In some aspects of the present disclosure, the diameter range of LNP is about 30 to about 500nm. In some aspects of the present disclosure, the diameter range of LNP is about 30 to about 500nm, about 50 to about 400nm, about 70 to about 300nm, about 100 to about 200nm, about 100 to about 175nm or about 100 to about 160nm. In some aspects of the present disclosure, the diameter range of LNP is 100-160nm. In some aspects of the present disclosure, the diameter of LNP can be about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 101nm, about 102nm, about 103nm, about 104nm, about 105nm, about 106nm, about 107nm, about 108nm, about 109nm, about 110nm, about 111nm, about 112nm, about 113nm, about 114nm, about 115nm, about 116nm, about 117nm, about 118nm, about 119nm, about 120nm, about 130nm, about 140nm, about 150nm or about 160nm. In some aspects, lipid nanoparticle has a diameter of about 140nm.

[0162] Zeta potential is a measure of the effective charge on the lipid nanoparticle surface. The size of the zeta potential provides information about particle stability. In some aspects, the zeta potential range of the nanoparticles described herein is about -20 to about 20mv. In some aspects of the present disclosure, the zeta potential range of LNP is about 3 to about 6mv. In some aspects of the present disclosure, the zeta potential of the LNP can be about 3 mv, about 3.1 mv, about 3.2 mv, about 3.3 mv, about 3.4 mv, about 3.5 mv, about 3.6 mv, about 3.7 mv, about 3.8 mv, about 3.9 mv, about 4 mv, about 4.1 mv, about 4.2 mv, about 4.3 mv, about 4.4 mv, about 4.5 mv, about 4.6 mv, about 4.7 mv, about 4.8 mv, about 4.9 mv, about 5 mv, about 5.1 mv, about 5.2 mv, about 5.3 mv, about 5.4 mv, about 5.5 mv, about 5.6 mv, about 5.7 mv, about 5.8 mv, about 5.9 mv or about 6 mv.In some aspects, the zeta potential of the nanoparticles described herein can be about -6.0 mv, about -5.9 mv, about -5.8 mv, about -5.7 mv, about -5.6 mv, about -5.5 mv, about -5.4 mv, about -5.3 mv, about -5.2 mv, about -5.1 mv, about -5.0 mv, about -4.9 mv, about -4.8 mv, about - 4.7mv, about -4.6mv, about -4.5mv, about -4.4mv, about -4.3mv, about -4.2mv, about -4.1mv, about -4.0mv, about -3.9mv, about -3.8mv, about -3.7mv, about -3.6mv, about -3.5mv, about -3.4mv, about -3.3mv, about -3.2mv, about -3 .1mv, about -3.0mv, about -2.9mv, about -2.8mv, about -2.7mv, about -2.6mv, about -2.5mv, about -2.4mv, about -2.3mv, about -2.2mv, about -2.1mv, about -2.0mv, about -1.9mv, about -1.8mv, about -1.7mv, about -1.6mv, about -1 .5mv, about -1.4mv, about -1.3mv, about -1.2mv, about -1.1mv, about -1.0mv, about -0.9mv, about -0.8mv, about -0.7mv, about -0.6mv, about -0.5mv, about -0.4mv, about -0.3mv, about -0.2mv, about -0.1mv, about 0.0mv, 0.1ab out mv, about 0.2mv, about 0.3mv, about 0.4mv, about 0.5mv, about 0.6mv, about 0.7mv, about 0.8mv, about 0.9mv, about 1.0mv, about 1.1mv, about 1.2mv, about 1.3mv, about 1.4mv, about 1.5mv, about 1.6mv, about 1.7mv, about 1.8mv, about 1.9mv, about 2.0mv, about 2.1mv, about 2.2mv, about 2.3mv, about 2.4mv, about 2.5mv, about 2.6mv, about 2.7mv, about 2.8mv, about 2.9mv, about 3.0mv, about 3 .1mv, about 3.2mv, about 3.3mv, about 3.4mv, about 3.5mv, about 3.6mv, about 3.7mv, about 3.8mv, about 3.9mv, about 4.0mv, about 4.1mv, about 4.2mv, about 4.3mv, about 4.4mv, about 4.5mv, about 4.6mv, about 4.7mv, about 4.8mv, about 4.9mv, about 5.0mv, about 5.1mv, about 5.2mv, about 5.3mv, about 5.4mv, about 5.5mv, about 5.6mv, about 5.7mv, about 5.8mv, about 5.9mv or about 6.0mv.

[0163] In some aspects, the disclosure relates to polynucleotides (e.g., VEE replicon mRNA) encapsulated with lipid nanoparticles (LNPs). In some aspects of the disclosure, the mass ratio between the lipids of the LNPs and the polynucleotides (e.g., VEE replicon mRNA) ranges from about 1:2 to about 15:1. In some aspects, the mass ratio between the lipids and the polynucleotides (e.g., VEE replicon mRNA) can be about 1:2, about 1:1.9, about 1:1.8, about 1:1.7, about 1:1.6, about 1:1.5, about 1:1.4, about 1:1.3, about 1:1.2, about 1:1.1, about 1:1, about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1 1, about 14: 1, about 14.5: 1, or about 15: 1. In some aspects of the present disclosure, the mass ratio between the lipid and the polynucleotide (e.g., VEE replicon mRNA) is about 10: 1.

[0164] IV. VEE Replicon

[0165] In addition to one or more types of lipids (e.g., those described above), the LNPs of the present disclosure comprise a VEE replicon comprising a nucleic acid sequence encoding a payload. As further described elsewhere in the present disclosure, Applicants have identified that the interaction between the VEE replicon and one or more lipids (e.g., lipids, e.g., TT3) provides certain improved properties for the LNPs described herein, such as tissue-specific tropism, which allows the LNPs to avoid the liver and / or reduce expression of the payload in the liver, compared to non-hepatic tissues (e.g., spleen and lung).

[0166] VEE virus is a viral pathogen usually carried by mosquitoes, which mainly causes VEE or encephalomyelitis in horse species. However, humans may also be infected with VEE, and people with weak immune systems (if infected with VEE) especially have the risk of serious complications. The virion of VEE is spherical and has a lipid membrane, in which glycoprotein surface proteins are scattered around the outer surface. The genome of VEE is about 11.45kb, does not include 5'-end cap and 3'-end poly (A) bundle, and comprises four kinds of nonstructural proteins (nsP) and five kinds of structural proteins. Nonstructural proteins include nsP1, nsP2, nsP3 and nsP4, and structural region encoding protein C, E3, E2, 6K and E1. In some aspects, self-amplification replicon RNA is the WT replicon RNA derived from VEE. The sequence of wild-type VEE virus replicon RNA is as shown in SEQ ID NO:186 (see Table 4).

[0167] In some aspects, VEE replicons useful in the present disclosure comprise a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the sequence shown in SEQ ID NO: 186 or a fragment thereof. In some aspects, VEE replicons useful in the present disclosure comprise a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the sequence shown in SEQ ID NO: 187. In some aspects, the VEE replicon comprises the nucleic acid sequence shown in SEQ ID NO: 187. In some aspects, the VEE replicon consists of the nucleic acid sequence set forth in SEQ ID NO: 187. In some aspects, the VEE replicon consists essentially of the nucleic acid sequence set forth in SEQ ID NO:187.

[0168] In some aspects, the VEE replicon that can be used for the present disclosure includes any VEE replicon known in the art. In some aspects, the VEE replicon includes one or more mutations. For example, in some aspects, the VEE replicon includes mutations with non-structural proteins nsP2 and nsP3, wherein the mutation promotes subgenomic expression in human cells. In some aspects, compared with the wild-type VEE replicon (SEQ ID NO:186), the VEE replicon includes mutations that allow the higher expression of the encoded payload. In some aspects, the self-amplification replicon RNA includes at least one point mutation at nucleic acid position 3936 and / or 4758 of the WT replicon of SEQ ID NO:186. In some aspects, the self-amplification replicon RNA includes at least one of the following point mutations: the guanine at position 3936 of the WT replicon sequence of SEQID NO:186 becomes cytosine (G3936C) and the adenine at position 4758 becomes guanine (A4758G). The G3936C mutation will result in a glycine change to arginine at amino acid residue 1309 (G1309R). The A4758G mutation will result in a serine change to glycine at amino acid residue 1583 (51583G). Additional disclosure about such VEE replicons is provided, for example, in US20200281994A1, which is incorporated herein by reference in its entirety.

[0169] Without being bound by any one theory, in some aspects, the interaction between the VEE replicon and one or more lipids (e.g., lipids, e.g., TT3) causes the LNP to not target the liver. And, because the LNP is not targeted to the liver, in some aspects, the payload is not expressed in the liver. In some aspects, the LNP described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload) is capable of targeting the liver, but expression of the payload is reduced compared to corresponding expression in non-hepatic tissues.

[0170] In some aspects, VEE replicons can induce activation of toll-like receptors (TLRs). TLR subsets TLR3, TLR7 / 8, and TLR9 participate in antiviral responses by triggering the production of antiviral cytokines such as type I interferons (IFNs). TLR3 responds to double-stranded RNA, which is a replication intermediate for many viruses. TLR7 / 8 recognizes viral single-stranded RNA, while TLR9 recognizes unmethylated CpG motifs within viral DNA. TLRs involved in viral recognition are expressed on endosomal membranes and can be separated based on their need for the adaptor protein MyD88: TLR3 activity is independent of MyD88, while TLRs7 / 8 / 9 is dependent on MyD88. Activation of TLR3 leads to the production of type I interferons (IFNs). Sustained Rip3 activation and necroptosis require type I interferon signaling through the ISGF3 (STAT1 / STAT2 / IRF9) complex. Without being bound by any one theory, in some aspects, induction of type I interferons can reduce and / or inhibit expression of the payload in the liver.

[0171] IV.A. Payload

[0172] As described herein, the VEE replicons useful in the present disclosure include a payload. As used herein, the term "payload" refers to any agent that can act on a target (e.g., a target cell) contacted with the LNP described herein. In some aspects, the term payload can be used interchangeably with the term "biologically active molecule" unless otherwise specified. Non-limiting examples of payloads useful in the present disclosure include: nucleotides (e.g., nucleotides containing a detectable portion or toxin or disrupting transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides (such as enzymes), or RNA molecules with regulatory functions, such as miRNA, dsDNA, lncRNA, siRNA, antisense oligonucleotides, phosphodiamidate morpholino oligonucleotides (PMOs), peptide-conjugated phosphodiamidate morpholino oligonucleotides (PPMOs), or combinations thereof), amino acids (e.g., amino acids containing a detectable portion or toxin or disrupting translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0173] In some aspects, the payload comprises a nucleic acid sequence encoding a protein (e.g., a heterologous protein). As will be apparent to those skilled in the art, any suitable protein may be encoded in the payload. In some aspects, the protein comprises a detectable protein (e.g., a fluorescent protein). Non-limiting examples of such detectable proteins include: luciferase, wt-GFP, green fluorescent protein (e.g., EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, etc.), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, mTagBFP, etc.), cyan fluorescent protein (e.g., ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), etc.), yellow fluorescent protein (e.g., EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, etc.), orange fluorescent protein (e.g., Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine, etc.) or red fluorescent protein (for example, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, etc.).

