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

Abstract

The present disclosure relates to lipid nanoparticles (LNPs) containing a VEE replicon, which are useful for selectively reducing and / or avoiding expression of a payload in the liver. The present disclosure also relates to the use of such LNPs to treat various diseases and disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 335,092, filed April 26, 2022, which is incorporated by reference in its entirety.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the Sequence Listing have been submitted electronically (Name: 4597_016PC01_SequenceListing_ST26.XML; Size: 410,263 bytes; and Creation Date: April 24, 2023) and submitted with this application, which is hereby incorporated by reference in its entirety. [Background technology]

[0003] Nucleic acid-based therapy (e.g., mRNA vaccines) is becoming an increasingly important approach for the treatment of various diseases. Upon administration, the encoded protein can be produced to induce an immune response (e.g., tumor antigens to induce anti-tumor immune responses) or to provide important functions to maintain a healthy state. However, current delivery platforms for delivering nucleic acid molecules are ineffective or have undesirable side effects, limiting the full potential of nucleic acid-based therapy. For example, when mRNA is involved, lipid nanoparticles (LNPs) are commonly used to encapsulate the mRNA. Also, to improve LNP delivery, much effort has been focused on identifying novel lipids or specific lipid compositions that can affect the intracellular delivery and / or expression of mRNA in various types of mammalian tissues, organs and / or cells (e.g., mammalian liver cells). However, these existing approaches are costly, time-consuming, and unpredictable. In addition, LNPs are also associated with hepatotoxicity when administered in vivo. Thus, there is a need in the art for improved LNPs that can provide a safer and more effective means of delivering nucleic acids to subjects. Summary of the Invention

[0004] Provided herein is a method of reducing or avoiding expression of a heterologous protein in the liver in a subject in need thereof, comprising administering to the subject (i) one or more lipids and (ii) a lipid nanoparticle comprising a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), where the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein. In some embodiments, reducing expression comprises (i) reducing the amount of heterologous protein expressed in the liver, (ii) reducing 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 administered the corresponding lipid nanoparticle, but where the replicon is not a VEE replicon).

[0005] In some embodiments, 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 that of the reference subject. In some embodiments, after administration, the liver does not express the heterologous protein.

[0006] In some embodiments, following 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 that of the reference subject.

[0007] In some embodiments, 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 embodiments, after administration, the amount of 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 that observed in the non-liver tissue of the reference subject.In some embodiments, the duration of expression of the heterologous protein 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% compared to that observed in the non-liver tissue of the reference subject.

[0008] Also provided herein is a method for selectively expressing a heterologous protein in non-liver tissue in a subject in need thereof, comprising administering to the subject (i) one or more lipids and (ii) lipid nanoparticles comprising a replicon derived from Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the heterologous protein.

[0009] In some embodiments, after administration, the amount of heterologous protein expressed in non-liver 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 greater than the corresponding amount observed in the liver of the subject. In some embodiments, after administration, the duration of expression of the heterologous protein in non-liver 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 greater than the corresponding duration observed in the liver of the subject. In some embodiments, the non-liver tissue comprises the spleen, the lung, or both.

[0010] Disclosed herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject lipid nanoparticles comprising (i) one or more 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, wherein following administration, the heterologous protein is preferentially expressed in non-liver tissues of the subject.

[0011] In some embodiments, following administration, the amount of heterologous protein expressed in non-liver 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 greater than the corresponding amount observed in the liver of the subject. In some embodiments, following administration, the duration of expression of the heterologous protein in the non-liver 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 longer than the corresponding duration observed in the liver of the subject. In some embodiments, the non-liver tissue comprises the spleen, lung, or both.

[0012] In some embodiments, the disease or disorder that can be treated with the present disclosure includes cancer, inflammatory disorder, single gene disorder, neurological disorder, psychiatric disorder, or a combination thereof. In some embodiments, the 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, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, head and neck cancer, or a combination thereof.

[0013] The present disclosure further provides a method of improving the tolerability of a lipid nanoparticle-based therapy in a subject in need thereof, comprising administering to the subject (i) one or more lipids and (ii) a lipid nanoparticle comprising a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), where the VEE replicon comprises a nucleic acid sequence encoding a heterologous protein.

[0014] In some embodiments, the tolerability of the lipid nanoparticle-based therapy 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 greater in a subject than the corresponding tolerability observed in a reference subject (e.g., a subject administered the corresponding lipid nanoparticle but where the replicon is not a VEE replicon).

[0015] In some embodiments, the improved tolerability of lipid nanoparticle-based therapeutics is associated with reduced hepatotoxicity.In some embodiments, after administration, the subject exhibits reduced hepatotoxicity compared to the corresponding hepatotoxicity observed in reference subjects.In some embodiments, 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 that of the reference subject.

[0016] In some embodiments, following administration, the subject exhibits reduced expression of the heterologous protein in the liver, compared to the corresponding expression observed in the liver of a reference subject.

[0017] In some embodiments, after administration, the amount of 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 that observed in the liver of the reference subject. In some embodiments, after administration, the duration of expression of the heterologous protein 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 that observed in the liver of the reference subject. In some embodiments, the amount of heterologous protein expressed in the non-liver 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 that observed in the non-liver tissue of the reference subject. In some embodiments, the duration of expression of the heterologous protein in the non-liver 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 that of the reference subject.

[0018] Provided herein is a method for producing a lipid nanoparticle-based therapy associated with reduced liver toxicity, the method comprising combining (i) a lipid nanoparticle comprising one or more lipids, and (ii) a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon is a nucleic acid sequence encoding a heterologous protein.

[0019] In any of the methods provided herein, in some embodiments, the VEE replicon has a nucleotide sequence set forth in SEQ ID NO: 187. In some embodiments, the heterologous protein comprises a cytokine, an antibody or an antigen-binding fragment thereof, a chimeric antigen receptor, or a combination thereof. In some embodiments, the cytokine comprises an interleukin (IL)-12 protein.

[0020] In some embodiments, the nucleic acid sequence of the VEE replicon (i) encodes an IL-12 β subunit and has at least about 7 amino acid sequence similar to the sequence set forth in 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. 5%, 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 (ii) a nucleotide sequence encoding an IL-12 α subunit having about 100% sequence identity 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: 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 (iii) a nucleotide sequence that is about 100% identical to (i) and (ii).

[0021] In some embodiments, the cytokine does not include an IL-12 protein.

[0022] In some embodiments, the one or more lipids include ionized lipids, cationic lipids, lipidoids, phospholipids, sterols, or combinations thereof. In some embodiments, the one or more lipids include TT3, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, C14-PEG2000, or combinations thereof. In some embodiments, the one or more lipids are TT3. In some embodiments, the C14-PEG2000 includes 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG2000), or both. In some embodiments, the C14-PEG2000 is embedded in lipid nanoparticles. In some embodiments, the C14-PEG2000 is added after the isolated polynucleotide is encapsulated in the lipid nanoparticle.

[0023] In some embodiments, the lipid nanoparticles have a diameter of about 30-500 nm. In some embodiments, the lipid nanoparticles have a diameter of about 50-400 nm. In some embodiments, the lipid nanoparticles have a diameter of about 70-300 nm. In some embodiments, the lipid nanoparticles have a diameter of about 100-200 nm. In some embodiments, the lipid nanoparticles have a diameter of about 100-175 nm. In some embodiments, the lipid nanoparticles have a diameter of about 100-160 nm.

[0024] In some embodiments, the one or more lipids and the VEE replicon have a mass ratio of about 1:2 to about 15: 1. In some embodiments, the one or more lipids and the VEE replicon have a mass ratio 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.

[0025] In some embodiments, the VEE replicon comprises a 5'-cap.

[0026] In some embodiments, the VEE replicon further comprises a regulatory element. In some embodiments, the regulatory element is selected from the group consisting of at least one translational enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or its seed, a 3' tailing region of linked nucleosides, an AU-rich element (ARE), a post-transcriptional regulatory modulator, a 5' UTR, a 3' UTR, and combinations thereof. In some embodiments, the 3' tailing region of linked nucleosides comprises a polyA tail, a polyAG quartet, or a stem-loop sequence.

[0027] In some embodiments, the VEE replicon comprises at least one modified nucleoside. In some embodiments, the at least one modified nucleoside is 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- Selected from the group consisting of guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 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, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, and combinations thereof.

[0028] In some embodiments, the one or more lipids and the VEE replicon have a mass ratio of about 10:1.

[0029] In some embodiments, the lipid nanoparticles are administered to a subject via intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration.

[0030] In any of the methods provided herein, in some embodiments, the method further comprises administering at least one additional therapeutic agent to the subject. In some embodiments, the at least one additional therapeutic agent comprises a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic agent, a cytotoxic agent, an immune system therapy, a cytokine, a surgical procedure, a radiation treatment, an activator of a costimulatory molecule, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. In some embodiments, 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. [Brief description of the drawings]

[0031] [Figure 1] A comparison of luminescence expression in the liver of mice after intravenous administration of one of the following: (1) RNA without any VEE replicon (RNA-No VEE), (2) RNA with the VEE replicon (RNA-VEE), and (3) PBS. For comparison, luminescence expression in the kidney, spleen, and lung are also shown. [Diagram 2] Comparison of the fluorescent expression of td-Tomato protein in the liver of mice after a single intravenous administration of LNP1 or LNP2. Both LNP1 and LNP2 contained a VEE replicon and encapsulated mRNA encoding Cre recombinase fused to a nuclear localization signal (Cre-NLS). Control animals received PBS. In A, the fluorescent expression is shown as % radiant efficiency (normalized to control). In B, the fluorescent expression is shown as mean radiant efficiency. In both figures, each symbol represents one organ measured and error bars represent standard error of the mean. Differences in fluorescent intensity between LNP and control groups were statistically analyzed using a two-tailed unpaired Student's t-test (N=3). [Diagram 3]Shows the comparison of fluorescence expression of td-Tomato protein in the liver of mice after single intravenous administration of LNP3, LNP4, LNP5, or LNP6. Each of LNP3-LNP6 contained no VEE replicon (RNA-VEE free) and encapsulated mRNA encoding Cre recombinase fused to a nuclear localization signal (Cre-NLS). PBS was administered to control animals. In A, fluorescence expression is shown as mean emission efficiency % (normalized to control). In B, fluorescence expression is shown as mean emission efficiency. [Figure 4] Shows the comparison of fluorescence expression of td-Tomato protein in the liver of mice after single intravenous administration of LNP7 or LNP3. Both LNP3 and LNP7 contained no VEE replicon (RNA-VEE free) and encapsulated mRNA encoding Cre recombinase fused to a nuclear localization signal (Cre-NLS). PBS was administered to control animals. In A, fluorescence expression is shown as emission efficiency % (normalized to control). In B, fluorescence expression is shown as mean emission efficiency. [Diagram 5] Shows the comparison of luciferase expression in the liver of mice after single administration of LNP8, LNP9, or LNP2, which all contained VEE replicon and encapsulated mRNA encoding firefly luciferase. LNP8 was administered to animals either intravenously (IV) or intratumorally (IT). LNP9 and LNP2 were administered to animals intravenously. PBS was administered to control animals. In A, luciferase expression is shown as emission % (normalized to control). In B, luciferase expression is shown as mean emission. In each figure, each symbol represents one measured organ, and error bars represent the standard error of the mean. [Figure 6]Comparison of firefly luciferase expression in mouse liver after a single intravenous administration of LNP8, LNP9, or LNP2, all of which do not contain any VEE replicon (no RNA-VEE) and are encapsulated with mRNA encoding firefly luciferase. Control animals received PBS. In A, luciferase expression is shown as % emission (normalized to control). In B, luciferase expression is shown as mean emission. In both figures, each symbol represents one organ measured and error bars represent standard error of the mean. Differences in bioluminescence intensity between LNP and control groups were statistically analyzed using a two-tailed unpaired Student's t-test (N=5). [Figure 7] Comparison of firefly luciferase expression in mouse liver after a single dose of either (a) LNP8 without any VEE replicon (no RNA-VEE), encapsulated with mRNA encoding firefly luciferase, or (b) LNP8 containing a VEE replicon and encapsulated with mRNA encoding firefly luciferase. In A, luciferase expression is shown as % emission (normalized to control). In B, luciferase expression is shown as mean emission. In both figures, each symbol represents one organ measured and error bars represent standard error of the mean. Differences in bioluminescence intensity between LNP and control groups were statistically analyzed using a two-tailed unpaired Student's t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure is directed to lipid nanoparticles (LNPs) and uses of the LNPs for selectively delivering a payload (e.g., a heterologous protein) in a subject. More specifically, the LNPs of the present disclosure include (i) one or more lipids, and (ii) a replicon ("VEE replicon") derived from the Venezuelan Equine Encephalitis (VEE) virus, where the VEE replicon includes a nucleic acid sequence encoding a payload. As provided herein, in some embodiments, the LNPs described herein, when administered to a subject, are capable of selectively avoiding and / or preventing expression of the encoded payload in the liver of the subject. Thus, in some embodiments, the LNPs of the present disclosure are associated with reduced hepatotoxicity compared to other delivery platforms available in the art. Additional aspects of the present disclosure are provided throughout the application.

[0033] Prior to describing the disclosure in more detail, it should be understood that the disclosure is not limited to specific compositions or process steps, as the specific compositions or process steps described can of course vary. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has separate components and features that can be readily separated from or combined with the features of any of the other aspects without departing from the scope or spirit of the disclosure. Any method described can be carried out in the order of events described or in any other order that is logically possible.

[0034] The headings provided herein are not limitations of various aspects of the disclosure, which may be defined by reference to the specification as a whole. It is also to 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.

[0035] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0036] Throughout this disclosure, the term "a" entity or "an" entity refers to one or more of that entity; for example, "a polynucleotide" is understood to refer to one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0037] Furthermore, as used herein, "and / or" shall be considered a specific disclosure of each of the two specified features or components, with or without the other. Thus, 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" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass 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 (single); B (single); and C (single).

[0038] As used herein, where an embodiment is described using the word "comprising," it is understood that similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0039] The term "about" is used herein to mean approximately, approximately, around, around, or within a range. When this 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, the term "about" is used herein to adjust a numerical value above or below the stated value by a variability of 10 percent above or below (high or low), unless otherwise indicated.

[0040] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included 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 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When at least is before a series of numbers or ranges, it is understood that "at least" can modify each number in the series or range. "At least" is not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, without considering the number of significant digits.

[0041] The term "derived from" as used herein refers to a component that is isolated or made 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 contain a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of a nucleotide or polypeptide, the derived species can be obtained, for example, by naturally occurring mutagenesis, artificial specific mutagenesis, or artificial random mutagenesis. The mutagenesis used to derive a nucleotide or polypeptide can be intentionally specific or intentionally random, or a combination of each. Mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from an initial nucleotide or polypeptide can be a random event (e.g., caused by polymerase infidelity), and the identification of the derived nucleotide or polypeptide can be performed by a suitable screening method, such as those described herein.In some embodiments, the nucleotide or amino acid sequence derived from the second nucleotide or amino acid sequence is 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%, or about 100% sequence identity, and wherein the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

[0042] "Nucleic acid", "nucleic acid molecule", "nucleotide sequence", "nucleic acid sequence", "polynucleotide", and grammatical variations thereof are used interchangeably and refer to a phosphate polymer of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), either in single-stranded form or in a double-stranded helix, or any phosphoester analogues thereof, such as phosphorothioates and thioesters. 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 terms nucleic acid molecule, and particularly DNA or RNA molecule, refer only to the primary and secondary structure of the molecule and are not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When describing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure includes one or more nucleic acids as described herein. As described herein, a polynucleotide of the present disclosure includes DNA, RNA, or both.In some embodiments, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), spliced ​​or unspliced, including tRNA, rRNA, shRNA, siRNA, miRNA, and mRNA, any other type of polynucleotide that is an N- or C-glycoside of purine or pyrimidine bases, as well as other polymers that contain non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other sequence-specific synthetic nucleic acid polymers, provided they contain nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA.