[0174] In some aspects, the payload comprises a nucleic acid sequence encoding a therapeutic protein. Non-limiting examples of such therapeutic proteins include cytokines. In some aspects, the cytokine comprises an interleukin (IL) -12 molecule. In some aspects, the IL-12 molecule comprises IL-12, an IL-12 subunit (e.g., an IL-12 β subunit or an IL-12 α subunit) or a mutant IL-12 molecule retaining an immunomodulatory function. In some aspects, the cytokine is not IL-12. In some aspects, the cytokine is selected from (i) a common γ chain family of cytokines; (ii) an IL-1 family of cytokines; (iii) hematopoietic cytokines; (iv) interferons (e.g., type I, type II, or type III); (v) a TNF family of cytokines; (vi) a cytokine IL-17 family; (vii) a damage-associated molecular pattern (DAMP); (viii) a tolerogenic cytokine; or (ix) a combination thereof. In some aspects, the cytokines include IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36ra, IL-37, IL-38, IL-3, IL-5, IL-6, IL-11, IL-13, IL-23, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), stem cell factor (SCFA), leukemia inhibitory factor (LIF), and leukemia inhibitory factor (LIF). Factor (SCF), thrombopoietin (TPO), macrophage-colony stimulating factor (M-CSF), erythropoietin (EPO), Flt-3, IFN-α, IFN-β, IFN-γ, IL-19, IL-20, IL-22, IL-24, TNF-α, TNF-β, BAFF, APRIL, lymphotoxin β (TNF-γ), IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-25, TSLP, IL-35, IL-27, TGF-β, or a combination thereof. Additional examples of other proteins that can be encoded are further provided below.

[0175] Where the payload comprises a nucleic acid sequence encoding an IL-12 protein, in some aspects, the IL-12 protein (e.g., encoded by a nucleic acid molecule described herein) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 182.

[0176] In some aspects, the IL-12 molecule comprises an IL-12 alpha and / or IL-12 beta subunit. In some aspects, the IL-12 alpha subunit comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 183.

[0177] In some aspects, the IL-12 β subunit comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 184.

[0178] In some aspects, the VEE replicon described herein comprises a codon-optimized nucleic acid sequence. Thus, in some aspects, the nucleotide sequence encoding the IL-12 protein (e.g., IL-12p35 subunit, IL-12p40 subunit, or heterodimer IL-12p70) disclosed herein is different from the nucleotide sequence of the wild-type nucleotide sequence (e.g., SEQ ID NO: 185).

[0179] In some aspects, the VEE replicon comprises a nucleic acid sequence encoding an IL-12 beta subunit, wherein the nucleic acid sequence is identical to SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. The sequence shown in any of NO:75 has at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity.

[0180] In some aspects, a VEE replicon useful in the present disclosure comprises a nucleic acid sequence encoding an IL-12 β subunit, wherein the nucleic acid sequence (i) has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the sequence shown in SEQ ID NO:51; (ii) has at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the sequence shown in SEQ ID NO:51; NO:52 has at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity; (iii) with SEQ ID 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO:54; (iv) at least 88%, at least 89%, 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% identical to the sequence shown in SEQ ID NO:55; (v) at least 89%, 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% identical to the sequence shown in SEQ ID NO:57; (v) at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence shown in SEQ ID NO:58. NO:55 has at least 88%, at least 89%, 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% sequence identity; (vi) has at least 87%, at least 88%, at least 89%, 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% sequence identity with the sequence shown in SEQ ID NO:56; (vii) has 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% sequence identity with the sequence shown in SEQ ID NO:57;(viii) has 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% sequence identity to the sequence shown in SEQ ID NO:58; (ix) has 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% sequence identity to the sequence shown in SEQ ID NO:59; (x) has 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% sequence identity to the sequence shown in SEQ ID NO:65, 69 or 74; (xi) has 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% sequence identity to the sequence shown in SEQ ID NO: NO:66, 70 or 75 has 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% sequence identity; (xii) has at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:62; (xiii) has at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:63; or (xiv) has at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:64.;

[0181] In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:51. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:52. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:53. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:54. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:55. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:56. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:57. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:58. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:59. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:65. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:66. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:67. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:68. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:69. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:70. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:71. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:72. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:73. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 74. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 75. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 62. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 63. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 64.In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 60. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 61.

[0182] In some aspects, the VEE replicon comprises a nucleic acid sequence encoding an IL-12 p35 subunit (also referred to herein as an IL-12 alpha subunit), wherein the nucleic acid sequence is identical to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 125. The sequences shown in any of NO:125 have at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity.

[0183] In some aspects, a VEE replicon useful in the present disclosure comprises a nucleic acid sequence encoding an IL-12p35 subunit, wherein the nucleic acid sequence (i) has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the sequence shown in SEQ ID NO: 101; (ii) has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the sequence shown in SEQ ID NO: 101; NO:102 has at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity; (iii) with SEQ ID 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO:104; (iv) at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence shown in SEQ ID NO:105; (v) at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to the sequence shown in SEQ ID NO:106; NO:105 has at least 88%, at least 89%, 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% sequence identity; (vi) has at least 88%, at least 89%, 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% sequence identity with the sequence shown in SEQ ID NO:106; (vii) has 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% sequence identity with the sequence shown in SEQ ID NO:107;(viii) has 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% sequence identity to the sequence shown in SEQ ID NO:108; (ix) has 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% sequence identity to the sequence shown in SEQ ID NO:109; (x) has 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% sequence identity to the sequence shown in SEQ ID NO:115, 119 or 124; (xi) has 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% sequence identity to the sequence shown in SEQ ID NO: NO:116, 120 or 125 has 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% sequence identity; (xii) has at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:112; (xiii) has at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:113; or (xiv) has at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO:114.;

[0184] In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 101. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 102. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 103. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 104. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 105. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 106. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 107. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 108. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 109. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 115. In some aspects, the nucleic acid molecule encoding the IL-12 alpha subunit comprises the sequence shown in SEQ ID NO: 116. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 117. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 118. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 119]. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 120. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 121. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 122. In some aspects, the nucleic acid molecule encoding the IL-12 beta subunit comprises the sequence shown in SEQ ID NO: 123. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 124. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 125. In some aspects, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence shown in SEQ ID NO: 112. In some aspects, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence shown in SEQ ID NO: 113. In some aspects, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence shown in SEQ ID NO: 114.In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO: 110. In some aspects, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence shown in SEQ ID NO:111.

[0185] In some aspects, the nucleic acid molecule encoding the IL-12p40 subunit and the nucleic acid molecule encoding the IL-12p35 subunit can be conjugated to each other. For example, in some aspects, the disclosure provides a kind of isolated polynucleotide, which comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid encodes the IL-12p40 subunit and the second nucleic acid encodes the IL-12p35 subunit. In some aspects, the IL-12α subunit and the IL-12β subunit are connected by a joint. In some aspects, the joint comprises at least about 2, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19 or at least about 20 amino acids of amino acid joints. In some aspects, the joint comprises a (GS) joint. In some aspects, the GS linker has the formula (Gly3Ser)n or S(Gly3Ser)n, wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100. In some aspects, the (Gly3Ser)n linker is (Gly3Ser)3 or (Gly3Ser)4.

[0186] In some aspects, the VEE replicon comprises a nucleic acid sequence encoding a chemokine. Non-limiting examples of chemokines include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10. In some aspects, the VEE replicon comprises a nucleic acid sequence encoding a growth factor. As used herein, the term "growth factor" refers to a naturally occurring substance that can perform signal transduction between cells and stimulate cell growth. Although a cytokine can be a growth factor, certain types of cytokines can also have an inhibitory effect on cell growth, thus distinguishing the two terms. Non-limiting examples of growth factors include adrenomedullin (AM), angiogenin (Ang), autotaxin, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin 6 (IL-6), macrophage colony stimulating factor (m-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin A6, ephrin A7, ephrin A8, ephrin A9, ephrin A10, ephrin A111, ephrin A12, ephrin A13, ephrin A14, ephrin A15, ephrin A16, ephrin A17, ephrin A18, ephrin A19, ephrin A20, ephrin A21, ephrin A22, ephrin A23, ephrin A24, ephrin A25, ephrin A26, ephrin A27, ephrin A28, ephrin A29, ephrin A30, ephrin A31, ephrin A32, ephrin A33, ephrin A34, ephrin A35, ephrin A36, ephrin A37, ephrin A40, ephrin A58, ephrin A60, ephrin A19, ephrin A20, ephrin A21, ephrin A22, ephrin A23, ephrin A24, ephrin A25 Ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 3 (FGF3), fibroblast growth factor 4 (FGF4), fibroblast growth factor 5 (FGF5), fibroblast growth factor 6 (FGF6), fibroblast growth factor 7 (FGF7), fibroblast growth factor 8 (FGF8), fibroblast growth factor fibroblast growth factor 1 (FGF11), fibroblast growth factor 12 (FGF12), fibroblast growth factor 13 (FGF13), fibroblast growth factor 14 (FGF14), fibroblast growth factor 15 (FGF15), fibroblast growth factor 16 (FGF16), fibroblast growth factor 17 (FGF17), fibroblast growth factor 18 (FGF18), Growth factor 19 (FGF19), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), fibroblast growth factor 22 (FGF22), fibroblast growth factor 23 (FGF23), fetal bovine growth hormone (FBS), glial cell line-derived neurotrophic factor (GDNF), neurotrophins, Persephin, Artemin, growth differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF),Insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 ( NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renin enzyme (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α) and vascular endothelial growth factor (VEGF).

[0187] In some aspects, the VEE replicon useful in the present disclosure comprises a nucleic acid sequence encoding a ligand binding protein. Non-limiting examples of ligand binding proteins include chimeric antigen receptors (CARs), T cell receptors (TCRs), chimeric antibody-T cell receptors (caTCRs), chimeric signaling receptors (CSRs), T cell receptor mimics (TCR mimics), and combinations thereof.

[0188] In some aspects, the VEE replicon includes a nucleic acid sequence encoding CAR. In some aspects, CAR is designed as standard CAR. In "standard CAR", different components (e.g., extracellular targeting domain, transmembrane domain and intracellular signal transduction / activation domain) are linearly constructed as a single fusion protein. In some aspects, CAR is designed as first generation CAR. "First generation" CAR consists of an extracellular binding domain, a hinge region, a transmembrane domain and one or more intracellular signal transduction domains. All first generation CARs include CD3 ζ chain domains as intracellular signal transduction domains. In some aspects, CAR is designed as second generation CAR. "Second generation" CAR additionally contains a costimulatory domain (e.g., CD28 or 4-1BB). In some aspects, CAR is designed as third generation CAR. "Third generation" CAR is similar to second generation CAR, except that they include multiple costimulatory domains (e.g., CD28-4-1BB or CD28-OX40). In some aspects, CAR is designed as fourth generation CAR. "Fourth generation" CAR (also referred to as TRUCK or armored CAR) additionally contains additional factors that can further improve function. For example, in some aspects, the fourth generation CAR additionally contains cytokines that can be released when CAR is signaled in targeted tumor tissue. In some aspects, the fourth generation CAR includes one or more additional elements, such as homing genes and suicide genes, which can help further regulate the activity of CAR. In some aspects, CAR is designed as a split CAR. In the "split CAR" system, one or more components of CAR (for example, extracellular targeting domain, transmembrane domain and intracellular signaling / activation domain) are divided into two or more parts, so that it depends on multiple inputs that promote the assembly of complete functional receptors. In some aspects, CAR is designed as a switchable CAR. Using "switchable CAR", CAR can be switched (for example, instantaneously) to open (open switch CAR) or close (close switch CAR) in the presence of stimulation. Additional examples of CARs that can be used with the present disclosure are described, for example, in US 2020 / 0172879A1 and US 2019 / 0183932 A1, each of which is incorporated herein by reference in its entirety.

[0189] In some aspects, the VEE replicon described herein comprises a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof (collectively referred to herein as an "antibody"). The antibody can be derived from a natural source, or partially or completely synthetically produced. In some aspects, the antibody is a monoclonal antibody. In some aspects, the monoclonal antibody is an IgG antibody. In some aspects, the monoclonal antibody is IgG1, IgG2, IgG3, or IgG4. In some aspects, the antibody is a polyclonal antibody. In some aspects, the antigen-binding fragment is selected from Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments. In some aspects, the antigen-binding fragment is a scFv or (scFv)2 fragment. In some aspects, the antibody or antigen-binding fragment is (Single domain antibody). In some aspects, the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

[0190] IV B. Additional Components

[0191] In some aspects, VEE replicons useful in the present disclosure comprise a nucleic acid sequence having one or more of the following additional components.