[0043] The term "replicon" as used herein refers to a polynucleotide that comprises an origin of replication that allows the polynucleotide to replicate in a cell of interest. As further described herein, in some embodiments, the replicon is a self-amplifying mRNA (i.e., capable of inducing its own amplification or replication in a target cell) (also referred to herein as "repRNA" or "RNA-VEE"). In order to induce its own amplification, the RNA molecule must encode the enzyme(s) required to catalyze RNA amplification (e.g., alphavirus nonstructural proteins nsP1, nsP2, nsP3, nsP4) and contain the cis-RNA sequences required for replication that are recognized and utilized by the encoded enzyme(s). An alphavirus RNA vector replicon must contain the following order of elements: 5' viral or cellular sequences required for nonstructural protein mediated amplification (which may also be referred to as 5'CSE, or 5' cis replication sequence, or 5' viral sequences in cis required for replication, or 5' sequences capable of initiating alphavirus transcription), sequences encoding alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4) that are biologically active when expressed, and 3' viral or cellular sequences required for nonstructural protein mediated amplification (which may also be referred to as 3'CSE, or 3' viral sequences in cis required for replication, or alphavirus RNA polymerase recognition sequence). An alphavirus RNA vector replicon may contain, in certain embodiments, a means for expressing one or more heterologous sequences, such as an IRES or viral (e.g., alphavirus) subgenomic promoter (e.g., junction region promoter), which may be modified to increase or decrease viral transcription of the subgenomic fragment or to decrease homology with deleted helper or structural protein expression cassettes, and the one or more heterologous sequences to be expressed. A replicon may also contain additional sequences, such as one or more heterologous sequences encoding one or more polypeptides (eg, a protein-encoding gene or a 3' proximal gene) and / or a polyadenylation region.The replicon should not contain sequences encoding all of the alphavirus structural proteins (capsid, E1, E2). Non-limiting examples of heterologous sequences that can be expressed by the replicon vector are described, for example, in U.S. Pat. No. 6,015,686 (incorporated herein by reference in its entirety), and include, for example, antigens, lymphokines, cytokines, and the like. As is evident from this disclosure, the VEE replicon provided herein is a repRNA. Thus, the terms "VEE replicon", "RNA-VEE", and "repRNA" are used interchangeably in this application. Furthermore, the terms "mRNA without any VEE replicon", "RNA without any VEE replicon", and "no RNA-VEE" are used interchangeably herein with the term "modRNA".

[0044] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", and grammatical variations thereof, include any of the agents approved by a regulatory agency of the U.S. Federal government for use in animals, including humans, or listed in the United States Pharmacopeia, as well as any carrier or diluent that does not produce undesirable physiological effects sufficient to prohibit the administration of the compound to a subject, and does not inhibit the biological activity and properties of the compound being administered. Included are excipients and carriers that are generally safe, non-toxic, and desirable, and are useful in the preparation of pharmaceutical compositions.

[0045] As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, such as, for example, the polynucleotides of the present disclosure, mixed or admixed with or suspended in one or more other chemical components, such as pharma- ceutically acceptable carriers and excipients.

[0046] 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 polymers may contain modified amino acids. These terms also encompass naturally modified amino acid polymers or amino acid polymers modified by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), and other modifications known in the art. The term "polypeptide" as used herein refers to proteins, polypeptides, and peptides of any size, structure, or function.

[0047] Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.

[0048] A polypeptide may be a single polypeptide or a multi-molecular complex such as a dimer, trimer or tetramer. It may include single chain polypeptides or multi-chain polypeptides. In multi-chain polypeptides, disulfide linkages are most commonly found. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a "peptide" may be about 50 amino acids or less in length, 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 in length.

[0049] The terms "coding sequence" or "encoding" sequence are used herein to mean a DNA or RNA region (transcribed region) that "encodes" a particular protein, such as IL-12. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide, either in vitro or in vivo, when placed under the control of appropriate regulatory regions, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic organisms, genomic DNA from prokaryotic or eukaryotic organisms, and synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0050] Kozak consensus sequence, Kozak consensus or Kozak sequence is known as a sequence present in eukaryotic mRNA and has the consensus (gcc)gccRccAUGG (SEQ ID NO: 174), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) followed by another "G". In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least about 95% or more, e.g., at least 99% sequence identity to the Kozak consensus sequence. In some embodiments, the polynucleotide comprises a Kozak consensus sequence.

[0051] The term "RNA" is used herein to mean a molecule that includes at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially or completely purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. The term "mRNA" means "messenger RNA" and relates to a "transcript" that is produced using a DNA template and encodes a peptide or protein. Typically, mRNA includes a 5'-UTR, a protein coding region and a 3'-UTR. mRNA has a limited half-life in cells and in vitro. In the context of the present disclosure, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. In some embodiments of the present disclosure, the RNA, preferably the mRNA, is modified with a 5'-cap structure.

[0052] As used herein, the term "identity" (e.g., sequence identity) refers to the overall monomer conservation between polymer molecules, e.g., between polynucleotide molecules. The term "identical" without any additional modifier, e.g., 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."

[0053] Calculation of percent identity of two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences to obtain optimal comparison performance (e.g., gaps can be introduced into one or both of the first and second polypeptide or polynucleotide sequences to obtain optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some embodiments, 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, or in the case of polynucleotides, bases at corresponding amino acid positions are then compared.

[0054] If a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences depends on the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be achieved using mathematical algorithms.

[0055] Suitable software programs that can be used to align different sequences (e.g., polynucleotide sequences) are available from various sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses the BLASTN or BLASTP algorithm to perform comparisons between two sequences. BLASTN is used to compare nucleic acid sequences, whereas BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are available from the European Bioinformatics Institute (EBI) at worldwideweb.ebi.ac.uk / Tools / psa.

[0056] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, and the like.

[0057] Different regions in a target sequence of a polynucleotide or polypeptide that is aligned with a reference sequence of a polynucleotide or polypeptide can have different sequence identity percentages.Please note that the sequence identity percentage value is rounded to one decimal place.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.Please also note that the length value is always an integer.

[0058] In some embodiments, the percentage 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), where Y is the number of amino acid residues (or nucleic acid bases) scored as identical matches in an alignment of the first and second sequences (either by visual inspection or by 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.

[0059] Those skilled in the art will understand that the generation of sequence alignment for calculating the sequence identity percentage is not limited to the comparison between two sequences that are exclusively derived by primary sequence data. It will also be understood that sequence alignment can be generated by integrating sequence data with data of different origins, such as structural data (e.g., protein crystal structure), functional data (e.g., mutation location), or phylogenetic data. A suitable program for integrating data of different origins to generate multiple sequence alignment is T-Coffee, available at www.tcoffee.org, or alternatively, for example, from EBI. It will also be understood that the final alignment used to calculate the sequence identity percentage can be curated either automatically or manually.

[0060] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably to refer to a state of preparation of a desired composition of the disclosure, e.g., a polynucleotide of the disclosure, that has been subjected to one or more purification processes. In some embodiments, isolation or purification, as used herein, is a process of removing, partially (e.g., a fraction of), a composition of the disclosure from a sample that contains contaminants.

[0061] The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product, e.g., an RNA or a polypeptide (e.g., a therapeutic protein, e.g., a coronavirus protein). Expression includes, but is not limited to, transcription of a polynucleotide into a microRNA binding site, a small hairpin RNA (shRNA), a short interfering RNA (siRNA), or any other RNA product. Expression includes, but is not limited to, transcription of a polynucleotide into a messenger RNA (mRNA), and translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be a nucleic acid, e.g., an RNA, produced by transcription of a gene. As used herein, a gene product can be a nucleic acid, RNA or miRNA, produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids that have undergone post-transcriptional modification, e.g., polyadenylation or splicing, or polypeptides that have undergone post-translational modification, e.g., phosphorylation, methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0062] "Heterologous" with respect to a polypeptide or polynucleotide portion that is part of a larger polypeptide or polynucleotide refers to a polypeptide or polynucleotide that is derived from a different polypeptide or polynucleotide, respectively, than the remainder of the polypeptide or polynucleotide molecule. The additional heterologous components of the polypeptide or polynucleotide may be derived from the same organism as the remaining polypeptide or polynucleotide, respectively, described herein, or the additional components may be derived from a different organism. For example, a heterologous polypeptide may be synthetic or derived from a different species, a different cell type of an individual, or the same or different cell type of a different individual. As described herein, a protein (or polypeptide) encoded by an ORF of a polynucleotide described herein is heterologous to the polynucleotide.

[0063] The term "half-life" refers to the time required to eliminate half of the activity, amount, or number of a molecule. In the context of this disclosure, the half-life of an RNA indicates the stability of said RNA. "Onset" or "progression" of a disease refers to the initial signs of a disease and / or its subsequent progression. Onset of a disease can be detected and assessed using standard clinical techniques, as is well known in the art. However, onset also refers to progression, which may be unpredictable. As used herein, onset or progression refers to the biological course of a symptom. Onset includes onset, recurrence, and manifestation. As used herein, manifestation or onset of a target disease or disorder includes initial onset and / or recurrence.

[0064] II. Methods of the Disclosure II.A. Methods of Selective Expression The present disclosure generally relates to the delivery of a payload (also referred to herein as a "biologically active molecule") to cells using lipid nanoparticles (LNPs). LNPs useful in the present disclosure are modified or engineered such that the LNPs exhibit tissue-specific tropism. As used herein, "tissue-specific tropism" refers to the ability of an LNP to target a particular tissue and not other tissues. For example, as shown herein, the LNPs described herein are capable of inducing expression of a payload in non-liver tissues (e.g., spleen and lung), but not in the liver. Without wishing to be bound by any theory, applicants have determined that modifying or engineering LNPs to include a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon") allows for selective delivery of the payload, thereby avoiding or reducing expression of the payload in the liver.

[0065] Thus, in some embodiments, provided herein is a method for reducing or avoiding expression of a payload (e.g., a heterologous protein) in the liver in a subject in need thereof, comprising administering to the subject (i) one or more lipids and (ii) a lipid nanoparticle comprising a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding the payload.

[0066] In some embodiments, expression of the payload is completely avoided (i.e., the payload is not expressed in the liver after administration). In some embodiments, expression of the payload in the liver is reduced compared to non-liver tissues (e.g., spleen and lung) in the subject. For example, in some embodiments, expression of the payload in the liver after administration 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 non-liver tissues. In some embodiments, expression of the payload in the liver is reduced compared to expression of the payload in the liver of a reference subject (e.g., a subject administered the corresponding lipid nanoparticle that does not contain the VEE replicon). In some embodiments, 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 corresponding expression in the liver of a reference subject.

[0067] As used herein, "reducing expression" refers to (i) reducing the total amount of payload expressed in a tissue (e.g., liver), (ii) reducing the time period (i.e., duration) that the payload is expressed in a tissue (e.g., liver), or (iii) both (i) and (ii).

[0068] Thus, in some embodiments, after administration of the 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 lung). In some embodiments, 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 tissues. In some embodiments, 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 administered a corresponding lipid nanoparticle that does not comprise a VEE replicon). In some embodiments, following administration of an LNP described herein (e.g., comprising a VEE replicon that includes 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.

[0069] As described herein, in some embodiments, following administration of a LNP described herein (e.g., a VEE replicon comprising a nucleic acid sequence encoding a payload), the payload is only transiently expressed in the liver, e.g., compared to the corresponding expression in a non-liver tissue and / or compared to the corresponding expression in the liver of a reference subject (e.g., a subject administered a corresponding lipid nanoparticle not comprising a VEE replicon). Thus, in some embodiments, administration of the LNP of the present disclosure reduces the duration of payload expression in the liver compared to non-liver tissues (e.g., spleen and lung). In some embodiments, the duration of payload expression 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 duration in a non-liver tissue. In some embodiments, after administration of a LNP described herein (e.g., comprising a VEE replicon that includes a nucleic acid sequence encoding a payload) to a subject in need thereof, the duration of payload expression in the liver of the subject is reduced compared to the duration of payload expression in the liver of a reference subject (e.g., a subject administered a corresponding lipid nanoparticle that does not include a VEE replicon). In some embodiments, the duration of payload expression 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 corresponding duration in the liver of the reference subject.

[0070] In some embodiments, after administration of the LNPs described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload), both (i) the total amount of payload expressed and (ii) the duration of payload expression in the liver are reduced compared to non-liver tissues (e.g., spleen and lung). In some embodiments, both (i) the total amount of payload expressed and (ii) the duration of 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 the corresponding values ​​in the non-liver tissues. In some embodiments, after administration of the LNPs described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload), both (i) the total amount of payload expressed and (ii) the duration of payload expression in the liver are reduced compared to the corresponding values ​​in the liver of a reference subject (e.g., a subject administered a corresponding lipid nanoparticle that does not comprise a VEE replicon). In some embodiments, both (i) the total amount of payload expressed and (ii) the duration of 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 the liver of a reference subject.

[0071] As is evident from the present disclosure, in some embodiments, the LNPs described herein have minimal effect on the expression of payload in non-lymphoid tissues compared to the corresponding expression in a reference subject (e.g., a subject administered with a corresponding lipid nanoparticle that does not contain a VEE replicon).Thus, in some embodiments, after administration, the total amount of payload expressed in the non-liver tissues 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 that observed in the non-liver tissues of the reference subject. In some embodiments, following administration of the LNP described herein (e.g., a subject administered a corresponding lipid nanoparticle that does not contain a VEE replicon), the duration of payload expression in non-liver tissues 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 that observed in non-liver tissues of a reference subject.

[0072] As apparent from at least the disclosure above, some aspects of the present disclosure are directed to a method of selectively expressing a payload (e.g., a heterologous protein) in a non-liver tissue of a subject in need thereof. In some embodiments, such a method comprises administering to a subject in need thereof lipid nanoparticles comprising (i) one or more lipids and (ii) a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), where the VEE replicon comprises a nucleic acid sequence encoding a payload, and following administration, payload expression in the liver is decreased compared to corresponding expression in the non-liver tissue. As further described herein, the non-liver tissue may include any suitable tissue other than the liver in the subject. Non-limiting examples of such non-liver tissues include the spleen, lung, or both. As used herein, "selective expression" or "preferential expression," or grammatical variations thereof, refers to increased expression (e.g., expression of a payload) in a first tissue (e.g., a non-liver tissue) compared to a second tissue (e.g., the liver). In some embodiments, a payload is selectively or preferentially expressed in a non-liver tissue if expression (e.g., total amount of payload and / or duration of payload expression) in the non-liver tissue is increased compared to the corresponding expression in the liver. In some embodiments, expression of the payload in the non-liver tissue is the same as the corresponding expression in other non-liver tissues of the subject. In some embodiments, expression of the payload in the non-liver tissue is increased compared to the corresponding expression in other non-liver tissues of the subject. In some embodiments, expression of the payload in the non-liver tissue is decreased compared to other non-liver tissues in the subject, but increased compared to the corresponding expression in the liver.

[0073] As shown herein, in some embodiments, the LNPs of the present disclosure are useful for inducing selective (or preferential) expression of payload in spleen compared to liver.Thus, in some embodiments, the total amount of payload expressed in 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 amount observed in liver. In some embodiments, 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 or more, at least about 75-fold or more, or at least about 100-fold or more, compared to the corresponding duration observed in the liver.

[0074] As also shown herein, in some embodiments, the LNP of the present disclosure is useful for inducing the selective expression of payload in lung compared to liver.Thus, in some embodiments, the total amount of payload expressed in 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 amount observed in liver. In some embodiments, 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 or more, at least about 75-fold or more, or at least about 100-fold or more, compared to the corresponding duration observed in the liver.