[0192] Terminal architecture modification: UTR

[0193] The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR begins at the transcription start site and continues to the start codon, but not including the start codon; while the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal. There is increasing evidence that UTRs play a regulatory role in the stability and translation of nucleic acid molecules. The regulatory features of UTRs can be incorporated into the RNA of the present disclosure to enhance the stability of the molecule. Specific features can also be incorporated to ensure controlled downregulation of transcripts to prevent them from being mistakenly directed to undesirable organ sites.

[0194] 5'UTR and translation initiation

[0195] Natural 5'UTRs have features that play a role in translation initiation. They contain features like the Kozak sequence, which is known to be involved in the process by which the ribosome initiates translation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another "G". The 5'UTR is also known to form secondary structures that are involved in the binding of elongation factors.

[0196] The 5'UTR secondary structure involved in the binding of elongation factors can interact with other RNA binding molecules in the 5'UTR or 3'UTR to regulate gene expression. For example, the binding of the elongation factor EIF4A2 to the secondary structural elements in the 5'UTR is essential for microRNA-mediated repression (Meijer HA et al., Science, 2013, 340, 82-85, which is incorporated herein by reference in its entirety). Different secondary structures in the 5'UTR can be incorporated into the flanking region to stabilize the mRNA in a specific tissue or cell or selectively destabilize it.

[0197] By engineering features that are typically found in genes that are abundantly expressed in specific target organs, the stability and protein yield of the nucleic acids or mRNAs disclosed herein can be enhanced. For example, the 5'UTR of mRNAs expressed in the liver (such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can be used to enhance the expression of nucleic acid molecules (such as mmRNA) in hepatocyte cell lines or liver. Likewise, it is possible to use 5'UTRs from other tissue-specific mRNAs to improve expression in that tissue—for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, Adiponectin), and for lung epithelial cells (SP-A / B / C / D).

[0198] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTR. For example, introns or portions of intronic sequences may be incorporated into the flanking regions of the nucleic acids or mRNAs of the present disclosure. Incorporation of intronic sequences may increase protein production as well as mRNA levels.

[0199] In some aspects of the present disclosure, at least one fragment of the IRES sequence from the GTX gene can be included in the 5'UTR. As a non-limiting example, the fragment can be 18 nucleotide sequences from the IRES of the GTX gene. As another non-limiting example, 18 nucleotide sequence fragments from the IRES sequence of the GTX gene can be repeated in series in the 5'UTR of the polynucleotide described herein. The 18 nucleotide sequences can be repeated in the 5'UTR at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times or more than ten times.

[0200] Nucleotide can be mutated, replaced and / or removed from 5' (or 3') UTR.For example, one or more nucleotides upstream of the start codon can be replaced with another nucleotide.The one or more nucleotides to be replaced can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,55,60 or more than 60 nucleotides upstream of the start codon.As another example, one or more nucleotides upstream of the start codon can be removed from UTR.

[0201] 5'UTR, 3'UTR and translational enhancer element (TEE)

[0202] In some aspects, the 5'UTR of the nucleotide sequence encoding IL-12 comprises at least one translation enhancer polynucleotide, a translation enhancer element, a translation enhancer element (collectively referred to as "TEE"). In some aspects, the TEE is located between the transcription promoter and the start codon. In some aspects, the RNA having at least one TEE in the 5'UTR comprises a cap at the 5'UTR. In some aspects, at least one TEE can be located in the 5'UTR of the nucleotide sequence encoding the IL-12 that undergoes cap-dependent or cap-independent translation.

[0203] The term "translation enhancer element" or "translation enhancer element" (collectively referred to herein as "TEE") refers to a sequence that increases the amount of a polypeptide or protein produced from an mRNA.

[0204] In one aspect, TEE is a conserved element in UTR that can promote the translation activity of nucleic acids, such as but not limited to cap-dependent or cap-independent translation. The conservation of these sequences has previously been shown by Panek et al. (Nucleic Acids Research, 2013, 1-10; incorporated herein by reference in its entirety) in 14 species including humans.

[0205] In some aspects, the nucleotide sequence encoding IL-12 has at least one TEE that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to that disclosed in U.S. Appl. No. 2014 / 0147454, which is incorporated herein by reference in its entirety. In some aspects, the RNA includes U.S. Patent Publication Nos. US20090226470, US20070048776, US20130177581, and US20110124100, International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886, and WO2007025008, European Patent Publication Nos. EP2610341A1 and EP2610340A1, U.S. Patent No. 6 , 310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, and U.S. Pat. No. 7,183,395, each of which is incorporated herein by reference in its entirety, to include at least one TEE having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity.

[0206] In some aspects, the 5'UTR of the nucleotide sequence encoding IL-12 can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In some aspects, the TEE sequences in the 5'UTR of RNA are identical or different TEE sequences. In some aspects, the TEE sequence is in a pattern that is repeated once, twice or more than three times, such as ABABAB or AABBAABBABB or ABCABCABC or variants thereof. In these patterns, each letter A, B or C represents a different TEE sequence at the nucleotide level.

[0207] In some aspects, the spacer separating two TEE sequences includes other sequences known in the art for regulating the translation of RNA, such as but not limited to miR sequences described herein (e.g., miR binding sites and miR seeds). In some aspects, each spacer for separating two TEE sequences includes different miR sequences or components of miR sequences (e.g., miR seed sequences).

[0208] In some aspects, the TEE used in the 5'UTR of the IL-12 encoding nucleotide sequences of the present disclosure is an IRES sequence, such as but not limited to those described in US Pat. No. 7,468,275 and International Patent Publication No. WO2001055369, each of which is herein incorporated by reference in its entirety.

[0209] In some aspects, the TEE described herein is located in the 5'UTR and / or 3'UTR of the nucleotide sequence encoding IL- 12. In some aspects, the TEE located in the 3'UTR is the same and / or different from the TEE located in the 5'UTR and / or described for incorporation into the 5'UTR.

[0210] In some aspects, the 3'UTR of the nucleotide sequence encoding IL-12 can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In some aspects, the TEE sequences in the 3'UTR of the nucleotide sequence encoding IL-12 disclosed herein are identical or different TEE sequences. The TEE sequence is in a pattern that is repeated once, twice, or more than three times, such as ABABAB or AABBAABBABB or ABCABCABC or variants thereof. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0211] In some aspects, the 3'UTR includes a spacer separating two TEE sequences. In some aspects, the spacer is a spacer of 15 nucleotides and / or other spacers known in the art. In some aspects, the 3'UTR can include a TEE sequence-spacer module that is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, and at least 9 times or more than 9 times in the 3'UTR.

[0212] In some aspects, the spacer separating the two TEE sequences includes other sequences known in the art that regulate the translation of the nucleotide sequence encoding IL-12, such as, but not limited to, miR sequences described herein (e.g., miR binding sites and miR seeds). In some aspects, each spacer for separating the two TEE sequences includes different miR sequences or components of miR sequences (e.g., miR seed sequences).

[0213] Incorporation of microRNA binding sites

[0214] In some aspects, the nucleotide sequence encoding IL-12 further comprises a sensor sequence. The sensor sequence includes, for example, a microRNA binding site, a transcription factor binding site, a structured mRNA sequence and / or motif, an artificial binding site engineered to act as a pseudoreceptor for an endogenous nucleic acid binding molecule. Non-limiting examples of polynucleotides comprising at least one sensor sequence are described in U.S. Application No. 2014 / 0147454, which is hereby incorporated by reference in its entirety.

[0215] In some aspects, microRNA (miRNA) profiling of a target cell or tissue is performed to determine the presence or absence of a miRNA in the cell or tissue.

[0216] MicroRNA (or miRNA) is a non-coding RNA of 19-25 nucleotides in length, which is bound to the 3'UTR of nucleic acid molecules and down-regulates gene expression by reducing nucleic acid molecule stability or by inhibiting translation. In some aspects, RNA comprises one or more microRNA target sequences, microRNA sequences or microRNA seeds. Such sequences may correspond to any known microRNA, such as those taught in US2005 / 0261218 and US2005 / 0059005 disclosed in the United States, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the seed sequences in known microRNA, its sequence and the human genome are described in U.S. Application No. 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0217] The microRNA sequence comprises a "seed" region, i.e., a sequence in the 2-8 region of a mature microRNA, which has perfect Watson-Crick complementarity with the miRNA target sequence. The microRNA seed comprises 2-8 or 2-7 of a mature microRNA. In some aspects, the microRNA seed comprises 7 nucleotides (e.g., nucleotides 2-8 of a mature microRNA), wherein the seed complementary site in the corresponding miRNA target is flanked by adenine (A) relative to microRNA position 1. In some aspects, the microRNA seed comprises 6 nucleotides (e.g., nucleotides 2-7 of a mature microRNA), wherein the seed complementary site in the corresponding miRNA target is flanked by adenine (A) relative to microRNA position 1. See, e.g., Grimson A, Farh K, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. July 6, 2007; 27 (1): 91-105. The bases of the microRNA seed have complete complementarity with the target sequence. By engineering microRNA target sequences into the 3'UTR of the nucleic acids or mRNAs of the present disclosure, the molecules can be targeted for degradation or reduced translation, provided that the microRNA in question is available. This process will reduce the risk of off-target effects when delivering nucleic acid molecules. Identification of microRNAs, microRNA target regions, their expression patterns, and roles in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (Dec 20, 2011. doi:10.1038 / leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al., Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393-403 and all references therein; each of which is incorporated herein by reference in its entirety).

[0218] For example, if mRNA is not intended to be delivered to the liver but ends there in the liver, when one or more target sites of miR-122 (a kind of microRNA abundant in the liver) are engineered into the 3'UTR of modified nucleic acid, enhanced modified RNA or ribonucleic acid, miR-122 can inhibit the expression of the gene of interest. The introduction of one or more binding sites of different microRNAs can be engineered to further reduce the life span, stability and protein translation of modified nucleic acid, enhanced modified RNA or ribonucleic acid. As used herein, the term "microRNA site" refers to any nucleotide sequence of microRNA target site or microRNA recognition site or microRNA binding or association. It should be understood that "binding" can follow traditional Watson-Crick hybridization rules, or can reflect any stable association of microRNA with target sequence at or near the microRNA site.

[0219] In contrast, for the purposes of the disclosed nucleotide sequences encoding IL-12, microRNA binding sites can be engineered from (i.e., removed from) their naturally occurring sequences to increase protein expression in specific tissues. For example, miR-122 binding sites can be removed to improve protein expression in the liver.

[0220] In some aspects, the nucleotide sequence encoding IL-12 comprises at least one miRNA binding site in the 3'UTR to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells, such as but not limited to normal and / or cancer cells (eg, HEP3B or SNU449).

[0221] Examples of tissues in which microRNAs are known to regulate mRNA and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).

[0222] Specifically, known microRNAs are differentially expressed in immune cells (also referred to as hematopoietic cells), such as antigen presenting cells (APC) (for example, dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell-specific microRNAs participate in immunogenicity, autoimmunity, immune responses to infection, inflammation, and immune responses that are not needed after gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also regulate many aspects of the development, proliferation, differentiation, and apoptosis of hematopoietic cells (immune cells). For example, miR-142 and miR-146 are only expressed in immune cells, and are particularly rich in bone marrow dendritic cells. It has been demonstrated in the art that the immune response to exogenous nucleic acid molecules is closed by adding the miR-142 binding site to the 3'UTR of the gene construct delivered, thereby enabling more stably transferred genes in tissues and cells. miR-142 effectively degrades exogenous mRNA in antigen-presenting cells and inhibits cytotoxic elimination of transduced cells (Annoni A et al., blood, 2009, 114, 5152-5161; Brown BD et al., Nat med. 2006, 12(5), 585-591; Brown BD et al., blood, 2007, 110(13):4144-4152, each of which is incorporated herein by reference in its entirety).

[0223] Many microRNA expression studies have been conducted in the art to analyze the differential expression of microRNAs in various cancer cells / tissues and other diseases. Some microRNAs are abnormally overexpressed in certain cancer cells, while other microRNAs are underexpressed. For example, microRNAs are differentially expressed in: cancer cells (WO2008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancer and diseases (US2009 / 0131348, US2011 / 0171646, US2010 / 0286232, U.S. Patent No. 8,389,210); asthma and inflammation (U.S. Patent No. 8,415,096); prostate cancer (US2013 / 0053264); hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, WO2008 / 054828, U.S. Patent No. 8,252,538); lung cancer cells (WO2011 / 076143, WO2013 / 033640, WO2009 / 070653, US2010 / 0323357); cutaneous T-cell lymphoma (WO2013 / 011378); colorectal cancer cells (WO2011 / 0281756, WO2011 / 076142); cancer-positive lymph nodes (WO2009 / 100430, US2009 / 0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US 2012 / 0264626, US2013 / 0053263); thyroid cancer (WO2013 / 066678); ovarian cancer cells (US2012 / 0309645, WO2011 / 095623); breast cancer cells (WO2008 / 154098, WO2007 / 081740, US2012 / 0214699), leukemia and lymphoma (WO2008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, WO2010 / 018563, the contents of each of which are incorporated herein by reference in their entirety.)

[0224] At least one microRNA site can be engineered into the 3'UTR of the nucleotide sequence encoding IL-12. In some aspects, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more microRNA sites can be engineered into the 3'UTR of the nucleotide sequence encoding IL-12. In some aspects, the microRNA sites incorporated into the nucleotide sequence encoding IL-12 are identical or different microRNA sites. In some aspects, the microRNA sites incorporated into the nucleotide sequence encoding IL-12 target the same or different tissues in vivo. As a non-limiting example, by introducing tissue-specific, cell type-specific or disease-specific microRNA binding sites in the 3'UTR of mRNA, the expression level in specific cell types (e.g., hepatocytes, bone marrow cells, endothelial cells, cancer cells, etc.) can be reduced.

[0225] In some aspects, the microRNA site is engineered near the 5' end of the 3'UTR, about halfway between the 5' end of the 3'UTR and the 3' end, and / or near the 3' end of the 3'UTR. In some aspects, the microRNA site is engineered near the 5' end of the 3'UTR and about halfway between the 5' end of the 3'UTR and the 3' end. In some aspects, the microRNA site is engineered near the 3' end of the 3'UTR and about halfway between the 5' end of the 3'UTR and the 3' end. In some aspects, the microRNA site is engineered near the 5' end of the 3'UTR and about halfway between the 5' end of the 3'UTR and the 3' end.

[0226] In some aspects, mRNA comprises and has 100% homology with known seed sequence or has the microRNA binding region site less than 100% homology with seed sequence.Seed sequence can be partially mutated to reduce microRNA binding affinity, thereby reduce the downward regulation of this mRNA transcript.Essentially, the matching or mismatch degree between target mRNA and microRNA seed can serve as rheostat, to adjust the ability of microRNA regulating protein expression more finely.In addition, the sudden change of non-seed region of microRNA binding site also can affect the ability of microRNA regulating protein expression.

[0227] RNA motifs of RNA binding proteins (RBPs)

[0228] RNA binding proteins (RBPs) can regulate many aspects of co-transcriptional and post-transcriptional gene expression, such as, but not limited to, RNA splicing, positioning, translation, turnover, polyadenylation, capping, modification, export and positioning. RNA binding domains (RBDs), such as, but not limited to, RNA recognition motifs (RRs) and hnRNP K-homology (KH) domains, typically regulate the sequence association between RBP and its RNA target (Ray et al. Nature 2013.499:172-177; incorporated herein by reference in its entirety). In some aspects, typical RBDs bind short RNA sequences. In some aspects, typical RBDs recognize RNA structures.

[0229] Non-limiting examples of RNA binding proteins and related nucleic acid and protein sequences are described in U.S. Appl. No. 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0230] In some aspects, in order to increase the stability of the mRNA of interest, the mRNA encoding HuR is co-transfected or co-injected into cells or tissues with the mRNA of interest. These proteins can also be tethered to the mRNA of interest in vitro and then applied to the cells together. The poly A tail binding protein PABP interacts with the eukaryotic translation initiation factor eIF4G to stimulate translation initiation. The mRNA encoding these RBPs is co-administered with mRNA drugs and / or these proteins are tethered to mRNA drugs in vitro and the protein-bound mRNA is applied to cells to increase the translation efficiency of mRNA. The same concept can be extended to co-administering mRNA with mRNA encoding various translation factors and promoters and with the protein itself to affect RNA stability and / or translation efficiency.

[0231] In some aspects, the nucleotide sequence encoding IL-12 comprises at least one RNA binding motif, such as but not limited to an RNA binding domain (RBD).

[0232] In some aspects, the first region of the connection nucleoside and / or at least one flanking region comprises at least one RBD. In some aspects, the first region of the connection nucleoside comprises an RBD associated with a splicing factor, and at least one flanking region comprises an RBD for stability and / or a translation factor.

[0233] Other regulatory elements in the 3'UTR

[0234] In addition to microRNA binding sites, other regulatory sequences that regulate mRNA stability and translation in different tissues and cells in the 3'-UTR of natural mRNA can be removed or introduced into RNA. Such cis-regulatory elements may include, but are not limited to, cis-RNP (ribonucleoprotein) / RBP (RNA binding protein) regulatory elements, AU-rich elements (AUE), structured stem loops, constitutive decay elements (CDE), GC richness and other structured mRNA motifs (Parker BJ et al., Genome Research, 2011, 21, 1929-1943, which are incorporated herein by reference in their entirety). For example, CDE is a class of regulatory motifs that mediate mRNA degradation by interaction with Roquin protein. In particular, CDE is present in many mRNAs that encode developmental and inflammatory regulatory factors to limit the production of cytokines in macrophages (Leppek K et al., 2013, Cell, 153, 869-881, which are incorporated herein by reference in their entirety).

[0235] In some aspects, RNA is auxotrophic. As used herein, the term "auxotrophic" refers to an mRNA comprising at least one feature that triggers, promotes or induces degradation or inactivation of mRNA in response to spatial or temporal clues so that protein expression is substantially prevented or reduced. Such spatial or temporal clues include the location of the mRNA to be translated, such as a specific tissue or organ or cell environment. Clues involving temperature, pH, ionic strength, moisture content, etc. are also contemplated.

[0236] 3'UTR and AU-rich elements

[0237] 3'UTR is known to have a stretch of adenosine and uridine embedded in it. These AU-rich features are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) 9-mers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples in this category. Most proteins that are known to bind to AREs destabilize the messenger, while members of the ELAV family, most notably HuR, have been shown to increase the stability of mRNA. HuR binds to all three categories of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and thus stabilization of the message in vivo.

[0238] The introduction, removal or modification of 3'UTR AU-rich elements (ARE) can be used to regulate the stability of nucleic acids or mRNAs disclosed herein. When engineering specific nucleic acids or mRNAs, one or more copies of AREs can be introduced to make nucleic acids or mRNAs disclosed herein less stable, thereby limiting translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing translation and the production of the resulting protein. Transfection experiments can be performed in relevant cell lines using nucleic acids or mRNAs disclosed herein, and protein yields can be measured at various time points after transfection. For example, cells can be transfected with different ARE engineered molecules, and the relevant proteins can be detected using an ELISA kit and the proteins produced at about 6 hours, about 12 hours, about 24 hours, about 48 hours and / or about 7 days after transfection are measured.

[0239] 3'UTR and triple helix

[0240] In some aspects, the nucleotide sequence encoding IL-12 comprises a triple helix at the 3' end of the nucleic acid, the enhancing nucleotide sequence encoding IL-12, or the ribonucleic acid. In some aspects, the 3' end of the nucleotide sequence encoding IL-12 comprises a triple helix alone or in combination with a Poly-A tail.

[0241] In some aspects, the nucleotide sequence encoding IL-12 comprises at least the first and second U-rich regions, the conserved stem-loop region between the first region and the second region, and the A-rich region. In some aspects, the first and second U-rich regions are associated with the A-rich region to form a triple helix on the 3' end of the nucleic acid. This triple helix can stabilize the nucleic acid, enhance the translation efficiency of the nucleic acid, and / or protect the 3' end from degradation. Exemplary triple helices include, but are not limited to, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN-β, and polyadenylated nuclear (PAN) RNA triple helical sequences (see Wilusz et al., Genes & Development 2012 26: 2392-2407; which is incorporated herein by reference in its entirety).

[0242] Stem-loop

[0243] In some aspects, the nucleotide sequence encoding IL-12 includes a stem loop, such as but not limited to a histone stem loop. In some aspects, the stem loop is a nucleotide sequence of about 25 or about 26 nucleotides in length, such as but not limited to SEQ ID NO: 7-17 as described in International Patent Publication No. WO2013103659, which is incorporated herein by reference in its entirety. The histone stem loop can be located at 3' (e.g., at the 3' end of the coding region) relative to the coding region. As a non-limiting example, the stem loop can be located at the 3' end of the nucleic acid described herein.

[0244] In some aspects, the nucleotide sequence encoding IL-12 comprising a histone stem-loop can be stabilized by adding at least one chain terminating nucleoside. Without wishing to be bound by theory, adding at least one chain terminating nucleoside can slow down the degradation of the nucleic acid and thus increase the half-life of the nucleic acid.

[0245] In some aspects, the chain-terminating nucleoside is those described in International Patent Publication No. WO2013103659, which is incorporated herein by reference in its entirety. In some aspects, the chain-terminating nucleoside is 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymidine, 2', 3'-dideoxynucleoside (such as 2', 3'-dideoxyadenosine, 2', 3'-dideoxyuridine, 2', 3'-dideoxycytosine, 2', 3'-dideoxyguanosine, 2', 3'-dideoxythymidine), 2'-deoxynucleoside or -O-methyl nucleoside.

[0246] In some aspects, the nucleotide sequence encoding IL-12 comprises a histone stem-loop, a polyA tail sequence and / or a 5' cap structure. In some aspects, the histone stem-loop is before and / or after the polyA tail sequence. The nucleic acid comprising the histone stem-loop and the polyA tail sequence may include a chain terminating nucleoside as described herein.

[0247] In some aspects, the nucleotide sequence encoding IL-12 comprises a histone stem-loop and a 5' cap structure. The 5' cap structure may include, but is not limited to, those described herein and / or known in the art.

[0248] 5' Cap

[0249] The 5' cap structure of mRNA participates in nuclear export, thereby increasing mRNA stability and binding mRNA cap binding protein (CBP), which is responsible for mRNA stability and translation competence in cells through the association of CBP with poly(A) binding protein to form mature circular mRNA species. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

[0250] Endogenous mRNA molecules can be 5'-capped to generate a 5'-ppp-5'-triphosphate connection between the terminal guanosine cap residue of the mRNA and the sense nucleotide transcribed at the 5'-end. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose of the nucleotides transcribed at the end and / or the front end of the 5' end of the mRNA can also be optionally 2'-O-methylated. 5'-decapping by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0251] Modification of the RNA disclosed herein can generate a non-hydrolyzable cap structure, thereby preventing decapping and thus increasing the mRNA half-life. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester connection, modified nucleotides can be used during the capping reaction. For example, vaccine capping enzymes from New England Biolabs (Ipswich, Mass.) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to produce thiophosphate connections in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonic acid and seleno-phosphate nucleotides, can be used.