[0075] II.B. Treatment method As is evident from the present disclosure, the LNPs described herein (e.g., comprising a VEE replicon comprising a nucleic acid sequence encoding a payload) can be useful in treating a wide variety of diseases or disorders. Thus, in some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a lipid nanoparticle comprising (i) one or more lipids and (ii) a replicon derived from the Venezuelan Equine Encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon comprises a nucleic acid sequence encoding a payload, and wherein following administration, the payload is preferentially expressed in non-liver tissues of the subject. In some embodiments, following administration of the LNP, the amount of payload expressed in non-liver 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 greater than the corresponding amount observed in the subject's liver. In some embodiments, following administration of the LNP, the duration of payload expression in non-liver tissues 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 longer than the corresponding duration observed in the subject's liver.

[0076] 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 disorder known in the art. Non-limiting examples of diseases and disorders that can be treated include cancer, inflammatory disorders, single gene disorders, neurological disorders, psychiatric disorders, or combinations thereof. As is evident from the present disclosure, in some embodiments, the LNPs described herein can be used to regulate (e.g., increase or decrease) an immune response. In some embodiments, the LNPs described herein can be used as a vaccine. In some embodiments, the disease or disorder comprises 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, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, head and neck cancer, or a combination thereof.

[0077] As further described elsewhere in this disclosure, in some embodiments, the LNPs described herein can reduce unwanted side effects and increase safety compared to other delivery platforms available in the art. For example, hepatotoxicity has been a major issue in many past LNP-based intratumoral therapeutic approaches (e.g., due to LNPs accumulating in the liver). See, e.g., Jain et al., Nucleic Acid Ther 28(5):285-296 (Oct. 2018), which is incorporated by reference in its entirety. Without wishing to be bound by any theory, in some embodiments, the LNPs described herein (e.g., VEE replicons comprising a nucleic acid sequence encoding a payload) are associated with reduced hepatotoxicity by avoiding or reducing expression of the payload in the liver. For example, in some embodiments, following administration of the LNPs provided herein, there is reduced hepatotoxicity in a subject compared to a reference subject (e.g., a subject administered the corresponding LNP but not comprising a VEE replicon). In some embodiments, 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 that of the reference subject. Hepatotoxicity can be measured using any suitable method available in the art. In some embodiments, hepatotoxicity can be evaluated by performing a general toxicity assessment of the subject (e.g., clinical tolerance and general health status). Non-limiting criteria that can be used to perform such an assessment include the subject's activity, food intake, body score index, hydration status, and combinations thereof. In some embodiments, a biological sample (e.g., blood or serum) can be taken from the subject, and hematology and / or clinical chemistry can be performed. For example, in some embodiments, the levels of liver transaminases (ALT / AST), total bilirubin, serum albumin, or combinations thereof can be measured.Such measurements can be compared to reference values ​​(e.g., corresponding amounts in healthy subjects) to assess hepatotoxicity. In some embodiments, more invasive methods (e.g., microscopic evaluation of the liver) can also be used.

[0078] As will be apparent to one of skill in the art, in some embodiments, reduced liver toxicity may improve the tolerability of the administered LNP. Thus, in some embodiments, provided herein is a method of improving the tolerability of an LNP-based therapy in a subject in need thereof, comprising administering to the subject (i) one or more lipids, and (ii) a lipid nanoparticle comprising a replicon derived from the 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 embodiments, the tolerability of the lipid nanoparticle-based therapy in a subject 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 greater than the corresponding tolerability observed in a reference subject (e.g., a subject administered the corresponding lipid nanoparticle but where the replicon is not a VEE replicon).

[0079] In some embodiments of the present disclosure, the polynucleotides, vectors, lipid nanoparticles, and / or pharmaceutical compositions (collectively referred to herein as "compositions") described herein are used to treat a disease or disorder. In some embodiments, the disease or disorder comprises cancer. Non-limiting examples of cancers that can be treated are provided elsewhere in this disclosure.

[0080] In some embodiments, an effective amount of any of the LNPs described herein or compositions comprising such LNPs (also collectively referred to herein as "compositions") is administered to a subject in need thereof via a suitable route, e.g., intratumoral administration, intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation, or topical route. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be nebulized, and lyophilized powders can be nebulized after reconstitution. In some embodiments, the pharmaceutical compositions described herein are aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized milled powders. In some embodiments, the pharmaceutical compositions described herein are formulated for intratumoral injection. In some embodiments, the pharmaceutical compositions described herein are administered to a subject via a topical route, e.g., injected into a local site, such as the site of a tumor or an infection. In some embodiments, the subject is a human.

[0081] As is evident from the present disclosure, in some embodiments, the compositions described herein are administered to a subject in an effective amount to impart a therapeutic effect, either alone or in combination with one or more other active agents. In some embodiments, the compositions are administered to a subject suffering from cancer, and the therapeutic effect includes a reduction in tumor burden, a reduction in cancer cells, an increase in immune activity, or a combination thereof. Whether the administered composition (e.g., lipid nanoparticles) has achieved a therapeutic effect can be determined using any suitable method known in the art (e.g., measuring tumor volume and / or T cell activity). The effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters such as age, physical condition, size, sex, and weight, duration of treatment, the nature of the concomitant therapy (if any), the particular route of administration, and similar factors within the expertise of the medical practitioner, as will be recognized by those skilled in the art.

[0082] Empirical considerations such as half-life generally contribute to the determination of dosage.Dosage frequency can be determined and adjusted over the course of treatment, but is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder.Alternatively, sustained continuous release formulations of the compositions described herein (e.g., lipid nanoparticles) may be appropriate.Various formulations and devices for achieving sustained release are known in the art.

[0083] In some embodiments of the present disclosure, the treatment is a single injection of the composition disclosed herein. In some embodiments, the single injection is administered intratumorally to a subject in need thereof.

[0084] In some embodiments of the present disclosure, the dosage of the compositions described herein can be empirically determined in individuals who have received one or more doses of the compositions (e.g., lipid nanoparticles described herein). In some embodiments, individuals are administered increasing doses of the compositions described herein. Disease / disorder indicators can be tracked to assess the effectiveness of the compositions described herein. In the case of repeated administration over several days or more, in some embodiments, treatment is continued until a desired suppression of symptoms occurs or until a sufficient therapeutic level is achieved to alleviate the target disease or disorder or its symptoms, depending on the condition.

[0085] In some embodiments 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 more. The administration regimen (e.g., dosage and / or administration frequency) of the compositions (e.g., lipid nanoparticles) described herein used may vary over time.

[0086] In some embodiments of the present disclosure, the methods include administering one or more doses of a composition described herein to a subject in need thereof.

[0087] The appropriate dosage of a composition (e.g., lipid nanoparticles described herein) depends on the particular 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 prophylactic or therapeutic purposes, prior treatment, the subject's medical history and response to the composition (e.g., lipid nanoparticles), and the discretion of the attending physician. In some embodiments, the clinician can administer a composition disclosed herein until a dosage is reached at which a desired result is achieved. In some embodiments, the desired result is a reduction in tumor burden, a reduction in cancer cells, or an increase in immune activity. Administration of one or more compositions described herein can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of a composition described herein can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0088] As used herein, alleviating a target disease / disorder includes delaying the onset or progression of the disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a target disease or disorder means advancing, hindering, decelerating, suppressing, stabilizing, and / or postponing the progression of the disease. This delay can be of various lengths of time, depending on the medical history and / or the subject being treated. A method of delaying or alleviating the onset of a disease, or delaying the manifestation of a disease, is a method that reduces the probability of developing one or more symptoms of the disease and / or reduces the degree of the symptoms within a given period of time as compared to not using the method. Such comparisons are typically based on clinical trials using a sufficient number of subjects to give statistically significant results.

[0089] In some embodiments, the compositions described herein are administered to a subject in need thereof in an amount sufficient to reduce tumor tissue mass 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 embodiments, the compositions described herein are administered in an amount effective 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.

[0090] In some embodiments, administration of the composition to a subject enhances immune activity, such as T cell activity, in the subject. In some embodiments, the immune activity is enhanced or increased by at least about 0.5-fold, 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 50-fold, or more, compared to the immune activity of a reference subject (e.g., the subject before administration of the composition or a corresponding subject that did not respond to administration of the composition).

[0091] In some embodiments, the subject is a human who has cancer, is suspected to have cancer, or is at risk of cancer.In some embodiments, the 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, digestive cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, and various types of head and neck cancer, including head and neck squamous cell carcinoma.In some embodiments, the cancer can be melanoma, lung cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma, or Hodgkin's lymphoma.

[0092] The subject with the target disease or disorder can be identified by routine medical examination, for example, clinical examination, organ function test, CT scan, or ultrasound.The subject suspected of having the target disease or disorder can show one or more symptoms of the disease or disorder.The subject at risk of the disease or disorder can be the subject with one or more risk factors associated with the disease or disorder.The subject at risk of the disease or disorder can also be identified by routine medical practice.

[0093] In some embodiments, the compositions described herein are co-administered with at least one additional suitable therapeutic agent. In some embodiments, the at least one additional suitable therapeutic agent includes an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, or other agent that serves to enhance and / or complement the immunostimulatory effect of the compositions described herein (e.g., lipid nanoparticles). Further examples of additional therapeutic agents that can be used in combination with the compositions described herein include chemotherapeutic agents, targeted anti-cancer therapies, oncolytic agents, cytotoxic agents, immune system therapies, cytokines, surgical procedures, radiation treatments, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof. In some embodiments, the compositions described herein and at least one additional therapeutic agent are administered to the subject sequentially, i.e., each therapeutic agent is administered at a different time. In some embodiments, the compositions described herein and at least one additional therapeutic agent are administered to the subject at about the same time.

[0094] Those skilled in the art will understand that any combination of the compositions described herein and other anti-cancer agents (e.g., chemotherapeutic agents) 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, effectiveness in reducing tumor formation or tumor growth, reducing cancer cells, increasing immune activity, and / or alleviating at least one symptom associated with cancer, or reducing the side effects of another agent in the combination. For example, the combination therapy described herein can reduce any of the side effects associated with each individual member of the combination, such as side effects associated with an anti-cancer agent.

[0095] In some embodiments, the other anticancer therapeutic agent is chemotherapy, radiation therapy, surgery, immunotherapy, or a combination thereof. In some embodiments, the chemotherapy agent is carboplatin, cisplatin, docetaxel, gemcitabine, nab-paclitaxel, pemetrexed, vinorelbine, or a combination thereof. In some embodiments, the radiation therapy is ionizing radiation, gamma radiation, neutron therapy, electron therapy, proton therapy, brachytherapy, systemic radioisotope, radiosensitizer, or a combination thereof. In some embodiments, the surgical therapy is curative surgery (e.g., tumor removal surgery), preventive surgery, laparoscopic surgery, laser surgery, or a combination thereof. In some embodiments, the immunotherapy is adoptive cell transfer, therapeutic cancer vaccine, or a combination thereof.

[0096] In some embodiments, the chemotherapeutic agent is a platinum agent, such as carboplatin, oxaliplatin, cisplatin, nedaplatin, satraplatin, lobaplatin, triplatin, tetranitrate, picoplatin, prolindac, aroplatin, and other derivatives; topoisomerase I inhibitors, such as camptothecin, topotecan, irinotecan / SN38, rubitecan, belotecan, and other derivatives; topoisomerase II inhibitors, such as etoposide (VP-16), daunorubicin, doxorubicin agents (e.g., doxorubicin, doxorubicin, doxorubicin HCl, doxorubicin analogs, or doxorubicin in liposomes and its salts or analogs), mitoxantrone, aclarubicin, epirubicin, idarubicin, amrubicin, amsacrine, pirarubicin, valrubicin, zorubicin, teniposide and other derivatives; antimetabolites, such as the folic acid family (methotrexate, pemetrexed, raltitrexed, aminopterin, and related); purine antagonists (thioguanine, fludarabine, cladribine, 6-mercaptopurine, pentostatin, clofarabine, and related) ) and pyrimidine antagonists (cytarabine, floxuridine, azacitidine, tegafur, carmofur, capacitabine, gemcitabine, hydroxyurea, 5-fluorouracil (5FU), and related); alkylating agents, such as nitrogen mustards (e.g., cyclophosphamide, melphalan, chlorambucil, mechlorethamine, ifosfamide, trofosfamide, prednimustine, bendamustine, uramustine, estramustine, and related); nitrosoureas (e.g., carmustine, lomustine, semustine, fote mustine, nimustine, ranimustine, streptozocin, and related; triazenes (e.g., dacarbazine, altretamine, temozolomide, and related); alkylsulfonates (e.g., busulfan, mannosulfan, treosulfan, and related); procarbazine; mitobronitol, and aziridines (e.g., carboquone, triaziquone, thiotepa, triethylenemalamine, and related); antibiotics, e.g., hydroxyurea, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and other derivatives);Anthracenediones (e.g., mitoxantrone and related); Streptomyces family (e.g., bleomycin, mitomycin C, actinomycin, plicamycin); ultraviolet light; and combinations thereof.

[0097] In some embodiments, the other anti-cancer therapeutic agent is an antibody. Antibodies (preferably monoclonal antibodies) achieve therapeutic effects on cancer cells through various mechanisms. Antibodies can have direct effects in causing apoptosis or programmed cell death. For example, they can block components of signal transduction pathways, such as growth factor receptors, effectively stopping tumor cell proliferation. In cells expressing monoclonal antibodies, the formation of anti-idiotypic antibodies can occur. Indirect effects include the recruitment of cytotoxic cells, such as monocytes and macrophages. This type of antibody-mediated cell killing is called antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind complement, causing direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Combining surgical techniques with immunotherapeutic or immunotherapeutic agents is a successful approach, as shown, for example, in Gadri et al. 2009: Synergistic effect of dendritic cell vaccination and anti-CD20 antibody treatment in the therapy of murine lymphoma. J Immunother. 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 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), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNT0888),Catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatoxin (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B-lymphoma cells), drotazobium (D-lymphoma cells), Zizumab (DR5), ecromeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH 900105), figitumumab (IGF-1 receptor), framvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (IL-1β), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatum Mabvedotin (GPNMB), Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovomab (CA-125), Indatuximab ravtansine (SDC1), Intetumumab (CD51), Inotuzumab ozogamicin (CD22), Ipilimumab (CD152), Iratumumab (CD30), Labetuzumab (CEA), lexatumumab (TRAIL-R2), ribivirumab (Hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitumomab (GD3 ganglioside), mogamulizumab (CCR4), moxetumomab passudotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), narunatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4),ofatumumab (CD20), olaratumab (PDGF-Rα), onartuzumab (human scatter factor receptor kinase), oportuzumab monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma) antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radletumab (fibronectin extra domain-B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samaryzumab (CD200), sibrotuzumab (FAP), siltuximab (IL-6), tabalumab (BAFF), tacatuzumab tetraxetan (alpha-fetoprotein), tapritumomab paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (C D20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1BB), vorociximab (integrin α5β1), votumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4).

[0098] In some embodiments, the other anti-cancer therapeutic agent is a cytokine, a chemokine, a costimulatory molecule, a fusion protein, or a combination thereof. Examples of chemokines include, but are not limited to, CCR7 and its ligands CCL19 and CCL21, as well as CCL2, CCL3, CCL5, and CCL16. Other examples are CXCR4, CXCR7, and CXCL12. In addition, costimulatory or regulatory molecules, such as B7 ligands (B7.1 and B7.2), are useful. Also, other cytokines such as, in particular, interleukins (e.g., IL-1 to IL17), interferons (e.g., IFNα1 to IFNα8, IFNα10, IFNα13, IFNα14, IFNα16, IFNα17, IFNα21, IFNβ1, IFNW, IFNE1 and IFNK), hematopoietic factors, TGFs (e.g., TGF-α, TGF-β and other members of the TGF family), and finally, receptors of members of the tumor necrosis factor family and their ligands, as well as 41BB, 41BB-L, CD137, CD137L, CTLA-4GITR, GITRL, Fas, Fas Other stimulatory molecules are also useful, including, but not limited to, CD40 / CD40L, TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, LT.β.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-associated protein, TNF receptor-associated apoptosis-mediating protein (TRAMP), and death receptor 6 (DR6). In particular, CD40 / CD40L and OX40 / OX40L are important targets for combination 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.