[0252] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose (as described above) of the 5'-terminal and / or 5'-preterminal nucleotide of the mRNA at the 2'-hydroxyl of the sugar ring.A variety of different 5'-cap structures can be used to generate a 5'-cap for a nucleic acid molecule, such as an mRNA molecule.

[0253] Cap analogs (also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs) differ in their chemical structure from the natural (i.e., endogenous, wild-type, or physiological) 5'-cap while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or attached to nucleic acid molecules.

[0254] For example, the anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, wherein one of the guanines contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which can be equivalently named 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA or mmRNA). The N7- and 3'-O-methylated guanines provide the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0255] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-β-methyl group on guanosine (ie, N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0256] In some aspects, the cap is a dinucleotide cap analog. In some aspects, the dinucleotide cap analog is modified with a boranephosphate group or a selenophosphate group at a different phosphate position, such as the dinucleotide cap analog described in U.S. Patent No. 8,519,110, the contents of which are incorporated herein by reference in their entirety.

[0257] In some aspects, the cap is a cap analog that is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide forms of cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., the various cap analogs and the methods of synthesizing cap analogs, described in Kor et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of the references are incorporated herein by reference in their entirety). In some aspects, the cap analog of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0258] Although cap analogs allow capping of nucleic acid molecules accompanying in vitro transcription reactions, up to about 20% of transcripts remain uncapped. This, as well as cap analogs, structural differences from the endogenous 5'-cap structure of nucleic acids produced by endogenous cellular transcription mechanisms can lead to reduced translational capacity and reduced cellular stability. Therefore, in some aspects, the methods provided herein (see, e.g., Examples 1-3) can increase the capping efficiency of the IL-12-expressing nucleotides described herein. In some aspects, using the methods provided herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 100% of the polynucleotides are capped. In some aspects, at least about 50% of the polynucleotides are capped using the methods provided herein. In some aspects, at least about 60% of the polynucleotides are capped. In some aspects, at least about 70% of the polynucleotides are capped. In some aspects, at least about 80% of the polynucleotides are capped. In some aspects, at least about 85% of the polynucleotides are capped. In some aspects, at least about 90% of the polynucleotides are capped. In some aspects, at least about 95% of the polynucleotides are capped. In some aspects, about 100% of the polynucleotides are capped. In some aspects, at least about 80% to about 100% of the polynucleotides are capped.

[0259] In some aspects, providing an RNA having a 5'-cap or a 5'-cap analog is accomplished by in vitro transcribing a DNA template in the presence of the 5'-cap or a 5'-cap analog, wherein the 5'-cap is co-transcriptionally incorporated into the resulting RNA strand,

[0260] In some aspects, RNA can be generated, for example, by in vitro transcription, and a capping enzyme (e.g., a capping enzyme of a vaccine virus) can be used to attach a 5'-cap to RNA after transcription. In some aspects, an enzyme is used to perform post-transcriptional capping of a nucleotide sequence encoding IL-12 to generate a more realistic 5' cap structure. As used herein, the phrase "more realistic" refers to a feature that closely reflects or simulates an endogenous or wild-type feature in structure or function. That is, compared to synthetic features or analogs of the prior art, "more realistic" features better represent endogenous, wild-type, natural or physiological cell functions and / or structures, or are superior to corresponding endogenous, wild-type, natural or physiological features in one or more aspects. Non-limiting examples of more realistic 5' cap structures disclosed herein are, among others, those with enhanced binding of cap-binding proteins, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' decapping compared to synthetic 5' cap structures known in the art (or wild-type, natural or physiological 5' cap structures). For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a typical 5'-5'-triphosphate connection between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide, wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl group. Such caps result in higher translational capacity and cellular stability and reduced activation of cellular proinflammatory cytokines compared to other 5' cap analog structures known in the art, for example. Cap structures include 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2mp, and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up.

[0261] In some aspects, the 5' terminal cap comprises an endogenous cap or a cap analog. In some aspects, the 5' terminal cap comprises a guanine analog. Useful guanine analogs include inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0262] In some aspects, the 5' cap comprises a 5' to 5' triphosphate connection. In some aspects, the 5' cap comprises a 5' to 5' triphosphate connection, including a thiophosphate modification. In some aspects, the 5' cap comprises a 2'-O or 3'-O-ribomethylated nucleotide. In some aspects, the 5' cap comprises a modified guanosine nucleotide or a modified adenosine nucleotide. In some aspects, the 5' cap comprises a 7-methylguanylate. Exemplary cap structures include m7G(5')ppp(5')G, m7,2`O-mG(5')ppSp(5')G, m7G(5')ppp(5')2`O-mG and m7,3`O-mG(5')ppp(5')2`O-mA.

[0263] In some aspects, the nucleotide sequence encoding IL-12 comprises a modified 5' cap. The modification on the 5' cap can increase the stability of the mRNA, increase the half-life of the mRNA, and can increase the efficiency of mRNA translation. In some aspects, the modified 5' cap comprises one or more of the following modifications: modification at the 2' and / or 3' position of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (which produces a carbocycle) with a methylene moiety (CH2), modification at the triphosphate bridge portion of the cap structure, or modification at the nucleobase (G) portion.

[0264] Modifiable 5' cap structures include, but are not limited to, the caps described in U.S. Application Nos. 2014 / 0147454 and WO2018 / 160540, which are incorporated herein by reference in their entirety.

[0265] IRES sequence

[0266] In some aspects, the nucleotide sequence encoding IL-12 comprises an internal ribosome entry site (IRES). IRES was originally identified as a characteristic small RNA virus RNA, and plays an important role in starting protein synthesis in the absence of a 5' cap structure. IRES can serve as a unique ribosome binding site, or can serve as one of multiple ribosome binding sites of mRNA. Nucleic acids or mRNA containing more than one functional ribosome binding site can encode several peptides or polypeptides ("polycistronic nucleic acid molecules") that are independently translated by ribosomes. When IRES is provided to nucleic acid or mRNA, a second translatable region is further optionally provided. Examples of IRES sequences that can be used according to the present disclosure include, but are not limited to, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0267] Terminal structure modification: Poly-A tail

[0268] During RNA processing, long chains of adenine nucleotides (poly-A tails) are usually added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3' end of the transcript is cleaved to release the 3' hydroxyl group. Poly-A polymerase then adds the adenine nucleotide chain to the RNA. The process (known as polyadenylation) adds a poly-A tail that is 100 to 250 residues long.

[0269] In some aspects, the length of the 3' tail is greater than about 30 nucleotides. In some aspects, the length of the poly-A tail is greater than about 35 nucleotides. In some aspects, the length is at least about 40 nucleotides. In some aspects, the length is at least about 45 nucleotides. In some aspects, the length is at least about 55 nucleotides. In some aspects, the length is at least about 60 nucleotides. In some aspects, the length is at least about 70 nucleotides. In some aspects, the length is at least about 80 nucleotides. In some aspects, the length is at least about 90 nucleotides. In some aspects, the length is at least about 100 nucleotides. In some aspects, the length is at least about 120 nucleotides. In some aspects, the length is at least about 140 nucleotides. In some aspects, the length is at least about 160 nucleotides. In some aspects, the length is at least about 180 nucleotides. In some aspects, the length is at least about 200 nucleotides. In some aspects, the length is at least about 250 nucleotides. In some aspects, the length is at least about 300 nucleotides. In some aspects, the length is at least about 350 nucleotides. In some aspects, the length is at least about 400 nucleotides. In some aspects, the length is at least about 450 nucleotides. In some aspects, the length is at least about 500 nucleotides. In some aspects, the length is at least about 600 nucleotides. In some aspects, the length is at least about 700 nucleotides. In some aspects, the length is at least about 800 nucleotides. In some aspects, the length is at least about 900 nucleotides. In some aspects, the length is at least about 1000 nucleotides. In some aspects, the length is at least about 1100 nucleotides. In some aspects, the length is at least about 1200 nucleotides. In some aspects, the length is at least about 1300 nucleotides. In some aspects, the length is at least about 1400 nucleotides. In some aspects, the length is at least about 1500 nucleotides. In some aspects, the length is at least about 1600 nucleotides. In some aspects, the length is at least about 1700 nucleotides. In some aspects, the length is at least about 1800 nucleotides. In some aspects, the length is at least about 1900 nucleotides. In some aspects, the length is at least about 2000 nucleotides. In some aspects, the length is at least about 2500 nucleotides. In some aspects, the length is at least about 3000 nucleotides.

[0270] In some aspects, the nucleotide sequence encoding IL-12 is designed to include a polyA-G quadruplex. The G-quadruplex is a cyclic hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this regard, the G-quadruplex is incorporated at the end of the poly-A tail. The stability, protein yield and other parameters of the resulting nucleic acid or mRNA, including half-life, can be measured at different time points. It has been found that the protein yield caused by the polyA-G quadruplex is equivalent to at least 75% of the protein yield seen using a 120-nucleotide poly-A tail alone.

[0271] In some aspects, the nucleotide sequence encoding IL-12 comprises a polyA tail and is stabilized by the addition of chain terminating nucleosides. In some aspects, the nucleotide sequence encoding IL-12 having a polyA tail further comprises a 5' cap structure.

[0272] In some aspects, the nucleotide sequence encoding IL-12 comprises a polyA-G quadruplex. In some aspects, the nucleotide sequence encoding IL-12 having a polyA-G quadruplex further comprises a 5' cap structure.

[0273] In some aspects, the nucleotide sequence encoding IL-12 comprising a polyA tail or a polyA-G quadruplex is stabilized by the addition of oligonucleotides terminating in 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3'-O-methyl nucleosides, 3'-O-ethyl nucleosides, 3'-arabinoside, and other modified nucleosides known in the art and / or described herein.

[0274] Modified Nucleosides

[0275] In some aspects, the nucleotide sequence encoding IL-12 comprises one or more modified nucleosides. In some aspects, the one or more modified nucleosides include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8 -Oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrole-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine and combinations thereof.

[0276] In some aspects, one or more uridines in the nucleotide sequence encoding IL-12 are replaced by modified nucleosides. In some aspects, the modified nucleosides replacing uridines are pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U).

[0277] In some aspects, the nucleotide sequence encoding IL-12 comprises a nucleotide sequence encoding IL-12 as described in U.S. Application No. 2014 / 0147454, International Application WO2018160540, International Application WO2015 / 196118, or International Application WO2015 / 089511, which are herein incorporated by reference in their entireties.

[0278] Cytotoxic nucleosides

[0279] In some aspects, the nucleotide sequence encoding IL-12 comprises one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into polynucleotides (such as bifunctional nucleotide sequences encoding IL-12s or mRNA). Cytotoxic nucleoside anticancer agents include, but are not limited to, adenosine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and 6-mercaptopurine.

[0280] Many cytotoxic nucleoside analogs are used clinically as anticancer agents or have been the subject of clinical trials. Examples of such analogs include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylene cytidine (DMDC), cladribine, clofarabine, 5-azacytidine, 4'-thio-arabino-cytarabine, cyclopentenylcytosine and 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine. Another example of such compounds is fludarabine phosphate. These compounds can be systemically administered and may have typical side effects of cytotoxic agents, such as, but not limited to, with little or no specificity for tumor cells relative to proliferating normal cells.

[0281] Many prodrugs of cytotoxic nucleoside analogs have also been reported in the art. Examples include, but are not limited to, N4-behenyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl-1-β-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-β-D-arabinofuranosyl)cytosine and P-4055 (cytosine arabinoside 5'-elidal ester). In general, these prodrugs can be converted into active drugs mainly in the liver and systemic circulation, and there is little or no selective release of active drugs in tumor tissue. For example, capecitabine (a prodrug of 5'-deoxy-5-fluorocytidine (and final 5-fluorouracil)) is metabolized in both liver and tumor tissue. Fujiu et al. (U.S. Patent No. 4,966,891) claim a series of capecitabine analogs containing "groups that are easily hydrolyzed under physiological conditions", and are incorporated herein by reference. Fujiu described a series including N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine and suggested that these compounds would be activated by hydrolysis under normal physiological conditions to provide 5'-deoxy-5-fluorocytidine.

[0282] Fadl et al. (Pharmazie.1995,50,382-7, incorporated herein by reference in its entirety) reported a series of cytarabine N4-carbamates, wherein the compound is designed to be converted into cytarabine in the liver and plasma. WO2004 / 041203 (incorporated herein by reference in its entirety) discloses prodrugs of gemcitabine, some of which are N4-carbamates. These compounds are intended to overcome the gastrointestinal toxicity of gemcitabine, and are intended to provide gemcitabine by hydrolyzing and releasing in the liver and plasma after absorbing the complete prodrug from the gastrointestinal tract. Nomura et al. (Bioorg Med.Chem.2003,11,2453-61, incorporated herein by reference in its entirety) describe acetal derivatives of 1-(3-C-ethynyl-β-D-ribose-pentofuranosyl) cytosine, which produce intermediates during bioreduction, and the intermediates need to be further hydrolyzed under acidic conditions to produce cytotoxic nucleoside compounds.

[0283] Cytotoxic nucleotides that may be chemotherapeutic also include, but are not limited to, pyrazolo[3,4-D]-pyrimidine, allopurinol, azathioprine, capecitabine, cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, vidarabine, ribavirin, 7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine, thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine and inosine, or combinations thereof.

[0284] V. Pharmaceutical Compositions

[0285] In some aspects, the disclosure relates to a pharmaceutical composition comprising a polynucleotide, a carrier and / or a lipid nanoparticle as described herein. In some aspects of the disclosure, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier (excipient). As used herein, "acceptable" means that the carrier must be compatible with the active ingredient of the composition and harmless to the subject to be treated. In some aspects, the carrier can stabilize the active ingredient. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Edition (2000) Lippincott Williams and Wilkoins, Editor: KE Hoover.

[0286] Pharmaceutical compositions for in vivo administration must be sterile. This is easily achieved by, for example, filtering through a sterile filtration membrane. The lipid nanoparticles can be placed in a container with a sterile access port (e.g., an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle).

[0287] In some aspects of the present disclosure, the pharmaceutical composition can be formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhaled, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, transdermal, sublingual, submucosal, transdermal or transmucosal administration. In some aspects of the present disclosure, the pharmaceutical composition can be formulated for intratumoral injection. As used herein, intratumoral injection refers to direct injection into a tumor. High concentrations of compositions can be obtained in situ while using small amounts of drugs. Local delivery of immunotherapy allows a variety of combination therapies while preventing significant systemic exposure and off-target toxicity.

[0288] In some aspects of the present disclosure, the pharmaceutical composition may be formulated for intramuscular injection, intravenous injection, or subcutaneous injection.

[0289] In some aspects of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, buffer, excipient, salt or stabilizer in the form of a lyophilized formulation or aqueous solution. See, for example, Remington: The Science and Practice of Pharmacy 20th Edition (2000) Lippincott Williams and Wilkins, ed. KE Hoover. Acceptable carriers and excipients or stabilizers are nontoxic to the recipient at the doses and concentrations used, and include: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0290] In some aspects, the pharmaceutical compositions described herein include lipid nanoparticles, which can be prepared by methods known in the art, such as those described in the following documents: Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545, which are incorporated herein by reference in their entirety. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556, which are incorporated herein by reference in their entirety. In some aspects, liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to produce liposomes with a desired diameter.

[0291] In some aspects of the present disclosure, the pharmaceutical composition is formulated in a sustained release form. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing lipid nanoparticles, the matrix being in the form of a shaped article, e.g., a film or microcapsule. Examples of sustained release matrices include, but are not limited to, polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPROM DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0292] In some aspects, suitable surfactants include, but are not limited to, nonionic agents such as polyoxyethylene sorbitan (e.g., TWEEN TM 20, 40, 60, 80 or 85) and other sorbitans (e.g., SPAN TM 20, 30, 60, 80 or 85). In some aspects, the composition with a surfactant comprises 0.05% to 5% of a surfactant. In some aspects, the composition comprises 0.1% and 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added if desired.

[0293] In some aspects, the pharmaceutical composition is in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0294] In order to prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier (e.g., conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum) and other pharmaceutical diluents (e.g., water) to form a solid preformulated composition containing a uniform mixture of a compound of the present disclosure or a nontoxic pharmaceutically acceptable salt thereof. When referring to these preformulated mixtures as uniform, it is meant that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulated composition is then subdivided into unit dosage forms of the above type containing about 0.1 to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that gives the advantage of prolonged action. For example, a tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of a film coating on the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0295] Suitable emulsions can be prepared using commercially available fat emulsions, such as INTRALI PID TM 、LIPOSYN TM 、INFONUTROL TM 、LIPOFUNDIN TM , and LIPI PHYSAN TM The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, the active ingredient can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with a phospholipid (e.g., lecithin, soybean lecithin, or soy lecithin) and water to form an emulsion. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the tension of the emulsion. Suitable emulsions will typically contain up to about 20% oil, for example, between about 5% and about 20%. The fat emulsion can contain fat droplets of a suitable size and can have a pH in the range of about 5.5 to about 8.0.

[0296] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some aspects, the compositions are administered by the oral or nasal respiratory route to produce local or systemic effects.

[0297] Compositions in pharmaceutically acceptable solvents can be nebulized by the use of gases. Nebulized solutions can be inhaled directly from the nebulizing device, or the nebulizing device can be attached to a mask tampons or intermittent positive pressure breathing machine. Solutions, suspensions or powder compositions can be administered from devices that deliver the formulation in an appropriate manner.

[0298] VI. Kits for use in therapy

[0299] The present disclosure also provides kits for immunotherapy and / or treating or reducing the risk of a disease or condition (e.g., cancer) for a disease or condition such as cancer (e.g., melanoma, lung cancer, colorectal cancer, or renal cell carcinoma). In some aspects, the kit includes one or more containers comprising a composition described herein.

[0300] In some aspects, the kit includes instructions for use according to any of the methods described herein. For example, the included instructions may include a description of administering a pharmaceutical composition as described herein to treat, delay onset, or alleviate a target disease. In some aspects, the instructions include a description of administering a composition as described herein to a subject at risk of a target disease / disorder (e.g., cancer).

[0301] In some aspects, the instructions include dosage information, dosing schedule, and route of administration. In some aspects, the container is a unit dose, bulk packaging (e.g., multi-dose packaging), or a subunit dose. In some aspects, the instructions are written instructions on a label or package insert (e.g., a sheet of paper included in a test kit). In some aspects, the instructions are machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk).

[0302] In some aspects, the label or package insert indicates that the compositions disclosed herein are used to treat, delay the onset of, and / or alleviate a disease or condition associated with cancer, such as those described herein.Instructions for practicing any of the methods described herein can be provided.

[0303] In some respects, test kit as described herein adopts suitable packaging. In some respects, suitable packaging includes vials, bottles, jars, soft packages (e.g., sealed Mylar or plastic bags) or combinations thereof. In some respects, packaging includes packaging used in combination with specific devices such as inhalers, nasal application devices (e.g., atomizers) or infusion devices such as miniature pumps. In some respects, test kit includes sterile access ports (e.g., containers can be intravenous solution bags or bottles with a stopper pierced by a hypodermic needle). In some respects, containers can also have sterile access ports (e.g., containers can be intravenous solution bags or bottles with a stopper pierced by a hypodermic needle). In some respects, at least one activating agent is a composition as described herein.

[0304] In some aspects, the kit further comprises additional components, such as buffer and interpretative information. In some aspects, the kit comprises a container and a label or package insert on or associated with the container. In some aspects, the disclosure provides an article comprising the contents of the kit described herein.

[0305] General techniques

[0306] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JBGiffiths and DG Newell, 1993-8) J. Wiley and Sons; Method of Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (FM Ausubel et al., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanette and JD Capra, ed., Harwood Academic Publishers, 1995). Without further elaboration, it is believed that based on the above description, one skilled in the art can utilize the present disclosure to the greatest extent. All publications cited herein (including those listed above and elsewhere in the present disclosure) are incorporated by reference in their entirety. .

[0307] Example

[0308] Example 1: Construction of LNPs containing VEE replicons

[0309] To construct the LNPs described herein, the following materials and methods were used:

[0310] Template preparation

[0311] The template DNA was linearized using the BspQI restriction enzyme. After linearization, the DNA was buffer exchanged on the filter for 6 diavolumes. This DNA was then subjected to IVT using T7 RNA polymerase and NTPs. After IVT, the remaining DNA was digested using DNaseI. The mRNA was then buffer exchanged prior to liquid chromatography. Subsequently, the mRNA was buffer exchanged again and then assayed for quality control purposes.

[0312] Preparation of conventional BL-1 LNP formulations with T-linkages

[0313] Lipid material is weighed and dissolved in ethanol separately.Ethanol phase is prepared by mixing all lipid materials according to the composition ratio of the following form.Water phase is prepared by diluting RNA molecule (for example, mRNA with VEE replicon or control mRNA without any VEE) with 20mM citrate buffer (pH 4.0), 300mM NaCl and water.Ethanol phase and water phase of mixed LNP are connected by T-with the flow rate ratio of 3:1 (aqueous phase: ethanol phase), then LNP is provided by online dilution with TBS buffer (pH 4.0) immediately connected via T-with the flow rate ratio of 1:1 (LNP phase: PEG phase).The following table 2 provides the relative amount of each component included in the LNP composition.

[0314] Table 2. BL-1 LNP composition

[0315]

[0316]

[0317] Preparation of micellar TT3 LNP formulations with T-linked PEG

[0318] Lipid material is weighed and dissolved in ethanol separately.Ethanol phase is prepared by mixing all lipid materials except DMG-PEG-2K according to the composition ratio of the following form.Water phase is prepared by diluting RNA molecule (for example, mRNA with VEE replicon or control mRNA without any VEE replicon) with 20mM citrate buffer (pH 4.0), 300mMNaCl and water.PEG micelle phase is prepared by adding DMG-PEG-2K of corresponding amount in TBS buffer and fully mixing via vortex.Finally, ethanol phase and aqueous phase of mixed LNP are connected by T-at first with the flow rate ratio of 3:1 (aqueous phase: ethanol phase), then with the flow rate ratio of 1:1 (LNP phase: PEG phase) via T-connection mixing immediately with PEG micelle phase and carry out online dilution to provide micelle TT3 LNP after PEG.Table 3 below provides the relative amount (as exemplary amount and weight ratio) of every kind of component included in LNP composition.

[0319] Table 3. TT3 LNP composition

[0320] Components Exemplary Amounts Weight Ratio mRNA 1.0mg 1.0 TT3 10mg 10.0 DOPE 8.0mg 8.0 cholesterol 5.6mg 5.6 DMG-PEG-2k 4.1mg 4.1

[0321] Table 3A.

[0322]

[0323]

[0324] Buffer exchange and freeze-thaw of BL-1 / TT3 LNPs

[0325] The obtained BL-1 / TT3 LNPs were buffer exchanged and concentrated via tangential flow filtration. Subsequently, 40% sucrose (W / V) in the TBS stock solution was added to all prepared BL-1 / TT3 LNPs to prepare a final solution of LNPs in 10% sucrose. The final RNA concentration of the LNPs was measured by dissociating the LNPs with 2% TE+Triton and further detected with Quant-itRiboGreen assay. The LNPs were divided into aliquots of 50 μg / tube and frozen at -80°C. Before administration with the LNPs, the LNPs were thawed at room temperature.