[0099] In some embodiments, another anti-cancer therapeutic is bacterial therapy. Researchers have used anaerobic bacteria, such as Clostridium novyi, to exhaust the interior of hypoxic tumors. These should then die when they come into contact with the oxygenated side of the tumor, meaning they are harmless to the rest of the body. Another strategy is to use anaerobic bacteria transformed to contain an enzyme that can convert a non-toxic prodrug into a toxic drug. The enzyme is expressed only in the tumor as the bacteria grow in the necrotic and hypoxic parts of the tumor. Thus, the systemically administered prodrug is metabolized into a toxic drug only in the tumor. This has been shown to be effective with the non-pathogenic anaerobic Clostridium sporogenes.

[0100] In some embodiments, the other anti-cancer therapeutic agent is a kinase inhibitor. The growth and survival of cancer cells are closely linked to the deregulation of kinase activity. A wide range of inhibitors have been used to restore normal kinase activity and thereby reduce tumor growth. Target kinases include receptor tyrosine kinases such as 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 such as c-SRC, c-YES, Abl, JAK-2, serine / threonine kinases such as ATM, Aurora A&B, CDK, mTOR, PKCi, PLK, b-Raf, S6K, STK11 / LKB1, and lipid kinases such as PI3K, SK1. Small molecule kinase inhibitors are, for example, PHA-739358, nilotinib, dasatinib, and PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sutent (SU11248), sorafenib (BAY43-9006) and leflunomide (SU101). For further information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

[0101] In some embodiments, the other anti-cancer therapeutic agent is a toll-like receptor. Members of the Toll-like receptor (TLR) family are important bridges between innate and adaptive immunity, and many adjuvant effects depend on the activation of TLRs. Many established vaccines against cancer incorporate TLR ligands to enhance the vaccine response. In addition to TLR2, TLR3, TLR4, in particular, TLR7 and TLR8 are being investigated for cancer therapy in passive immunotherapy approaches. The closely related TLR7 and TLR8 contribute to the anti-tumor response by affecting immune cells, tumor cells, and the tumor microenvironment and can be activated by nucleoside-like structures. All TLRs are used as single immunotherapeutic agents or cancer vaccine adjuvants and can be combined synergistically with the formulations and methods of the present disclosure. For further information, see van Duin et al. 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55.

[0102] In some embodiments, the other anti-cancer therapeutic agent is an angiogenesis inhibitor. Angiogenesis inhibitors inhibit the extensive blood vessel growth (angiogenesis) required for tumor survival. For example, angiogenesis promoted by tumor cells to meet the increasing demand for nutrients and oxygen can be blocked by targeting different molecules. Non-limiting examples of angiogenesis mediating or inhibitory molecules that can be combined with the present disclosure include 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, TIMPs and CDAI, Meth-1 and Meth-2, IFN-α, IFN-β and IFN-γ, CXCL10, IL-4, IL-12 and IL-18, prothrombin (kringle domain-2), antithrombin III fragments, Prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, as well as drugs such as bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-α, platelet factor 4, suramin, SU5416, thrombospondin, VEGFR antagonists, antiangiogenic steroids plus heparin, cartilage-derived angiogenesis inhibitor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin Vβ3 inhibitors, linomide, tasquinimod, etc., for a review see Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccin. (9): 976-81.

[0103] In some embodiments, the other anti-cancer therapeutic agent is a viral vaccine. There are many viral cancer vaccines available or in development, which can be used in combination treatment approaches with the formulations of the present disclosure. One advantage of using such viral vectors is their inherent ability to initiate immune responses, and viral infection results in inflammatory reactions and danger signals necessary for immune activation. An ideal viral vector should be safe, not generate anti-vector immune responses, and be able to raise anti-tumor specific responses. Recombinant viruses such as vaccinia virus, herpes simplex virus, adenovirus, adeno-associated virus, retrovirus, and avipox virus have been used in animal tumor models, and based on the promising results, human clinical trials have been initiated. A particularly important viral vaccine is the virus-like particle (VLP), which is a small particle that contains certain proteins derived from the viral envelope. Virus-like particles do not contain any genetic material of the virus and do not cause infection, but can be constructed to present tumor antigens on their envelope. VLPs can be derived from a variety of viruses, such as, for example, Hepatitis B virus or other viral families including Parvoviridae (eg, adeno-associated viruses), Retroviridae (eg, HIV), and Flaviviridae (eg, Type C viruses).For a review see Sorensen and Thompsen 2007: “Virus-based immunotherapy of cancer: what do we know and where are we going?” APMIS 115(11):1177-93, and virus-like particles for cancer are reviewed in Buonaguro et al. 2011: Developments in virus-like particle-based vaccines for infectious diseases and cancer. Expert Rev Vaccines 10(11):1569-83; and Guillen 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.

[0104] In some embodiments, the other anti-cancer therapeutic agent is a peptide-based targeted therapy. The peptides can bind to cell surface receptors or to the diseased extracellular matrix surrounding the tumor. Radionuclides bound to these peptides (e.g., RGD) ultimately kill the cancer if the nuclide decays in the vicinity of the cell. In particular, oligomers or multimers of these binding motifs are of great interest because they can lead to improved 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.

[0105] III. Lipid Nanoparticles As described herein, the LNPs of the present disclosure exhibit tissue-specific tropism that allows the LNPs to selectively deliver payloads (e.g., heterologous proteins) to cells (e.g., cells in non-liver tissues). In some embodiments, delivery can occur in vivo (e.g., by administering the LNPs described herein to a subject) or ex vivo (e.g., by culturing the LNPs described herein with cells in vitro). "Lipid nanoparticles" (LNPs), as used herein, refer to vesicles, such as spherical vesicles, that have a continuous lipid bilayer. Lipid nanoparticles can be used in methods of delivering drug therapies to a target location. Non-limiting examples of LNPs include liposomes, bolaamphiphiles, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), unilamellar membrane structures (e.g., archaeosomes and micelles), lipid-like nanoparticles (LLNs), polymeric nanoparticles (PNPs), lipid-polymer hybrid nanoparticles (LPNs), or combinations thereof.

[0106] In some embodiments, the lipid nanoparticles include one or more lipids. "Lipid" as used herein refers to a group of organic compounds, including but not limited to fatty acid esters, which in some embodiments are characterized by being insoluble in water but soluble in many organic solvents. Thus, unless otherwise stated, lipids include natural lipids and lipid-like substances (e.g., lipidoids). Lipids are generally classified into at least three classes: (1) "simple lipids," including fats and oils and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids. Non-limiting examples of lipids include triglycerides (e.g., tristearin), diglycerides (e.g., glycerol behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). In some embodiments, lipids useful in the present disclosure include ionizable lipids, cationic lipids, lipidoids, phospholipids, sterols, or combinations thereof.

[0107] In some embodiments, one or more lipids in the LNP comprises an ionizable lipid. Thus, in some embodiments, the LNPs useful in the present disclosure comprise a VEE replicon that comprises (i) an ionizable lipid and (ii) a nucleic acid sequence encoding a payload.

[0108] Non-limiting examples of ionizable lipids include ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)ethyl))-1,2-dihydro ... N-(2-(di-methylamino)butanoyl)oxy)heptadecanedioate (L319), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), Nl-[2(didodecylamino)ethyl]-N1,N4,N4-tridodecyl 1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethyl Aminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12 Z)-Octadeca-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)-octadeca-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)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)).12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), or any combination thereof.

[0109] In some embodiments, one or more lipids in the LNP comprises a cationic lipid. In some embodiments, the LNPs useful in the present disclosure comprise (i) a cationic lipid and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some embodiments, the LNPs (i.e., comprising a cationic lipid and a VEE replicon) further comprise an ionizable lipid.

[0110] "Cationic lipid," as used herein, refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Non-limiting examples of cationic lipids include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleyloxy-N-[2(sperminecarboxamide) N,N-dimethyl-l-propaneaminium trifluoroacetate (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(l,2-dioleoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N -Dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-dilauroyl-sn-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), 1,2-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 (14:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.Additional examples are provided throughout this disclosure.

[0111] In some embodiments, one or more lipids in the LNP comprises a lipidoid. Thus, in some embodiments, the LNPs described herein comprise (i) a lipidoid and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some embodiments, the LNPs (i.e., comprising a lipidoid and a VEE replicon) further comprise an ionizable lipid, a cationic lipid, or both.

[0112] As used herein, the term "lipidoid" refers to a molecule that has one or more characteristics of a lipid, e.g., a synthetic cationic lipid. In some embodiments, the lipidoid can have a series of secondary and tertiary amines, which increases the net positive charge of the LNP. Non-limiting examples of lipid mimetics and / or lipidoids 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), 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-hydroxyoctadeca9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(butane-4,1-diyl) (9Z,9'Z,9''Z,9''''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate) -9,12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tris(3-(didodecylamino)propyl)benzene 1,3,5-tricarboxamide (TT3), hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-(((benzene-1,3,5-tricarbonyl)ris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate (FTT5), PL-1 [Nature Communications,12-7264(2021) and incorporated herein by reference], 98N12-5 [disclosed in Molecular Therapy vol.17 no.5 May 2009 and incorporated herein by reference], ethyl 5,5-di((Z)-heptadec-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.

[0113] In some embodiments, the lipid comprises a sterol. In some embodiments, the LNPs useful in the present disclosure comprise a VEE replicon that comprises (i) a sterol and (ii) a nucleic acid sequence encoding a payload. In some embodiments, the LNPs (i.e., comprising a sterol and a VEE replicon) further comprise an ionizable lipid, a cationic lipid, a lipidoid, or any combination thereof.

[0114] As used herein, "sterol" refers to cholesterol or cholesterol analogs that can be used to fill gaps in the packing of lipid membranes and provide structural integrity.Non-limiting examples of sterols include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and combinations thereof.In some embodiments, the sterol is cholesterol.

[0115] In some embodiments, the lipid comprises a phospholipid. In some embodiments, the 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 embodiments, the LNPs (i.e., comprising a phospholipid and a VEE replicon) further comprise an ionizable lipid, a cationic lipid, a lipidoid, a sterol, or any combination thereof.

[0116] Non-limiting examples of phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di Undecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and any combination thereof. In some embodiments, the phospholipid is 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (14:0-16:0 PC, MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (14:0-18:0PC, MSPC), 1-palmitoyl 2-acetyl-sn-glycero-3-phosphocholine (16:0-02:0 PC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (16:0-14:0 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-18:0 PC, PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 PC, POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC, PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC, PMPC), 1-palmitoyl-2-stearo ...20:4 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC, PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (16:0-20:4 PC, PMPC), 1-palmitoyl-2-stearoyl-sn PC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (14:0-22:6 PC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:0-14:0 PC, SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:0-16:0 PC, SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1 PC, SOPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (18:0-20:4 PC), PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6 PC), 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine (18:1-14:0 PC, OMPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (18:1-16:0 PC, OPPC), 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (18:1-18:0 PC, OSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE, POPE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:2PE), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (16:0-20:4 PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6 PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0-18:1 PE), 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 (18:0-22:6 PE), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and any combination thereof.

[0117] Thus, in some embodiments, LNPs useful in the present disclosure include a VEE replicon that includes (i) one or more natural lipids (e.g., natural cationic lipids) and (ii) a nucleic acid sequence encoding a payload. In some embodiments, LNPs useful in the present disclosure include a VEE replicon that includes (i) one or more synthetic lipids (e.g., lipidoids) and (ii) a nucleic acid sequence encoding a payload. In some embodiments, LNPs for use in the present disclosure include a VEE replicon that includes (i) both natural lipids (e.g., cationic lipids) and synthetic lipids (e.g., lipidoids), and (ii) a nucleic acid sequence encoding a payload.

[0118] Examples of lipids that can be used with the LNPs of the present disclosure include N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); Lipofectamine; 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA); Dioctadecyldimethylammonium (DODMA), Distearyldimethylammonium (DIAM), and Distearyldimethylammonium (DDM). N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N-N-distearyl-N,N-dimethylammonium bromide (DDAB); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0119] In some embodiments, the lipid has Formula I: [ka] and salts thereof, wherein each R is independently an unsubstituted alkyl; and each R 2 is independently unsubstituted alkyl, and 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 embodiments, each R 1 is independently unsubstituted alkyl, and each R 2 is independently unsubstituted alkyl; R 3 is hydrogen and each m is 3. In some embodiments, at least one R 1 is unsubstituted C 1-24 In some embodiments, at least one R 1is unsubstituted C 1-18 In some embodiments, at least one R 1 is unsubstituted C 1-12 In some embodiments, at least one R 1 is unsubstituted C 6-18 In some embodiments, at least one R 1 is unsubstituted C 6-12 In some embodiments, at least one R 1 is unsubstituted C 8-12 In some embodiments, at least one R 1 is unsubstituted C 10-12 In some embodiments, at least one R 1 is unsubstituted C 11 It is an alkyl.

[0120] In some embodiments, at least one R 2 is unsubstituted C 1-24 In some embodiments, at least one R 2 is unsubstituted C 1-18 In some embodiments, at least one R 2 is unsubstituted C 1-12 In some embodiments, at least one R 2 is unsubstituted C 6-18 In some embodiments, at least one R 2 is unsubstituted C 6-12 In some embodiments, at least one R 2 is unsubstituted C 8-12 In some embodiments, at least one R 2 is unsubstituted C 10-12 In some embodiments, at least one R 2 is unsubstituted C 11 It is an alkyl.

[0121] In some embodiments, at least two R 1 is unsubstituted C1-24 In some embodiments, at least two R 1 is unsubstituted C 1-18 In some embodiments, at least two R 1 is unsubstituted C 1-12 In some embodiments, at least two R 1 is unsubstituted C 6-18 In some embodiments, at least two R 1 is unsubstituted C 6-12 In some embodiments, at least two R 1 is unsubstituted C 8-12 In some embodiments, at least two R 1 is unsubstituted C 10-12 In some embodiments, at least two R 1 is unsubstituted C 11 It is an alkyl.

[0122] In some embodiments, at least two R 2 is unsubstituted C 1-24 In some embodiments, at least two R 2 is unsubstituted C 1-18 In some embodiments, at least two R 2 is unsubstituted C 1-12 In some embodiments, at least two R 2 is unsubstituted C 6-18 In some embodiments, at least two R 2 is unsubstituted C 6-12 In some embodiments, at least two R 2 is unsubstituted C 8-12 In some embodiments, at least two R 2 is unsubstituted C 10-12 In some embodiments, at least two R 2 is unsubstituted C 11 It is an alkyl.

[0123] In some embodiments, R 1 In all cases, unsubstituted C 1-24 In some embodiments, R 1 In all cases, unsubstituted C 1-18 In some embodiments, R 1 In all cases, unsubstituted C 1-12 In some embodiments, R 1 In all cases, unsubstituted C 6-18 In some embodiments, R 1 In all cases, unsubstituted C 6-12 In some embodiments, R 1 In all cases, unsubstituted C 8-12 In some embodiments, R 1 In all cases, unsubstituted C 10-12 In some embodiments, R 1 In all cases, unsubstituted C 11 It is an alkyl.

[0124] In some embodiments, R 2 In all cases, unsubstituted C 1-24 In some embodiments, R 2 In all cases, unsubstituted C 1-18 In some embodiments, R 2 In all cases, unsubstituted C 1-12 In some embodiments, R 2 In all cases, unsubstituted C 6-18 In some embodiments, R 2 In all cases, unsubstituted C 6-12 In some embodiments, R 2 In all cases, unsubstituted C 8-12 In some embodiments, R 2 In all cases, unsubstituted C 10-12 In some embodiments, R 2 In all cases, unsubstituted C 11It is an alkyl.

[0125] In some embodiments, at least one R 3 is hydrogen. In some embodiments, at least one R 3 is substituted or unsubstituted alkyl. In some embodiments, at least one R 3 is a substituted or unsubstituted C 1-18 In some embodiments, at least one R 3 is a substituted or unsubstituted C 1-12 In some embodiments, at least one R 3 is a substituted or unsubstituted C 1-6 In some embodiments, at least one R 3 is a substituted or unsubstituted C 1-4 In some embodiments, at least one R 3 is a substituted or unsubstituted C 2-4 In some embodiments, at least one R 3 is substituted or unsubstituted methyl.

[0126] In some embodiments, at least one R 3 is a substituted alkyl, wherein the substituted alkyl is substituted with a halogen. In some embodiments, at least one R 3 is a substituted alkyl, wherein the substituted alkyl is substituted with fluorine. In some embodiments, at least one R 3 is a substituted alkyl, where the substituted alkyl is substituted with an alkyl halide.

[0127] In some embodiments, at least two R 3 is hydrogen. In some embodiments, at least two R 3 is substituted or unsubstituted alkyl. In some embodiments, at least two R 3 is a substituted or unsubstituted C 1-18 In some embodiments, at least two R3 is a substituted or unsubstituted C 1-12 In some embodiments, at least two R 3 is a substituted or unsubstituted C 1-6 In some embodiments, at least two R 3 is a substituted or unsubstituted C 1-4 In some embodiments, at least two R 3 is a substituted or unsubstituted C 2-4 In some embodiments, at least two R 3 is substituted or unsubstituted methyl.

[0128] In some embodiments, at least two R3 are substituted alkyl, where the substituted alkyl is substituted with halogen. In some embodiments, at least two R3 are substituted alkyl, where the substituted alkyl is substituted with fluorine. In some embodiments, at least two R3 are substituted alkyl, where the substituted alkyl is substituted with alkyl halide.

[0129] In some embodiments, R 3 In some embodiments, R 3 is, in all cases, substituted or unsubstituted alkyl. In some embodiments, R 3 In all cases, substituted or unsubstituted C 1-18 In some embodiments, R 3 In all cases, substituted or unsubstituted C 1-12 In some embodiments, R 3 In all cases, substituted or unsubstituted C 1-6 In some embodiments, R 3 In all cases, substituted or unsubstituted C 1-4 In some embodiments, R 3 In all cases, substituted or unsubstituted C 2-4 In some embodiments, R 3is in all cases substituted or unsubstituted methyl.

[0130] In some embodiments, R 3 In all cases, R is a substituted alkyl, where the substituted alkyl is substituted with a halogen. 3 In all cases, R is a substituted alkyl, where the substituted alkyl is substituted with fluorine. In some embodiments, R 3 is in all cases a substituted alkyl, where the substituted alkyl is substituted with an alkyl halide.

[0131] In some embodiments, at least one m is 3. In some embodiments, at least one m is 4. In some embodiments, at least one m is 5. In some embodiments, at least one m is 6. In some embodiments, at least one m is 7. In some embodiments, at least one m is 8. In some embodiments, at least two m are 3. In some embodiments, at least two m are 4. In some embodiments, at least two m are 5. In some embodiments, at least two m are 6. In some embodiments, at least two m are 7. In some embodiments, at least two m are 8.

[0132] In some embodiments, m is 3 at all occurrences. In some embodiments, m is 4 at all occurrences. In some embodiments, m is 5 at all occurrences. In some embodiments, m is 6 at all occurrences. In some embodiments, m is 7 at all occurrences. In some embodiments, m is 8 at all occurrences.

[0133] In some embodiments of the present disclosure, the lipid is TT3, represented by: [ka]

[0134] wherein m is 3 in all cases. The composition, synthesis, and use of Formula I and TT3 are described in WO2016187531A1, which is incorporated herein by reference in its entirety.

[0135] Thus, in some embodiments, the LNPs described herein comprise a VEE replicon that includes a nucleic acid sequence encoding (i) TT3 and (ii) a payload. As used herein, "TT3" is capable of forming lipid nanoparticles for delivering various biologically active agents (e.g., payloads described herein) to cells. In addition, the present disclosure also shows that unencapsulated TT3-LNPs can induce immunogenic cell death (ICD) of cancer cells in vivo and in vitro. Immunogenic cell death, as described herein, refers to a form of cell death that can induce an effective immune response through activation of dendritic cells (DCs) and the resulting activation of specific T cell responses. In some embodiments, the cells undergoing immunogenic cell death are tumor cells.

[0136] In some embodiments, the lipid is DOTAP. Thus, in some embodiments, the LNP of the present disclosure comprises a VEE replicon that comprises (i) DOTAP and (ii) a nucleic acid sequence encoding a payload. As used herein, "DOTAP" can also form lipid nanoparticles. DOTAP can be used to transfect DNA, including yeast artificial chromosomes (YACs), into eukaryotic cells with high efficiency, resulting in transient or stable gene expression, and is also suitable for efficiently introducing other negatively charged molecules, such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes, and proteins into mammalian cell research samples.

[0137] In some embodiments of the present disclosure, the lipid is lipofectamine. In some embodiments, the LNP described herein comprises (i) lipofectamine and (ii) a VEE replicon comprising a nucleic acid sequence encoding a payload. As used herein, "lipofectamine" is a common transfection reagent used in molecular and cell biology, manufactured and sold by Invitrogen. Lipofectamine is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine contains lipid subunits and can form liposomes or lipid nanoparticles in an aqueous environment to encapsulate the transfection payload. RNA-containing liposomes (whose surfaces are positively charged) can fuse with the negatively charged plasma membrane of live cells, with neutral co-lipids mediating fusion of the liposome with the cell membrane, thereby allowing the nucleic acid cargo molecule to be translocated into the cytoplasm and replicated or expressed.

[0138] In some embodiments, the LNPs described herein comprise one type of lipid (e.g., all lipidoids, e.g., all TT3). In some embodiments, the LNPs of the present disclosure comprise multiple types of lipids. For example, in some embodiments, the LNPs comprise a lipidoid (e.g., TT3) along with other lipid components. These typically include, but are not limited to, other lipid molecules belonging to the 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 formulation of exemplary LNPs TT3-LNP and DOTAP-LNP. [Table 1]

[0139] In some embodiments, the LNP comprises C14-PEG2000. Thus, in some embodiments, the LNP useful in the present disclosure comprises a VEE replicon comprising (i) C14-PEG2000 and (ii) a nucleic acid sequence encoding a payload. In some embodiments, the C14-PEG2000 comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG2000), or both. In some embodiments, the C14-PEG2000 (or other lipid moieties disclosed herein) can be embedded in the LNP prior to packaging of the VEE replicon. In some embodiments, the C14-PEG2000 (or other lipid moieties disclosed herein) can be added to the LNP after packaging of the VEE replicon. For example, in some embodiments, the VEE replicon is encapsulated in a LNP, and then C14-PEG2000 (or other lipid moieties disclosed herein) is attached to the LNP, eg, using micelles.

[0140] In some embodiments, the LNPs useful in the present disclosure comprise a polymeric material. Thus, in some embodiments, the LNPs provided herein comprise a VEE replicon comprising (i) a polymeric material and (ii) a nucleic acid sequence encoding a payload. In some embodiments, the LNPs comprise (i) one or more lipids, (ii) a polymeric material, and (iii) a VEE replicon comprising a nucleic acid sequence encoding a payload. In some embodiments, the one or more lipids comprise an ionizable lipid, a cationic lipid, a lipidoid, a phospholipid, a sterol, or a combination thereof.

[0141] In some embodiments, the polymeric material comprises a cationic polymer or a non-cationic polymer, such as those described herein. Non-limiting examples of polymeric materials are disclosed in Jiang et al. Reference Module in Materials Science and Materials Engineering. (2021) and Byun et al. BioChip J (2022), which are incorporated herein by reference. Non-limiting examples of polymeric materials include polyethyleneimine (PEI), poly(amidoamine) (PAMAM), poly(β-aminoester) (PBAE), poly(2-N,N-dimethylaminoethyl methacrylate) (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-co-glycolic acid) (PLGA), and polycaprolactone (PCL).

[0142] As is apparent from at least the disclosure above, in some embodiments, 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 lipid-like substance (e.g., lipidoid), (iv) a phospholipid, (v) a sterol, (vi) a pegylated lipid, and (vii) a polymeric material. In some embodiments, 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 embodiments, LNPs described herein comprise a VEE replicon comprising a nucleic acid sequence encoding a payload, and one or more of the following: (i) a lipid-like substance (e.g., lipidoid), (ii) a phospholipid, (iii) a sterol, and (iv) a pegylated lipid. In some embodiments, the LNPs described herein comprise a VEE replicon comprising a nucleic acid sequence encoding a payload, and one or more of the following: (i) a polymeric material, (ii) a phospholipid, (iii) a sterol, and (iv) a pegylated lipid. In some embodiments, the LNPs described herein comprise a VEE replicon comprising a nucleic acid sequence encoding a payload, and one or more of the following: (i) a polymeric material, (ii) a lipid, (iii) a phospholipid, (iv) a sterol, and (v) a pegylated lipid.

[0143] The particle size of lipid nanoparticles can affect drug release rate, biodistribution, mucoadhesion, intracellular uptake of water and buffer exchange into the nanoparticle interior, and protein diffusion. In some embodiments of the present disclosure, the diameter of the LNP is in the range of about 30 to about 500 nm. In some embodiments of the present disclosure, the diameter of the LNP is in the range of about 30 to about 500 nm, about 50 to about 400 nm, about 70 to about 300 nm, about 100 to about 200 nm, about 100 to about 175 nm, or about 100 to about 160 nm. In some embodiments of the present disclosure, the diameter of the LNP is in the range of 100 to 160 nm. In some embodiments of the present disclosure, the diameter of the LNP can be about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm, about 115 nm, about 116 nm, about 117 nm, about 118 nm, about 119 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, or about 160 nm. In some embodiments, the lipid nanoparticle has a diameter of about 140 nm.

[0144] Zeta potential is a measure of the effective charge on the lipid nanoparticle surface. The magnitude of the zeta potential provides information about the stability of the particle. In some embodiments, the zeta potential of the nanoparticles described herein ranges from about -20 to about 20 mv. In some embodiments of the present disclosure, the zeta potential of the LNP ranges from about 3 to about 6 mv. In some embodiments of the present disclosure, the zeta potential of the LNP can be about 3mv, 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 4mv, 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 5mv, 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 6mv.In some embodiments, the zeta potential of the nanoparticles described herein is about -6.0mv, about -5.9mv, about -5.8mv, about -5.7mv, about -5.6mv, about -5.5mv, about -5.4mv, about -5.3mv, about -5.2mv, about -5.1mv, about -5.0mv, about -4.9mv, about -4.8mv, 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, approx. -3.4mv, approx. -3.3mv, approx. -3.2mv, approx. -3.1mv, approx. -3.0mv, approx. -2.9mv, approx. -2.8mv, approx. -2.7mv, approx. -2.6mv, approx. -2.5mv, approx. -2.4mv, approx. -2.3mv, approx. -2.2mv, approx. -2.1mv, approx. -2.0mv, approx. -1.9mv, approx. -1.8mv, approx. -1.7mv, approx. -1.6mv, approx. -1.5mv, approx. -1.4mv, approx. -1.3mv, approx. -1.2mv, approx. -1.1mv, approx. -1.0mv, approx. -0.9mv, approx. -0.8mv, approx. -0.7mv, approx. -0.6mv, -0.5mv, -0.4mv, -0.3mv, -0.2mv, -0.1mv, 0.0mv, 0.1mv, 0.2m v, approx. 0.3mv, approx. 0.4mv, approx. 0.5mv, approx. 0.6mv, approx. 0.7mv, approx. 0.8mv, approx. 0.9mv, approx. 1.0mv, approx. 1.1mv, about 1.2mv, about 1.3mv, about 1.4mv, about 1.5mv, about 1.6mv, about 1.7mv, about 1.8mv, about 1.9 mv, approx. 2.0mv, approx. 2.1mv, approx. 2.2mv, approx. 2.3mv, approx. 2.4mv, approx. 2.5mv, approx. 2.6mv, approx. 2.7mv, approx. It can be 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.

[0145] In some embodiments, the present disclosure relates to a polynucleotide (e.g., VEE replicon mRNA) encapsulated in a lipid nanoparticle (LNP). In some embodiments of the present disclosure, the mass ratio between the lipids and the polynucleotide (e.g., VEE replicon mRNA) of the LNP ranges from about 1:2 to about 15:1. In some embodiments, the mass ratio between the lipids and the polynucleotide (e.g., VEE replicon mRNA) ranges from 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, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, about 10:1, about 10.5:1, about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, or about 15:1. In some embodiments of the present disclosure, the mass ratio between lipid and polynucleotide (e.g., VEE replicon mRNA) is about 10:1.

[0146] IV. VEE Replicon In addition to one or more lipids (e.g., those described above), the LNPs of the present disclosure include a VEE replicon that includes a nucleic acid sequence encoding a payload. As further described elsewhere in this disclosure, Applicants have determined that interactions between the VEE replicon and one or more lipids (e.g., lipidoids, e.g., TT3) provide the LNPs described herein with certain improved properties, such as tissue-specific tropism, such that the LNPs can avoid the liver and / or have reduced expression of the payload in the liver compared to non-liver tissues (e.g., spleen and lung).

[0147] The VEE virus is a viral pathogen that is typically transmitted by mosquitoes and causes VEE or encephalomyelitis primarily in equine species. However, humans can also be infected with VEE, and those with weakened immune systems are at particular risk of having severe complications if infected with VEE. VEE virions are spherical and have a lipid membrane with glycoprotein surface proteins extending across the outer surface. VEE has a genome of approximately 11.45 kb, excluding the 5'-end cap and 3'-end poly(A) regions, and includes four nonstructural proteins (nsPs) and five structural proteins. The nonstructural proteins include nsP1, nsP2, nsP3, and nsP4, and the structural region encodes proteins C, E3, E2, 6K, and E1. In some embodiments, the self-amplifying replicon RNA is a wild-type replicon RNA derived from VEE. The sequence of the wild-type VEE virus replicon RNA is set forth in SEQ ID NO: 186 (see Table 4).

[0148] In some embodiments, a VEE replicon useful in the present disclosure comprises 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 set forth in SEQ ID NO: 186 or a fragment thereof. In some embodiments, a VEE replicon useful in the present disclosure comprises 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 set forth in SEQ ID NO: 187. In some embodiments, a VEE replicon comprises a nucleic acid sequence set forth in SEQ ID NO: 187. In some embodiments, the VEE replicon consists of the nucleic acid sequence set forth in SEQ ID NO: 187. In some embodiments, the VEE replicon consists essentially of the nucleic acid sequence set forth in SEQ ID NO:187.

[0149] In some embodiments, the VEE replicon useful in the present disclosure includes any VEE replicon known in the art. In some embodiments, the VEE replicon includes one or more mutations. For example, in some embodiments, the VEE replicon includes a mutation in the nonstructural proteins nsP2 and nsP3, which promote subgenomic expression in human cells. In some embodiments, the VEE replicon includes a mutation that allows for greater expression of the encoded payload compared to the wild-type VEE replicon (SEQ ID NO: 186). In some embodiments, the self-amplifying replicon RNA includes at least one point mutation at nucleic acid positions 3936 and / or 4758 of the WT replicon of SEQ ID NO: 186. In some embodiments, the self-amplifying replicon RNA includes at least one of the following point mutations: a guanine to cytosine point mutation at position 3936 (G3936C) and an adenine to guanine point mutation at position 4758 (A4758G) of the WT replicon sequence of SEQ ID NO: 186. The G3936C mutation results in a change from glycine to arginine at amino acid residue 1309 (G1309R). The A4758G mutation results in a change from serine to glycine at amino acid residue 1583 (51583G). Further disclosure regarding such VEE replicons is provided, for example, in US20200281994A1, which is incorporated by reference herein in its entirety.