[0326] Example 2: Analysis of liver expression

[0327] To evaluate the ability of the LNPs constructed in Example 1 to target the liver, biodistribution analysis was performed in mice. Briefly, animals were administered the following and protein expression was assessed using a bioluminescence assay: (1) PBS (negative control); (2) LNPs loaded with control mRNA without any VEE replicon (RNA-no VEE); or (3) LNPs loaded with mRNA with VEE replicon (RNA-VEE).

[0328] like Figure 1 As shown, in animals receiving RNA-no VEE, there was significant protein expression in the liver. In contrast, in animals receiving RNA-VEE, almost no detectable protein expression was observed in the liver. Protein expression in other tissues (e.g., spleen and lung) was comparable in animals from the RNA-no VEE group and the RNA-VEE group, demonstrating the specificity of the effects observed in the liver.

[0329] The above results demonstrate that the LNPs described herein can selectively avoid and / or reduce expression in the liver, suggesting that the LNPs may allow for greater tolerability (eg, reduced hepatotoxicity).

[0330] Example 3: Assessment of Hepatotoxicity

[0331] To further evaluate the in vivo effects, the LNPs of the present disclosure will be administered to animals (e.g., mice) as described in Example 2. Hepatotoxicity will then be assessed in animals via various methods. For example, in some aspects, liver enzymes (ALT / AST) levels, such as in the blood of the animals, will be measured. In some aspects, the general toxicity of the animals (e.g., activity, food consumption, body score index, hydration status, or a combination thereof) will be assessed. In some aspects, microscopic assessment of the liver will be performed to detect any LNP-related effects on the liver.

[0332] Example 4: Construction of Additional Lipid Nanoparticles

[0333] To further evaluate the VEE replicons described herein, additional LNPs were constructed as described in Example 1 to include different lipids described herein (e.g., ionizable lipids, cationic lipids, or lipidoid materials, e.g., lipids). A total of nine different LNPs (referred to herein as LNP1-LNP9) were constructed and used to encapsulate mRNA with a VEE replicon (described herein) or a control mRNA without any VEE replicon. The ability of these LNPs to induce expression of the payload was evaluated as described in subsequent examples.

[0334] Example 5: Analysis of liver expression using lipid nanoparticles loaded with RNA bearing the VEE replicon

[0335] To further evaluate the ability of the VEE replicon described herein to selectively avoid and / or reduce expression in the liver, B6.Cg Gt (ROSA) 26Sortm14 (CAG-tdTomato) Hze / J (Ai reporter gene) mice received a single intravenous administration of LNP1 or LNP2. Both LNP1 and LNP2 were loaded with 20 μg of VEE replicon containing a nucleic acid sequence encoding a nuclear localization signal (NLS)-Cre. Control animals received administration of phosphate buffered saline (PBS). The Cre gene encodes the Cre protein, which is a site-specific DNA recombinase that catalyzes DNA recombination between specific sites in a DNA molecule. These sites are called loxP sequences and contain specific binding sites for Cre around a directional core sequence where recombination can occur. Cre induces tdTomato fluorescence, which indicates Cre-mediated recombination in liver tissue. About 72 hours (hrs) after LNP administration, tdTomato fluorescence expression in mouse liver was measured using fluorescent IVIS imaging.

[0336] like Figure 2A and 2B As shown, the fluorescence signals observed in the livers of mice receiving LNP1 and LNP2 were not significantly different from those observed in control animals. In addition, there was no significant difference in fluorescence expression in the liver between animals receiving LNP1 and LNP2. These results highlight the ability of the VEE replicon described herein to selectively avoid and / or reduce expression in the liver. The results further indicate that the liver avoidance properties of the VEE replicon are at least independent of any differences between LNP1 and LNP2.

[0337] Example 6: Analysis of liver expression using lipid nanoparticles loaded with RNA without VEE replicon

[0338] In order to confirm that LNP as described herein can indeed target liver, B6.Cg Gt (ROSA) 26Sortm14 (CAG-tdTomato) Hze / J (Ai reporter gene) mice received a single intravenous administration of LNP3, LNP4, LNP5 or LNP6. Each of the LNPs is loaded with 20 μg of mRNA (RNA-without VEE) without any VEE replicon and encoding nuclear localization signal (NLS)-Cre. Control animals receive the administration of PBS. About 72 hours after LNP administration, the tdTomato fluorescence expression in the mouse liver is measured again using fluorescent IVIS imaging. As described in Example 4, it is expected that the td-Tomato fluorescence expression in the liver will indicate the recombination mediated by Cre in the tissue.

[0339] like Figure 3A and 3B As shown, in control animals, no detectable fluorescent signal was observed in the liver of mice from the control group. In contrast, a strong fluorescent signal was observed in the liver of mice that received administration of one of the tested LNPs (each loaded with RNA-without VEE).

[0340] For further analysis, LNP7 (also loaded with mRNA without any VEE replicon, wherein the mRNA encodes NLS-Cre; RNA-VEE) was intravenously administered to tdTomato reporter mice. For comparison, some animals received the LNP3 used above (i.e., loaded with mRNA without any VEE replicon and encoding NLS-Cre). Control mice were treated with a single PBS. Similarly, about 72 hours after LNP administration, td-Tomato fluorescence was quantified using fluorescent IVIS imaging.

[0341] like Figure 4A and 4B As shown, a significant fluorescent signal was observed in the liver of animals receiving LNP7 compared to control animals. The fluorescent signal was comparable to that observed in mice treated with LNP3.

[0342] The results presented in this example collectively demonstrate that the LNPs described herein can indeed be targeted to the liver, suggesting that the liver avoidance and / or reduced liver expression observed in earlier examples is specific to the VEE replicon itself.

[0343] Example 7: Analysis of firefly luciferase expression in the liver after administration of lipid nanoparticles loaded with RNA with VEE replicon or RNA without any VEE replicon

[0344] In order to confirm the above results in different fluorescence models, C57BL / 6J mice received a single administration of one of the following LNPs: LNP8, LNP9 and LNP2. LNP9 and LNP2 were administered intravenously to animals. LNP8 was administered intravenously or intratumorally to animals. Each of the LNPs (i.e., LNP8, LNP9 and LNP2) was loaded with 40 μg of mRNA including VEE replicons and encoding FLuc, which is a firefly luciferase gene that encodes the enzyme that catalyzes the oxygenation of d-luciferin to oxyluciferin, and the reaction produces visible light (530nm-640nm). Control animals were administered with PBS. Approximately 24 hours after administration, the luciferase expression in the liver was measured using bioluminescent IVIS imaging.

[0345] As observed with td-Tomato reporter mouse above, compared with control animals, there is no significant difference in the luciferase expression in the liver of C57BL / 6J mice receiving LNP2 administration. Similar results were observed in C57BL / 6J mice receiving LNP8 or LNP9. In addition, the route of administration (at least between intravenous administration and intratumoral administration) does not seem to have any effect, because no significant difference in luciferase expression was observed in animals receiving LNP8 intravenously or intratumorally.

[0346] Again, to confirm that lipid nanoparticles themselves can indeed target the liver, LNP2, LNP8, and LNP9 were loaded with 10 μg of mRNA encoding firefly luciferase without any VEE replicon. A single dose of LNPs was then administered intravenously to C57BL / 6J mice, and about 24 hours later, luciferase expression in the liver was measured using bioluminescent IVIS imaging.

[0347] like Fig. 6A and 6B As shown, the bioluminescent signals in the liver were significantly higher for the LNP groups (including LNP8, LNP9, and LNP2) compared to the control, indicating that for each LNP tested, FLuc-mod RNA was specifically expressed in the liver.

[0348] The above results confirm that the LNPs described herein are capable of targeting the liver. The above results also help to confirm the liver avoidance properties of the VEE replicon described herein.

[0349] Example 8: Comparison of liver-specific delivery of lipid nanoparticles loaded with RNA with VEE replicon or RNA without any VEE replicon

[0350] To provide a direct comparison of liver specificity between mRNA without any VEE replicon (RNA-no VEE) and mRNA with VEE replicon (RNA-VEE), C57BL / 6J mice received a single intravenous administration of LNP8 loaded with 2 mg / kg (1) RNA-no VEE encoding firefly luciferase or (2) RNA-VEE encoding firefly luciferase. Then, approximately 48 hours after administration, luciferase expression in the liver of the animals was measured using bioluminescent IVIS imaging.

[0351] like Fig. 7A and 7B As shown, the bioluminescent signal in the liver of the RNA-no VEE group was significantly higher than that of the RNA-VEE replicon group. This result further confirms that RNA-no VEE allows expression in the liver, while RNA-VEE prevents expression in the liver. In addition, the results further demonstrate that the liver avoidance properties of the VEE replicon described herein are independent of the lipid nanoparticles used.

[0352] Example 9: Lipid Nanoparticles Comprising RNA with VEE Replicon and Ionizable Lipids

[0353] To evaluate the liver avoidance properties of the VEE replicons described herein, lipid nanoparticles comprising ionizable lipids will be constructed to prepare lipid nanoparticles, which will then be used to encapsulate mRNA encoding the VEE replicon and the payload (RNA-VEE). The ionizable lipid will be selected from the following: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)octanoic acid heptadecan-9-yl ester (SM-102), 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid heptadecan-9-yl ester (Lipid 5), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-(eicosyl)-1,2-dimethylamino)butanoyl)oxy)heptadecano ... 1,2-Dilinoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 6,9-heptadecanoic acid-6,9-dimethylaminopropane (DLin-DMA), 1,2-di ... 28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA ), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)) or any combination thereof.

[0354] The above LNPs (i.e., comprising ionizable lipids and RNA-VEE) will be administered in vivo, and the expression of the encoded payload will then be assessed in the liver (e.g., as described in Examples 4-7). In some aspects, hepatotoxicity will also be assessed in animals (e.g., as described in Example 3).

[0355] Example 10: Lipid Nanoparticles Comprising RNA with VEE Replicon and Cationic Lipids

[0356] To evaluate the liver avoidance properties of the VEE replicon described herein, lipid nanoparticles comprising cationic lipids will be constructed to prepare lipid nanoparticles, which will then be used to encapsulate mRNA (RNA-VEE) with the VEE replicon and encoding the payload. The cationic lipid will be selected from the following: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl ammonium bromide (DMRIE), N-(l,2-dioleoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), l,2-dilauroyl-sn- Glycerol-3-ethylphosphocholine (DLePC), l,2-distearoyl-3-trimethylammonium-propane (DSTAP), l,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), l,2-dilinoleoyl-3-trimethylammonium-propane (DLTAP), l,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-distearoyl-sn-glycerol-3-ethylphosphocholine (DSePC), l,2-dipalmitoyl-sn- Glycero-3-ethylphosphocholine (DPePC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC), 1,2-dioleoyl sn-glycero-3-ethylphosphocholine (DOePC), 1,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1EPC), or any combination thereof.

[0357] The above LNPs (i.e., comprising cationic lipids and RNA-VEE) will be administered in vivo, and the expression of the encoded payload will then be assessed in the liver (e.g., as described in Examples 4-7). In some aspects, hepatotoxicity will also be assessed in animals (e.g., as described in Example 3).