[0150] Without being bound by any theory, in some embodiments, the interaction between the VEE replicon and one or more lipids (e.g., lipidoids, e.g., TT3) results in the LNP not targeting the liver. Also, since the LNP does not target the liver, in some embodiments, the payload is not expressed in the liver. In some embodiments, the LNP described herein (e.g., includes a VEE replicon that includes a nucleic acid sequence that encodes a payload) can target the liver, but the expression of the payload is reduced compared to the corresponding expression in non-liver tissues.

[0151] In some embodiments, the VEE replicon is capable of inducing activation of toll-like receptors (TLRs). TLR subsets, TLR3, TLR7 / 8, and TLR9, are involved in antiviral responses by inducing the production of antiviral cytokines such as type I interferon (IFN). TLR3 responds to double-stranded RNA, which is a replication intermediate of many viruses. TLR7 / 8 recognizes viral single-stranded RNA, whereas TLR9 recognizes unmethylated CpG motifs in viral DNA. TLRs involved in virus recognition are expressed on endosomal membranes and can be divided according to the requirement of the adaptor protein MyD88, with TLR3 activity being MyD88-independent, whereas TLR7 / 8 / 9 are MyD88-dependent. Activation of TLR3 leads to the production of type I interferon (IFN). Type I interferon signaling through the ISGF3 (STAT1 / STAT2 / IRF9) complex requires sustained Rip3 activation and necroptosis. Without being bound by any theory, in some embodiments, induction of type I interferon may reduce and / or suppress expression of the payload in the liver.

[0152] IV.A. Payload As described herein, the VEE replicon useful in the present disclosure includes a payload. As used herein, the term "payload" refers to any agent capable of acting on a target (e.g., a target cell) contacted with the LNP described herein. In some embodiments, unless otherwise stated, the term payload may be used interchangeably herein with the term "biologically active molecule." Non-limiting examples of payloads useful in the present disclosure include nucleotides (e.g., nucleotides that contain a detectable moiety or toxin or inhibit transcription), nucleic acids (e.g., DNA or mRNA molecules that code for polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, siRNA, antisense oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), peptide-linked phosphorodiamidate morpholino oligomers (PPMOs), or combinations thereof), amino acids (e.g., amino acids that contain a detectable moiety or toxin or inhibit translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0153] In some embodiments, the payload comprises a nucleic acid sequence encoding a protein (e.g., a heterologous protein). As will be apparent to one skilled in the art, any suitable protein can be encoded into the payload. In some embodiments, the protein comprises a detectable protein (e.g., a fluorescent protein). Non-limiting examples of such detectable proteins include luciferase, wt-GFP, green fluorescent proteins (e.g., EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, etc.), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mTagBFP, etc.), cyan fluorescent proteins (e.g., ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1(Teal), etc.), yellow fluorescent proteins (e.g., EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, etc.), orange fluorescent proteins (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 proteins (e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRedl, mRaspberry, dKeima-T and em, HcRed-T and em, mPlum, AQ143, etc.).

[0154] In some embodiments, the payload comprises a nucleic acid sequence encoding a therapeutic protein. Non-limiting examples of such therapeutic proteins include cytokines. In some embodiments, the cytokine comprises an interleukin (IL)-12 molecule. In some embodiments, the IL-12 molecule comprises IL-12, an IL-12 subunit (e.g., an IL-12β subunit or an IL-12α subunit), or a mutant IL-12 molecule that retains immunomodulatory function. In some embodiments, the cytokine is not IL-12. In some embodiments, the cytokine is selected from (i) the common gamma chain family of cytokines, (ii) the IL-1 family of cytokines, (iii) a hematopoietic cytokine, (iv) an interferon (e.g., type I, type II, or type III), (v) the TNF family of cytokines, (vi) the IL-17 family of cytokines, (vii) a damage-associated molecular pattern (DAMP), (viii) a tolerogenic cytokine, or (ix) a combination thereof. In some embodiments, the cytokine is 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 ( 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 combinations thereof. Additional examples of other proteins that can be encoded are provided further below.

[0155] If the payload comprises a nucleic acid sequence encoding an IL-12 protein, in some embodiments, 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.

[0156] In some embodiments, the IL-12 molecule comprises an IL-12 alpha subunit and / or an IL-12 beta subunit. In some embodiments, 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.

[0157] In some embodiments, 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.

[0158] In some embodiments, the VEE replicon described herein comprises a codon-optimized nucleic acid sequence. Thus, in some embodiments, the nucleotide sequence encoding the IL-12 protein disclosed herein (e.g., IL-12 p35 subunit, IL-12 p40 subunit, or heterodimeric IL-12 p70) differs from the wild-type nucleotide sequence (e.g., SEQ ID NO: 185).

[0159] In some embodiments, the VEE replicon comprises a nucleic acid sequence encoding an IL-12 β subunit, wherein the nucleic acid sequence has at least about 7 amino acid residues to the sequence set forth in any one of 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. 5%, 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.

[0160] In some embodiments, a VEE replicon useful in the present disclosure comprises a nucleic acid sequence encoding an IL-12 β subunit, wherein the nucleic acid sequence has a sequence similar to that of SEQ ID NO:51, (i) 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 set forth in SEQ ID NO:52; (ii) 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 (iii) 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 set forth in SEQ ID NO:53; 5%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; (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% sequence identity to the sequence set forth in SEQ ID NO:54; (v) at least 88%, at least 89%, at least 90% to the sequence set forth in SEQ ID NO:55;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) 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 set forth in SEQ ID NO:56. (vii) 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 set forth in SEQ ID NO:57; (viii) 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 set forth in SEQ ID NO:58; (ix) 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 set forth in SEQ ID NO:59. (x) 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 a sequence set forth in SEQ ID NO: 65, 69, or 74; (xi) 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 a sequence set forth in SEQ ID NO: 66, 70, or 75. (xii) at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:62; (xiii) at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:63; or (xiv) to the sequence set forth in SEQ ID NO:64.have at least 98%, at least 99%, or 100% sequence identity.

[0161] In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:51. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:52. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:53. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:54. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:55. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:56. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:57. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:58. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:59. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:65. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:66. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:67. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:68. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:69. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:70. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:71. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:72. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO:73.In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 74. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 75. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 62. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 63. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 64. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 60. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit comprises the sequence set forth in SEQ ID NO: 61.

[0162] In some embodiments, 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 any of 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. Any one of the sequences has 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 to any one of the sequences set forth.

[0163] In some embodiments, a VEE replicon useful in the present disclosure comprises a nucleic acid sequence encoding an IL-12 p35 subunit, wherein the nucleic acid sequence is (i) 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 set forth in SEQ ID NO: 102; (ii) 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%, at least 100% sequence identity to the sequence set forth in SEQ ID NO: 102; 9%, or 100% sequence identity, (iii) 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% to the sequence set forth in SEQ ID NO: 103. , at least 99%, or 100% sequence identity; (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% sequence identity to the sequence set forth in SEQ ID NO:104; (v) at least 88%, at least 89%, at least 90% to the sequence set forth in SEQ ID NO:105;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 set forth in SEQ ID NO: 106; (vi) 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 set forth in SEQ ID NO: 106; (vii) (viii) 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 set forth in SEQ ID NO:108; (ix) 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 set forth in SEQ ID NO:109; (x) 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 set forth in SEQ ID NO:115, 119, or 124; (xi) 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 set forth in SEQ ID NO:116, 120, or (xii) at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:112; (xiii) at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:113; or (xiv) to the sequence set forth in SEQ ID NO:114.have at least 98%, at least 99%, or 100% sequence identity.

[0164] In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 102. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 103. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 104. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 105. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 106. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 108. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 109. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 115. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 116. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 117. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 118. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 119. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 120. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 121. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 122. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 123.In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 124. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 125. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 112. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 113. In some embodiments, the nucleic acid molecule encoding the IL-12 α subunit comprises the sequence set forth in SEQ ID NO: 114. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 110. In some embodiments, the nucleic acid molecule encoding the IL-12 β subunit comprises the sequence set forth in SEQ ID NO: 111.

[0165] In some embodiments, the nucleic acid molecule encoding the IL-12 p40 subunit and the nucleic acid molecule encoding the IL-12 p35 subunit can be conjugated to each other. For example, in some embodiments, the disclosure provides an isolated polynucleotide comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid encodes the IL-12 p40 subunit and the second nucleic acid encodes the IL-12 p35 subunit. In some embodiments, the IL-12 α subunit and the IL-12 β subunit are linked by a linker. In some embodiments, the linker comprises an amino acid linker of 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. In some embodiments, the linker comprises a (GS) linker. In some embodiments, the GS linker has the formula (Gly3Ser)n or S(Gly3Ser)n, where 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 embodiments, the (Gly3Ser)n linker is (Gly3Ser)3 or (Gly3Ser)4.

[0166] In some embodiments, 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 embodiments, 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 transmit signals between cells and stimulate cell growth. Although cytokines can be growth factors, the two terms are differentiated because certain cytokines also have an inhibitory effect on cell growth. Non-limiting examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, 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 B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 3 (FGF3), and 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 9 (FGF9), fibroblast growth factor 10 (FGF10), fibroblast growth factor 11 (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), fibroblast 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 serum (FBS), glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, artemin, growth differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), hepatocellular carcinoma-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 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), renalase (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF).

[0167] In some embodiments, 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 signal transduction receptors (CSRs), T cell receptor mimics (TCR mimics), and combinations thereof.

[0168] In some embodiments, the VEE replicon comprises a nucleic acid sequence encoding a CAR. In some embodiments, the CAR is designed as a standard CAR. In a "standard CAR", different components (e.g., an extracellular targeting domain, a transmembrane domain, and an intracellular signaling / activation domain) are linearly assembled as a single fusion protein. In some embodiments, the CAR is designed as a first generation CAR. A "first generation" CAR is composed of an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains. All first generation CARs contain a CD3 zeta chain domain as an intracellular signaling domain. In some embodiments, the CAR is designed as a second generation CAR. A "second generation" CAR further contains a costimulatory domain (e.g., CD28 or 4-1BB). In some embodiments, the CAR is designed as a third generation CAR. A "third generation" CAR is similar to a second generation CAR, except that it contains multiple costimulatory domains (e.g., CD28-4-1BB or CD28-OX40). In some embodiments, the CAR is designed as a fourth generation CAR. "Fourth generation" CARs (also known as TRUCK or enhanced CARs) further contain additional factors that can further improve function. For example, in some embodiments, fourth generation CARs further contain cytokines that can be released upon CAR signaling in the targeted tumor tissue. In some embodiments, fourth generation CARs include one or more additional elements, such as homing genes and suicide genes, that can help to further control the activity of the CAR. In some embodiments, the CAR is designed as a split CAR. In a "split CAR" system, one or more components of the CAR (e.g., extracellular targeting domain, transmembrane domain, and intracellular signaling / activation domain) are split into two or more parts such that they depend on multiple inputs to promote the assembly of a complete functional receptor. In some embodiments, the CAR is designed as a switchable CAR. In a "switchable CAR", the CAR can be switched on (switch-on CAR) or off (switch-off CAR) in the presence of a stimulus (e.g., transiently).Additional examples of CARs that can be used with the present disclosure are described, for example, in US2020 / 0172879A1 and US2019 / 0183932A1, each of which is incorporated by reference in its entirety.

[0169] In some embodiments, the VEE replicon described herein comprises a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof (collectively referred to herein as "antibodies"). The antibodies may be derived from natural sources or may be partially or wholly synthetically produced. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is an IgG antibody. In some embodiments, the monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antigen-binding fragment is a Fab, Fab', or F(ab') 2 , F(ab1) 2 In some embodiments, the antigen-binding fragment is selected from an scFv or (scFv) 2 In some embodiments, the antibody or antigen-binding fragment is a NANOBODY® (single domain antibody). In some embodiments, the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

[0170] IV.B. Additional Components In some embodiments, a VEE replicon useful in the present disclosure comprises a nucleic acid sequence that includes one or more of the following additional components:

[0171] Terminal structural modification: UTR The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR starts at the transcription initiation site and continues up to, but not including, the initiation codon. Meanwhile, the 3'UTR starts immediately after the stop codon and continues up to the transcription termination signal. There is a lot of evidence that UTRs play a regulatory role in the stability and translation of nucleic acid molecules. The regulatory function of UTRs can be incorporated into the RNA of the present disclosure to increase the stability of the molecule. Specific functions can also be incorporated to ensure regulated downregulation of the transcript in case of misdirection to undesired organ sites.

[0172] 5'UTR and translation start Natural 5'UTRs function to play a role in translation initiation. They have signatures such as the Kozak sequence, which is commonly known to be involved in the translation process of many genes initiated by the ribosome. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) followed by another "G". 5'UTRs are also known to form secondary structures involved in the binding of elongation factors.

[0173] 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 control gene expression. For example, microRNA-mediated repression requires the elongation factor EIF4A2 to bind to secondary structured elements in the 5'UTR (Meijer HA et al., Science, 2013, 340, 82-85, incorporated herein by reference in its entirety). Different secondary structures of the 5'UTR can be engineered into the flanking regions to stabilize or selectively destabilize mRNA in specific tissues or cells.

[0174] By engineering the features typically found in genes that are abundantly expressed in a particular target organ, the stability and protein production of the nucleic acid or mRNA of the present disclosure can be increased. For example, introducing the 5'UTR of liver-expressed mRNA 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 mRNA in hepatic cell lines or the liver. Similarly, by using the 5'UTR of other tissue-specific mRNAs, it is possible to improve expression in the tissues of muscle (MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (Tie-1, CD36), bone marrow cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (SP-A / B / C / D).

[0175] Other non-UTR sequences can also be incorporated into the 5' (or 3' UTR) UTR. For example, introns or portions of intron sequences can be incorporated into the flanking regions of the nucleic acids or mRNAs of the present disclosure. Incorporation of intron sequences can increase protein production and mRNA levels.

[0176] In some embodiments 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 an 18 nucleotide sequence from the IRES of the GTX gene.As another non-limiting example, the 18 nucleotide sequence fragment from the IRES sequence of the GTX gene can be repeated in tandem in the 5'UTR of the polynucleotide described herein.The 18 nucleotide sequence can be repeated 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 ten times or more in the 5'UTR.

[0177] Nucleotides of the 5' (or 3') UTR may be mutated, substituted and / or removed. For example, one or more nucleotides upstream of the start codon can be replaced with other nucleotides. The replaced nucleotide(s) are 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 the UTR.

[0178] 5'UTR, 3'UTR and translational enhancer elements (TEEs) In some embodiments, the 5'UTR of a nucleotide sequence encoding IL-12 comprises at least one translation enhancer polynucleotide, translation enhancer element, or translation enhancer element (collectively referred to as "TEE"). In some embodiments, the TEE is located between the transcription promoter and the start codon. In some embodiments, an RNA comprising at least one TEE in its 5'UTR comprises a cap in the 5'UTR. In some embodiments, at least one TEE can be located in the 5'UTR of a nucleotide sequence encoding IL-12 that undergoes cap-dependent or cap-independent translation.

[0179] The term "translation enhancer element" or "translation enhancer elements" (collectively referred to herein as "TEEs") refers to a sequence that increases the amount of a polypeptide or protein produced from an mRNA.

[0180] In one embodiment, TEE is a conserved element of UTR that can promote the translation activity of nucleic acid, such as, but not limited to, cap-dependent or cap-independent translation. The conservation of these sequences has been previously shown by Panek et al. (Nucleic Acids Research, 2013, 1-10; the entire contents of which are incorporated herein by reference) across 14 species, including humans.

[0181] In some embodiments, the nucleotide sequence encoding IL-12 has at least one TEE that has 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 to that disclosed in U.S. Application No. 2014 / 0147454, which is incorporated by reference in its entirety. In some embodiments, the RNA is synthesized by any method as described in 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 ... Nos. 6,849,405, 7,456,273, 7,183,395, each of which is incorporated herein by reference in its entirety.

[0182] In some embodiments, the 5'UTR of the nucleotide sequence encoding IL-12 may comprise 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 embodiments, the TEE sequences in the 5'UTR of the RNA are the same or different TEE sequences. In some embodiments, the TEE sequences are in a pattern such as ABABAB or AABBABBAABB or ABCABCABC or variations thereof, repeated once, twice, or more than three times. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0183] In some embodiments, the spacer separating the two TEE sequences comprises other sequences known in the art that control translation of RNA, including, but not limited to, miR sequences described herein (e.g., miR binding sites and miR seeds). In some embodiments, each spacer used to separate the two TEE sequences comprises a different miR sequence or component of a miR sequence (e.g., a miR seed sequence).

[0184] In some embodiments, the TEE used in the 5'UTR of a nucleotide sequence encoding IL-12 of the present disclosure is an IRES sequence, such as, but not limited to, those described in U.S. Pat. No. 7,468,275 and International Patent Publication No. WO2001055369, each of which is incorporated by reference in its entirety.

[0185] In some embodiments, the TEE described herein is located in the 5'UTR and / or the 3'UTR of a nucleotide sequence encoding IL-12. In some embodiments, the TEE located in the 3'UTR is the same and / or different from the TEE located in the 5'UTR and / or the TEE described for incorporation into the 5'UTR.

[0186] In some embodiments, the 3'UTR of the nucleotide sequence encoding IL-12 may contain 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 embodiments, the TEE sequences in the 3'UTR of the nucleotide sequence encoding IL-12 of the present disclosure are the same or different TEE sequences. The TEE sequences are in a pattern such as ABABAB or AABBABBAABB or ABCABCABC or variations thereof, repeated once, twice, or more than three times. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0187] In some embodiments, the 3'UTR comprises a spacer separating the two TEE sequences. In some embodiments, the spacer is a 15 nucleotide spacer and / or other spacers known in the art. In some embodiments, the 3'UTR may comprise a TEE sequence-spacer module that is repeated 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, and at least nine times or more in the 3'UTR.

[0188] In some embodiments, the spacer separating the two TEE sequences comprises other sequences known in the art that control translation of a nucleotide sequence encoding IL-12, including, but not limited to, miR sequences described herein (e.g., miR binding sites and miR seeds). In some embodiments, each spacer used to separate the two TEE sequences comprises a different miR sequence or component of a miR sequence (e.g., a miR seed sequence).

[0189] Incorporation of microRNA binding sites In some embodiments, the nucleotide sequence encoding IL-12 further comprises a sensor sequence. Sensor sequences include, for example, microRNA binding sites, transcription factor binding sites, structured mRNA sequences and / or motifs, artificial binding sites designed to act as analogous receptors for endogenous nucleic acid binding molecules. Non-limiting examples of polynucleotides comprising at least one sensor sequence are described in U.S. Application No. 2014 / 0147454, the entire contents of which are incorporated herein by reference.

[0190] In some embodiments, microRNA (miRNA) profiling of target cells or tissues can be performed to determine the presence or absence of miRNAs in the cells or tissues.

[0191] MicroRNAs (or miRNAs) are non-coding RNAs, 19-25 nucleotides long, that downregulate gene expression by binding to the 3'UTR of a nucleic acid molecule and either reducing the stability of the nucleic acid molecule or inhibiting translation. In some embodiments, the 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 U.S. Publication No. US2005 / 0261218 and U.S. Publication No. US2005 / 0059005, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, known microRNAs in the human genome, their sequences, and seed sequences are described in U.S. Application No. 2014 / 0147454, the contents of which are incorporated herein by reference in their entirety.

[0192] MicroRNA sequences include a "seed" region, i.e., the sequence in the region of positions 2-8 of the mature microRNA, and this sequence has perfect Watson-Crick complementarity to the miRNA target sequence. The microRNA seed includes positions 2-8 or 2-7 of the mature microRNA. In some embodiments, the microRNA seed includes 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), and the seed complementary site of the corresponding miRNA target has an adenine (A) adjacent to the position 1 of the microRNA. In some embodiments, the microRNA seed includes 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), and the seed complementary site of the corresponding miRNA target has an adenine (A) adjacent to the position 1 of the microRNA. See, for example, Grimson A, Farh K, Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul. 6;27(1):91-105. The bases of the microRNA seed have perfect complementarity to the target sequence. By engineering a microRNA target sequence into the 3' UTR of a nucleic acid or mRNA of the present disclosure, if the microRNA is available, the molecule can be targeted for degradation or translational reduction. This process reduces the risk of off-target in nucleic acid molecule delivery.Identification of microRNAs, their target regions, and 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(2011 Dec.20.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).

[0193] For example, if an mRNA is not intended for delivery to the liver but does end up there, expression of the gene of interest can be inhibited by miR-122, a microRNA that is abundant in the liver, if one or more target sites for miR-122 are engineered into the 3'UTR of the modified nucleic acid, enhanced modified RNA, or ribonucleic acid. The life span, stability, and protein translation of the modified nucleic acid, enhanced modified RNA, or ribonucleic acid can be further reduced by introducing one or more binding sites for different microRNAs. As used herein, the term "microRNA site" refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It is understood that "binding" may follow conventional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with a target sequence at or adjacent to the microRNA site.

[0194] Conversely, for purposes of the disclosed IL-12-encoding nucleotide sequences, microRNA binding sites can be engineered out (i.e., removed) from the naturally occurring sequence to increase protein expression in a particular tissue. For example, the miR-122 binding site can be removed to improve protein expression in the liver.

[0195] In some embodiments, the nucleotide sequence encoding IL-12 contains at least one miRNA binding site in the 3'UTR to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells, including, but not limited to, normal cells and / or cancerous cells (e.g., HEP3B or SNU449).

[0196] 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), bone marrow 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).

[0197] Specifically, microRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, and natural killer cells. Immune cell-specific microRNAs are involved in immunogenicity, autoimmunity, immune responses to infections, inflammation, and undesirable immune responses after gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also control many aspects of hematopoietic cell (immune cell) development, proliferation, differentiation, and apoptosis. For example, miR-142 and miR-146 are expressed exclusively in immune cells and are particularly abundant in myeloid dendritic cells. It has been shown in the art that by adding miR-142 binding sites to the 3'UTR of the gene construct to be delivered, immune responses to exogenous nucleic acid molecules are blocked, allowing for more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous mRNA in antigen-presenting cells and prevents 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).

[0198] Many microRNA expression studies have been conducted in the art to profile the differential expression of microRNAs in various cancer cells / tissues and other diseases. Some microRNAs are abnormally overexpressed and some are underexpressed in certain cancer cells. 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. respiratory and inflammation (U.S. Patent No. 8,415,096); prostate cancer (U.S. Patent No. 2013 / 0053264); hepatocellular carcinoma (WO2012 / 151212, U.S. Patent No. 2012 / 0329672, WO2008 / 054828, U.S. Patent No. 8,252,538); lung cancer cells (WO2011 / 076143, WO2013 / 033640, WO2009 / 070653, U.S. Patent No. 2010 / 0323357); cutaneous T-cell lymphoma ( WO2013 / 011378; colon cancer cells (WO2011 / 0281756, WO2011 / 076142); cancer positive lymph nodes (WO2009 / 100430, US2009 / 0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012 / 0264626, US2013 / 0053263); thyroid cancer (WO2013 / 066678); ovarian cancer cells (US2012 / 030 9645, 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).

[0199] At least one microRNA site may be engineered into the 3'UTR of the nucleotide sequence encoding IL-12. In some embodiments, 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 or more microRNA sites may be engineered into the 3'UTR of the nucleotide sequence encoding IL-12. In some embodiments, the microRNA sites incorporated into the nucleotide sequence encoding IL-12 are the same or different microRNA sites. In some embodiments, the microRNA sites incorporated into the nucleotide sequence encoding IL-12 target the same or different tissues in the body. As a non-limiting example, the degree of expression in a particular cell type (e.g., hepatocytes, bone marrow cells, endothelial cells, cancer cells, etc.) can be reduced by introducing tissue, cell type, or disease-specific microRNA binding sites into the 3'UTR of the mRNA.

[0200] In some embodiments, the microRNA site is engineered near the 5' end of the 3'UTR, approximately halfway between the 5' and 3' ends of the 3'UTR, and / or near the 3' end of the 3'UTR. In some embodiments, the microRNA site is engineered near the 5' end of the 3'UTR and approximately halfway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is engineered near the 3' end of the 3'UTR and approximately halfway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is engineered near the 5' end of the 3'UTR and near the 3' end of the 3'UTR.

[0201] In some embodiments, the mRNA comprises a microRNA binding region site that either has 100% identity to a known seed sequence or has less than 100% identity to the seed sequence. The seed sequence may be partially mutated to reduce the binding affinity of the microRNA, resulting in a reduced downregulation of its mRNA transcript. In essence, the degree of match or mismatch between the target mRNA and the microRNA seed can act as a rheostat to finely adjust the ability of the microRNA to regulate protein expression. In addition, mutations in the non-seed region of the microRNA binding site can also affect the ability of the microRNA to regulate protein expression.

[0202] RNA motifs in RNA-binding proteins (RBPs) RNA binding proteins (RBPs) can control many aspects of co-transcriptional and post-transcriptional gene expression, including but not limited to RNA splicing, localization, translation, turnover, polyadenylation, capping, modification, export and localization. RNA binding domains (RBDs), including but not limited to RNA recognition motifs (RR) and hnRNP K homology (KH) domains, typically control the sequence association between RBPs and their RNA targets (Ray et al. Nature 2013.499:172-177; incorporated herein by reference in its entirety). In some embodiments, canonical RBDs bind to short RNA sequences. In some embodiments, canonical RBDs recognize RNA structures.

[0203] Non-limiting examples of RNA binding proteins and related nucleic acid and protein sequences are described in U.S. Application No. 2014 / 0147454, which is incorporated by reference in its entirety.

[0204] In some embodiments, mRNA encoding HuR is co-transfected or coinjected into cells or tissues with the mRNA of interest to increase the stability of the mRNA of interest. These proteins can also be tethered to the mRNA of interest in vitro and then administered together to cells. The polyA tail binding protein PABP interacts with the eukaryotic translation initiation factor eIF4G to stimulate translation initiation. Co-administration of mRNA encoding these RBPs with mRNA drugs and / or tethering these proteins to mRNA drugs in vitro and administering the protein-bound mRNA to cells can increase the translation efficiency of the mRNA. The same concept can be extended to co-administration of mRNA with mRNAs encoding various translation factors and enhancers as well as the proteins themselves to affect RNA stability and / or translation efficiency.

[0205] In some embodiments, the nucleotide sequence encoding IL-12 contains at least one RNA binding motif, such as, but not limited to, an RNA binding domain (RBD).

[0206] In some embodiments, the first region of the linked nucleosides and / or at least one flanking region comprises at least one RBD, hi some embodiments, the first region of the linked nucleosides comprises an RBD associated with a splicing factor and at least one flanking region comprises an RBD associated with a stability and / or translation factor.

[0207] Other regulatory elements in the 3'UTR In addition to microRNA binding sites, other regulatory sequences in the 3'-UTR of native mRNAs that control mRNA stability and translation in different tissues and cells may be removed or introduced into the RNA. Such cis-regulatory elements include, but are not limited to, Cis-RNP (ribonucleoprotein) / RBP (RNA-binding protein) regulatory elements, AU-rich elements (AUEs), structured stem-loops, constitutive decay elements (CDEs), GC-rich and other structured mRNA motifs (Parker BJ et al., Genome Research, 2011, 21, 1929-1943, incorporated herein by reference in its entirety). For example, CDEs are a type of regulatory motif that mediates mRNA degradation by interacting with Roquin proteins. In particular, CDEs are found in many mRNAs that code for regulators of development and inflammation, limiting cytokine production in macrophages (Leppek K et al., 2013, Cell, 153, 869-881, incorporated herein by reference in its entirety).

[0208] In some embodiments, the RNA is auxotrophic.As used herein, the term "auxotrophic" refers to an mRNA that contains at least one feature that induces, promotes, or induces the degradation or inactivation of the mRNA in response to spatial or temporal cues, such that protein expression is substantially prevented or reduced.Such spatial or temporal cues include the location of the mRNA to be translated, for example, a specific tissue or organ or cellular environment.Cues related to temperature, pH, ionic strength, water content, etc. are also contemplated.

[0209] 3'UTR and AU-rich elements 3'UTRs are known to be embedded with stretches of adenosines and uridines. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on sequence features and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995). Class I AREs contain several copies of the AUUUA motif dispersed 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) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III AREs are less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize messengers, but members of the ELAV family, particularly HuR, have been shown to enhance mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding, which stabilizes the messenger in vivo.

[0210] The introduction, removal or modification of 3'UTR AU-rich elements (AREs) can be used to modulate the stability of the nucleic acids or mRNAs of the present disclosure. When engineering a particular nucleic acid or mRNA, one or more copies of AREs can be introduced to reduce the stability of the nucleic acid or mRNA of the present disclosure, thereby reducing translation and resulting protein production. Similarly, to increase intracellular stability, AREs can be identified and removed or mutated, thereby increasing translation and resulting protein production. The nucleic acids or mRNAs of the present disclosure can be used to perform transfection experiments in relevant cell lines and assay protein production at various times after transfection. For example, different ARE engineered molecules can be transfected into cells and ELISA kits for the relevant proteins can be used to assay protein produced at about 6 hours, about 12 hours, about 24 hours, about 48 hours, and / or about 7 days after transfection.

[0211] 3'UTR and triple helix In some embodiments, the nucleotide sequence encoding IL-12 comprises a triple helix at the 3' end of the nucleic acid, enhanced nucleotide sequence encoding IL-12, or ribonucleic acid. In some embodiments, the 3' end of the nucleotide sequence encoding IL-12 comprises a triple helix alone or in combination with a polyA tail.

[0212] In some embodiments, the nucleotide sequence encoding IL-12 comprises at least a first and a second U-rich region, a conserved stem-loop region between the first and the second region, and an A-rich region. In some embodiments, the first and the second U-rich region and the A-rich region associate to form a triple helix at the 3' end of the nucleic acid. This triple helix can stabilize the nucleic acid, increase the translation efficiency of the nucleic acid, and / or protect the 3' end from degradation. Exemplary triple helices include, but are not limited to, triple helix sequences of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN-β, and polyadenylated nuclear (PAN) RNA (see Wilusz et al., Genes & Development 2012 26:2392-2407, which is incorporated herein by reference in its entirety).

[0213] Stem Loop In some embodiments, the nucleotide sequence encoding IL-12 includes a stem loop, such as, but not limited to, a histone stem loop. In some embodiments, the stem loop is a nucleotide sequence of about 25 or about 26 nucleotides in length, such as, but not limited to, SEQ ID NOs: 7-17 in International Patent Publication No. WO2013103659, which is incorporated by reference in its entirety. The histone stem loop can be located 3' to the coding region (e.g., the 3' end of the coding region). As a non-limiting example, the stem loop can be located at the 3' end of the nucleic acid described herein.

[0214] In some embodiments, IL-12-encoding nucleotide sequences containing histone stem loops can be stabilized by the addition of at least one chain-terminating nucleoside. Without wishing to be bound by theory, the addition of 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.

[0215] In some embodiments, the chain-terminating nucleoside is one described in International Patent Publication No. WO2013103659, the entire contents of which are incorporated herein by reference. In some embodiments, the chain-terminating nucleoside is 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleoside, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleoside, or -O-methylnucleoside.

[0216] In some embodiments, the nucleotide sequence encoding IL-12 comprises a histone stem loop, a polyA tail sequence and / or a 5' cap structure. In some embodiments, the histone stem loop precedes and / or follows the polyA tail sequence. Nucleic acids comprising a histone stem loop and a polyA tail sequence can include chain-terminating nucleosides as described herein.