[0358] Example 10: Lipid Nanoparticles Comprising RNA with VEE Replicon and Lipidoid Material

[0359] To evaluate the liver avoidance properties of the VEE replicons described herein, lipid nanoparticles comprising lipidoid materials will be constructed to prepare lipidoid nanoparticles (LLNs), which will then be used to encapsulate mRNA encoding a payload (RNA-VEE) with the VEE replicon. The lipidoid material will be selected from the following: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine 2,5-dione (cKK-E12), tetrakis(8-methylnonyl) 3,3',3",3"'-((( methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10), G0-C14, 5A2-SC8, 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecadien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(ethane-2,1 -diyl)(9Z,9'Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetra(octadec-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))tetra(azanetriyl))tetra(butane-4,1-diyl)(9Z,9′Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetra(octadec-9,12-dienoate) -9,12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tri(3-(docosylamino)propyl)benzene 1,3,5-tricarboxamide (TT3), hexa(oct-3-yl) 9,9′,9″,9″′,9″″,9″″′-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))nonanoic acid hexyl ester (FTT5), PL-1 [published in Nature Communications, 12-7264 (2021), which is hereby incorporated by reference], 98N12-5 [published in Molecular Therapy Vol. 17 No. 5 May 2009, which is hereby incorporated by reference], ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18(A2)) and A12-Iso5-2DC18(A12), or any combination thereof.

[0360] The above LLPs (i.e., comprising lipidoid material and RNA-VEE) will be administered in vivo, and the expression of the encoded payload will then be assessed in the liver (e.g., as described in Examples 4-7). In some aspects, hepatotoxicity will also be assessed in animals (e.g., as described in Example 3).

[0361] Example 12: Lipid Nanoparticles Comprising RNA with VEE Replicon and Polymer Material

[0362] To evaluate the liver avoidance properties of the VEE replicons described herein, lipid nanoparticles comprising polymeric materials will be constructed to prepare polymeric nanoparticles (PNPs), which will then be used to encapsulate mRNA (RNA-VEE) encoding the VEE replicon. The polymeric material will be selected from the following: polyethyleneimine (PEI), poly(amidoamine) (PAMAM), poly(β-amino ester) (PBAE), poly(2-N,N-dimethylaminoethyl methacrylate) (PDMAEMA), poly(amino acids) (PAA), chitosan dextran, cyclodextrin, cellulose, hyaluronic acid, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL).

[0363] The above-described PNPs (i.e., comprising a polymeric material and RNA-VEE) will be administered in vivo, and the expression of the encoded payload will then be assessed in the liver (e.g., as described in Examples 4-7). In some aspects, hepatotoxicity will also be assessed in animals (e.g., as described in Example 3).

[0364] Table 4. Exemplary VEE replicon sequences

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] It should be understood that the Detailed Description section, rather than the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventors, and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0377] The present disclosure has been described above with the aid of functional building blocks that illustrate implementation of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may also be defined as long as the specified functions and their interrelationships are appropriately performed.

[0378] The foregoing description of specific aspects will fully reveal the general nature of the present disclosure so that others, by applying knowledge in the art, can easily modify and / or change such specific aspects for various applications without undue experimentation and without departing from the general concepts of the present disclosure. Therefore, based on the teachings and guidance presented herein, such changes and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects. It should be understood that the phrases or terms herein are for descriptive and non-limiting purposes, so that the terms or phrases of this specification will be interpreted by those skilled in the art according to teachings and guidance.

[0379] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. A method for reducing or avoiding expression of a heterologous protein in the liver of a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the heterologous protein.

2. The method of claim 1, wherein reducing the expression comprises (i) reducing the amount of the heterologous protein expressed in the liver, (ii) shortening the duration of expression of the heterologous protein in the liver, or (iii) both (i) and (ii), compared to a reference subject (e.g., a subject receiving the corresponding lipid nanoparticles but wherein the replicon is not a VEE replicon).

3. The method of claim 2, wherein after said administration, the amount of heterologous protein expressed in said liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to said reference subject.

4. The method of claim 3, wherein after said administering, said liver does not express said heterologous protein.

5. The method of claim 2 or 3, wherein after said administration, the duration of expression of said heterologous protein in said liver is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to said reference subject.

6. The method of any one of claims 1 to 5, wherein after said administration, said heterologous protein is expressed in a non-hepatic tissue of said subject, wherein said non-hepatic tissue is selected from the group consisting of spleen, lung, tumor, or a combination thereof.

7. The method of claim 6, wherein after said administration, the amount of the heterologous protein expressed in the non-hepatic tissue is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% or more compared to the amount observed in the non-hepatic tissue of the reference subject.

8. The method of claim 6 or 7, wherein after said administration, the duration of expression of said heterologous protein in said non-hepatic tissue is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% as compared to the duration observed in said non-hepatic tissue of said reference subject.

9. A method for selectively expressing a heterologous protein in a non-liver tissue of a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the heterologous protein.

10. The method of claim 9, wherein after the administration, the amount of the heterologous protein expressed in the non-hepatic tissue is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold the corresponding amount observed in the liver of the subject.

11. The method of claim 9 or 10, wherein after the administration, the duration of expression of the heterologous protein in the non-hepatic tissue is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or longer than the corresponding duration observed in the liver of the subject.

12. The method of any one of claims 9 to 11, wherein the non-hepatic tissue comprises spleen, lung, or both.

13. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein, and wherein following said administration, the heterologous protein is preferentially expressed in a non-hepatic tissue of the subject.

14. The method of claim 13, wherein after the administration, the amount of the heterologous protein expressed in the non-hepatic tissue is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold or more of the corresponding amount observed in the liver of the subject.

15. The method of claim 13 or 14, wherein after the administration, the duration of expression of the heterologous protein in the non-hepatic tissue is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or longer than the corresponding duration observed in the liver of the subject.

16. The method of any one of claims 13 to 15, wherein the non-hepatic tissue comprises spleen, lung, or both.

17. The method of any one of claims 13 to 16, wherein the disease or disorder comprises cancer, an inflammatory disorder, a monogenic disorder, a neurological disorder, a psychiatric disorder, or a combination thereof.

18. The method of any one of claims 17, wherein the cancer comprises melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver tumors, stomach cancer, head and neck cancer, or a combination thereof.

19. A method for increasing tolerability of a lipid nanoparticle-based therapy in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle comprising (i) one or more types of lipids, and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein.

20. The method of claim 19, wherein the tolerability of the lipid nanoparticle-based therapy is increased in the subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold or more compared to the corresponding tolerability observed in a reference subject (e.g., a subject receiving the corresponding lipid nanoparticle but wherein the replicon is not a VEE replicon).

21. The method of claim 20, wherein the increased tolerability of the lipid nanoparticle-based therapy is associated with reduced liver toxicity.

22. The method of claim 21, wherein following said administering, said subject exhibits reduced hepatotoxicity compared to corresponding hepatotoxicity observed in said reference subject.

23. The method of claim 22, wherein the hepatotoxicity in the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the hepatotoxicity in the reference subject.

24. The method of any one of claims 19 to 23, wherein following said administration, said subject exhibits reduced expression of said heterologous protein in said liver compared to corresponding expression observed in said liver of said reference subject.

25. The method of claim 24, wherein following the administration, the amount of the heterologous protein expressed in the liver of the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% as compared to the amount observed in the liver of the reference subject.

26. The method of claim 24 or 25, wherein after said administration, the duration of expression of said heterologous protein in said liver of said subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% as compared to the duration observed in said liver of said reference subject.

27. The method of any one of claims 19 to 26, wherein the amount of heterologous protein expressed in the non-hepatic tissue of the subject is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% or more compared to the amount observed in the non-hepatic tissue of the reference subject.

28. The method of any one of claims 19 to 27, wherein the duration of expression of the heterologous protein in the non-hepatic tissue of the subject is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% compared to the reference subject.

29. A method of producing a lipid nanoparticle-based therapy associated with reduced liver toxicity, the method comprising combining (i) lipid nanoparticles comprising one or more types of lipids and (ii) a replicon derived from Venezuelan equine encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein.

30. The method of any one of claims 1 to 29, wherein the VEE replicon has the nucleotide sequence shown in SEQ ID NO:

187.

31. The method of any one of claims 1 to 30, wherein the heterologous protein comprises a cytokine, an antibody or antigen-binding fragment thereof, a chimeric antigen receptor, or a combination thereof.

32. The method of claim 31, wherein the cytokine comprises interleukin (IL)-12 protein.

33. The method of claim 32, wherein the nucleic acid sequence of the VEE replicon comprises: (i) a nucleic acid sequence encoding an IL-12 β subunit and covalently linked to SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:

75. The sequence shown in NO:75 has a nucleotide sequence of at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity;(ii) Encoding IL-12 alpha subunit and corresponding to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124 or SEQ ID The sequence shown in NO:125 has a nucleotide sequence that is at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical; or (iii) both of (i) and (ii). ; 34. The method of claim 33, wherein the cytokines do not comprise IL-12 protein.

35. The method of any one of claims 1 to 34, wherein the one or more types of lipids comprise ionizable lipids, cationic lipids, lipids, phospholipids, sterols, or combinations thereof.

36. The method of claim 35, wherein the one or more types of lipids include TT3, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, C14-PEG2000, or a combination thereof.

37. The method of claim 36, wherein the one or more types of lipids are TT3.

38. The method of claim 36 or 37, wherein the C14-PEG2000 comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), or both.

39. The method of any one of claims 36 to 38, wherein the C14-PEG2000 is embedded in the lipid nanoparticles.

40. The method of any one of claims 36 to 38, wherein the C14-PEG2000 is added after the isolated polynucleotide has been encapsulated in the lipid nanoparticle.

41. The method of any one of claims 1 to 40, wherein the lipid nanoparticle has a diameter of about 30-500 nm.

42. The method of any one of claims 1 to 41, wherein the lipid nanoparticle has a diameter of about 50-400 nm.

43. The method of any one of claims 1 to 42, wherein the lipid nanoparticle has a diameter of about 70-300 nm.

44. The method of any one of claims 1 to 43, wherein the lipid nanoparticle has a diameter of about 100-200 nm.

45. The method of any one of claims 1 to 44, wherein the lipid nanoparticle has a diameter of about 100-175 nm.

46. ​​The method of any one of claims 1 to 45, wherein the lipid nanoparticle has a diameter of about 100-160 nm.

47. The method of any one of claims 1 to 46, wherein the one or more types of lipids have a mass ratio to the VEE replicon of about 1:2 to about 15:

1.

48. The method of claim 47, wherein the one or more types of lipids have a mass ratio to the VEE replicon of 1:2, 1:1.5, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:

1.

49. The method of any one of claims 1 to 48, wherein the VEE replicon comprises a 5'-cap.

50. The method of any one of claims 1 to 49, wherein the VEE replicon further comprises a regulatory element.

51. The method of claim 50, wherein the regulatory element is selected from the group consisting of: at least one translation enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or a seed thereof, a 3' tailing region of linked nucleosides, an AU-rich element (ARE), a post-transcriptional control regulator, a 5'UTR, a 3'UTR, and a combination thereof.

52. The method of claim 51, wherein the 3' tailing region of linked nucleosides comprises a poly-A tail, a polyA-G quadruplex, or a stem-loop sequence.

53. The method of any one of claims 1 to 52, wherein the VEE replicon comprises at least one modified nucleoside.

54. The method of claim 53, wherein the at least one modified nucleoside is selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrole-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine and combinations thereof.

55. The method of any one of claims 1 to 54, wherein the one or more types of lipids have a mass ratio of about 10:1 to the VEE replicon.

56. The method of any one of claims 1 to 55, wherein the lipid nanoparticles are administered to the subject via intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, transdermal, sublingual, submucosal, transdermal, or transmucosal administration.

57. The method of any one of claims 1 to 56, further comprising administering to the subject at least one additional therapeutic agent.

58. The method of claim 57, wherein the at least one additional therapeutic agent comprises a chemotherapeutic drug, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of a co-stimulatory molecule, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.

59. The method of claim 58, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM3 antibody, or any combination thereof.

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