[0217] In some embodiments, the nucleotide sequence encoding IL-12 comprises a histone stem loop and a 5' cap structure, including but not limited to those described herein and / or known in the art.

[0218] 5' Capping The 5' cap structure of mRNA is involved in nuclear export, enhances mRNA stability, and binds to mRNA cap-binding protein (CBP). CBP is responsible for mRNA stability and translational competence in cells by associating with poly(A)-binding protein to form mature cyclic mRNA species. The cap also aids in the removal of the 5' adjacent intron during mRNA splicing.

[0219] Endogenous mRNA molecules may be 5'-end capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA. This 5'-guanylic acid cap may then be methylated to generate an N7-methylguanylic acid residue. The ribose sugar of the terminal and / or pre-terminal transcribed nucleotide at the 5' end of the mRNA may also be optionally 2'-O-methylated. 5'-capping removal via hydrolysis and cleavage of the guanylic acid cap structure may target nucleic acid molecules, such as mRNA molecules, for degradation.

[0220] Modifications to RNA of the present disclosure may generate non-hydrolyzable cap structures that increase mRNA half-life by preventing decapping. Because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond, modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to generate phosphorothioate bonds in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, can also be used.

[0221] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5'-terminus and / or penultimate nucleotide of an mRNA at the 2'-hydroxyl group of the sugar ring (as described above).Several different 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.

[0222] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, have a different chemical structure from the natural (i.e., endogenous, wild-type or physiological) 5'-cap but retain cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or attached to a nucleic acid molecule.

[0223] For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by 5'-5'-triphosphate groups, where one guanosine contains an N7 methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which can equivalently be represented as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other, unmodified guanosine is linked to the 5' terminal nucleotide of a capped nucleic acid molecule (e.g., an mRNA or mmRNA). The N7- and 3'-O-methylated guanine provide the terminal portion of the capped nucleic acid molecule (e.g., an mRNA or mmRNA).

[0224] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-β-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0225] In some embodiments, the cap is a dinucleotide cap analog in which different phosphate positions are modified with boranophosphate or phosphoroselenoate groups, such as the dinucleotide cap analogs described in U.S. Patent No. 8,519,110, the contents of which are incorporated herein by reference in their entirety.

[0226] In some embodiments, the cap is a cap analog and is an N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of cap analog N7-(4-chlorophenoxyethyl) substituted dinucleotide forms include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for various cap analogs and methods of synthesizing cap analogs; the contents of which are incorporated herein by reference in their entirety). In some embodiments, the cap analog of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0227] Although cap analogs can simultaneously cap nucleic acid molecules in in vitro transcription reactions, up to 20% of the transcripts remain uncapped. This, in addition to the structure of the cap analog being different from the endogenous 5' cap structure of the nucleic acid generated by the endogenous cellular transcription machinery, can lead to reduced translational capacity and reduced cellular stability. Thus, in some embodiments, the methods provided herein (see, e.g., Examples 1-3) can increase the capping efficiency of the IL-12 expression nucleotides produced as described herein. In some embodiments, the methods provided herein result in 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%, or about 100% of the polynucleotides being capped. In some embodiments, at least about 50% of the polynucleotides are capped by the methods provided herein. In some embodiments, at least about 60% of the polynucleotides are capped. In some embodiments, at least about 70% of the polynucleotides are capped. In some embodiments, at least about 80% of the polynucleotides are capped. In some embodiments, at least about 85% of the polynucleotides are capped. In some embodiments, at least about 90% of the polynucleotides are capped. In some embodiments, at least about 95% of the polynucleotides are capped. In some embodiments, about 100% of the polynucleotides are capped. In some embodiments, at least about 80% to about 100% of the polynucleotides are capped.

[0228] In some embodiments, providing an RNA comprising a 5'-cap or 5'-cap analog is accomplished by in vitro transcribing a DNA template in the presence of the 5'-cap or 5'-cap analog, wherein the 5'-cap is co-transcriptionally incorporated into the generated RNA strand.

[0229] In some embodiments, the RNA can be produced, for example, by in vitro transcription, and a 5' cap can be post-transcriptionally attached to the RNA using a capping enzyme, for example, vaccinia virus capping enzyme. In some embodiments, the IL-12-encoding nucleotide sequence is post-transcriptionally capped using an enzyme to generate a more authentic 5'-cap structure. As used herein, the term "more authentic" refers to a feature that closely reflects or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a "more authentic" feature is one that better represents an endogenous, wild-type, natural or physiological cellular function and / or structure, as compared to prior art synthetic features or analogs, or that exceeds the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5' cap structures of the present disclosure include, inter alia, those that enhance binding of cap-binding proteins, extend half-life, reduce susceptibility to 5' endonucleases, and / or reduce 5' capping removal, as compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can generate a standard 5'-5'-triphosphate linkage between the 5' terminal nucleotide of an mRNA and a guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5' terminal nucleotide of the mRNA contains a 2'-O-methyl. This cap results in higher translational competence and cellular stability, as well as reduced activation of cellular pro-inflammatory cytokines, as compared to, for example, other 5' cap analog structures known in the art. 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.

[0230] In some embodiments, the 5' end cap comprises an endogenous cap or a cap analog. In some embodiments, the 5' end 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.

[0231] In some embodiments, the 5' cap comprises a 5'-5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5'-5' triphosphate linkage comprising a phosphorothioate modification. In some embodiments, the 5' cap comprises a 2'-O or 3'-O-ribose-methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine or adenosine nucleotide. In some embodiments, the 5' cap comprises 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.

[0232] In some embodiments, the nucleotide sequence encoding IL-12 comprises a modified 5' cap. Modifications on the 5' cap can increase mRNA stability, increase the half-life of the mRNA, and increase mRNA translation efficiency. In some embodiments, the modified 5' cap comprises one or more of the following modifications: a modification at the 2' and / or 3' position of the capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (forming a carbon ring) with a methylene moiety (CH2), a modification in the triphosphate bridge portion of the cap structure, or a modification in the nucleobase (G) portion.

[0233] 5' cap structures that may be modified include, but are not limited to, the caps described in U.S. Application No. 2014 / 0147454 and WO2018 / 160540, which are incorporated by reference in their entireties.

[0234] IRES sequence In some embodiments, the nucleotide sequence encoding IL-12 contains an internal ribosome entry site (IRES). IRES, first identified as a feature of picornavirus RNA, plays an important role in initiating protein synthesis in the absence of a 5' cap structure. IRES can function alone as a ribosome binding site for an mRNA or as one of several ribosome binding sites. A nucleic acid or mRNA containing several functional ribosome binding sites can code for several peptides or polypeptides that are translated independently by the ribosome ("multicistronic nucleic acid molecule"). When a nucleic acid or mRNA comprises an IRES, a second translatable region is optionally further provided. Examples of IRES sequences that can be used in accordance with the present disclosure include, but are not limited to, those derived from picornaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0235] Terminal structural modification: polyA tail Messenger RNA (mRNA) molecules typically have a long chain of adenine nucleotides (poly-A tail) added to them during RNA processing to increase the stability of the molecule. Immediately after transcription, the 3' end of the transcript is cleaved, liberating a 3' hydroxyl group. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. A process called polyadenylation adds a poly-A tail that is 100-250 residues long.

[0236] In some embodiments, the length of the 3' tail is greater than about 30 nucleotides long. In some embodiments, the polyA tail is greater than about 35 nucleotides long. In some embodiments, the length is at least about 40 nucleotides. In some embodiments, the length is at least about 45 nucleotides. In some embodiments, the length is at least about 55 nucleotides. In some embodiments, the length is at least about 60 nucleotides. In some embodiments, the length is at least about 70 nucleotides. In some embodiments, the length is at least about 80 nucleotides. In some embodiments, the length is at least about 90 nucleotides. In some embodiments, the length is at least about 100 nucleotides. In some embodiments, the length is at least about 120 nucleotides. In some embodiments, the length is at least about 140 nucleotides. In some embodiments, the length is at least about 160 nucleotides. In some embodiments, the length is at least about 180 nucleotides. In some embodiments, the length is at least about 200 nucleotides. In some embodiments, the length is at least about 250 nucleotides. In some embodiments, the length is at least about 300 nucleotides. In some embodiments, the length is at least about 350 nucleotides. In some embodiments, the length is at least about 400 nucleotides. In some embodiments, the length is at least about 450 nucleotides. In some embodiments, the length is at least about 500 nucleotides. In some embodiments, the length is at least about 600 nucleotides. In some embodiments, the length is at least about 700 nucleotides. In some embodiments, the length is at least about 800 nucleotides. In some embodiments, the length is at least about 900 nucleotides. In some embodiments, the length is at least about 1000 nucleotides. In some embodiments, the length is at least about 1100 nucleotides. In some embodiments, the length is at least about 1200 nucleotides.In some embodiments, the length is at least about 1300 nucleotides. In some embodiments, the length is at least about 1400 nucleotides. In some embodiments, the length is at least about 1500 nucleotides. In some embodiments, the length is at least about 1600 nucleotides. In some embodiments, the length is at least about 1700 nucleotides. In some embodiments, the length is at least about 1800 nucleotides. In some embodiments, the length is at least about 1900 nucleotides. In some embodiments, the length is at least about 2000 nucleotides. In some embodiments, the length is at least about 2500 nucleotides. In some embodiments, the length is at least about 3000 nucleotides.

[0237] In some embodiments, the nucleotide sequence encoding IL-12 is designed to include a poly A-G cassette. The G cassette is a sequence in which four guanine nucleotides are hydrogen-bonded cyclically and can be formed by a G-rich sequence in both DNA and RNA. In this embodiment, the G cassette is incorporated at the end of the poly A tail. The resulting nucleic acid or mRNA can be assayed for other parameters including stability, protein production, and half-life at various time points. The poly A-G cassette has been found to result in protein production equivalent to at least 75% of the protein production seen when using a 120-nucleotide poly A tail alone.

[0238] In some embodiments, the nucleotide sequence encoding IL-12 includes a poly A tail and is stabilized by the addition of a chain-terminating nucleoside. In some embodiments, the nucleotide sequence encoding IL-12 that includes a poly A tail further includes a 5' cap structure.

[0239] In some embodiments, the nucleotide sequence encoding IL-12 comprises a poly-AG quartet. In some embodiments, the nucleotide sequence encoding IL-12 comprising a poly-AG quartet further comprises a 5' cap structure.

[0240] In some embodiments, nucleotide sequences encoding IL-12 containing poly-A tails or poly-AG quartets are stabilized by the addition of oligonucleotides terminating in 3'-deoxynucleosides, 2',3'-dideoxynucleosides 3'-0-methylnucleosides, 3'-0-ethylnucleosides, 3'-arabinosides, and other modified nucleosides known in the art and / or described herein.

[0241] Modified Nucleosides In some embodiments, the nucleotide sequence encoding IL-12 comprises one or more modified nucleosides. In some embodiments, the one or more modified nucleosides are 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-o-cytidine, 5-amino-cytidine, 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-amino-cytidine, 5 ... These include xo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 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, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, and combinations thereof.

[0242] In some embodiments, one or more uridines in a nucleotide sequence encoding IL-12 are replaced by a modified nucleoside. In some embodiments, the modified nucleoside replacing the uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0243] In some embodiments, the nucleotide sequence encoding IL-12 comprises a nucleotide sequence encoding IL-12 described in U.S. Application No. 2014 / 0147454, International Application No. WO2018160540, International Application No. WO2015 / 196118, or International Application No. WO2015 / 089511, each of which is incorporated by reference in its entirety.

[0244] Cytotoxic nucleosides In some embodiments, the nucleotide sequence encoding IL-12 comprises one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into a polynucleotide, such as a bifunctional nucleotide sequence or mRNA encoding IL-12. Cytotoxic nucleoside anticancer drugs include, but are not limited to, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and 6-mercaptopurine.

[0245] Many cytotoxic nucleoside analogues are in clinical use or are the subject of clinical trials as anticancer drugs. Examples of such analogues include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), cladribine, clofarabine, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine and 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine. Another example of such a compound is fludarabine phosphate. These compounds can be administered systemically and may have side effects typical of cytotoxic drugs, including but not limited to little or no specificity for tumor cells over proliferating normal cells.

[0246] Many prodrugs of cytotoxic nucleoside analogues have also been reported in the art. Examples include, but are not limited to, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidic acid ester). In general, these prodrugs can be circulated into active drugs mainly in the liver and systemic circulation, and show little or no selective release of active drugs in tumor tissue. For example, capecitabine, a prodrug of 5'-deoxy-5-fluorocytidine (and ultimately 5-fluorouracil), is metabolized in both the liver and tumor tissue. A series of capecitabine analogs containing "radicals readily hydrolyzable under physiological conditions" have been claimed by Fujiu et al. (U.S. Pat. No. 4,966,891) and is incorporated herein by reference. The series described by Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine, which are suggested to be activated by hydrolysis under normal physiological conditions to yield 5'-deoxy-5-fluorocytidine.

[0247] A series of cytarabine N4-carbamates have been reported by Fadl et al (Pharmazie. 1995, 50, 382-7, incorporated herein by reference in its entirety), and the compounds are designed to convert to 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 designed to overcome the gastrointestinal toxicity of gemcitabine, and are intended to provide gemcitabine by hydrolysis release in the liver and plasma after the intact prodrug is absorbed from the gastrointestinal tract. Nomura et al. (Bioorg Med. Chem. 2003, 11, 2453-61, incorporated herein by reference in its entirety) described acetal derivatives of 1-(3-C-ethynyl-β-D-ribo-pentopharanosyl)cytosine, which upon bioreduction produce intermediates that require further hydrolysis under acidic conditions to afford cytotoxic nucleoside compounds.

[0248] Cytotoxic nucleotides that may be chemotherapeutic agents include, but are not limited to, pyrazolo[3,4-D]-pyrimidines, allopurinol, azathioprine, capecitabine, cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, 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.

[0249] V. Pharmaceutical Compositions In some embodiments, the present disclosure relates to a pharmaceutical composition comprising the polynucleotide, vector, and / or lipid nanoparticle described herein. In some embodiments of the present disclosure, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier (excipient). "Acceptable" as used herein means that the carrier must be compatible with the active ingredient of the composition and not deleterious to the subject being treated. In some embodiments, the carrier is capable of stabilizing the active ingredient. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkoins, Ed. KE Hoover.

[0250] The pharmaceutical composition used for in vivo administration must be sterile.This can be easily achieved by, for example, filtration through a sterile filtration membrane.Lipid nanoparticles can be placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0251] In some embodiments of the present disclosure, the pharmaceutical composition can be formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration. In some embodiments of the present disclosure, the pharmaceutical composition can be formulated for intratumoral injection. Intratumoral injection, as used herein, refers to injection directly into the tumor. High concentrations of the composition can be obtained in situ while using small amounts of drug. Local delivery of immunotherapy allows for multiple combination therapies while avoiding significant systemic exposure and off-target toxicity.

[0252] In some embodiments of the present disclosure, the pharmaceutical compositions can be formulated for intramuscular, intravenous, or subcutaneous injection.

[0253] In some embodiments of the present disclosure, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, buffer, excipient, salt, or stabilizer in the form of a lyophilized formulation or an aqueous solution. th See, Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. Acceptable carriers and excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, 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 10 residues) polypeptides; serum albumin, proteins such as 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 dextran; 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 non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0254] In some embodiments, the pharmaceutical compositions described herein comprise lipid nanoparticles that can be prepared by methods known in the art, such as those described in 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 entireties. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556, which is incorporated herein by reference in its entirety. In some embodiments, liposomes can be made by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0255] In some embodiments of the present disclosure, the pharmaceutical composition is formulated into sustained release form.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing lipid nanoparticles, and the matrices are in the form of molded articles, such as films or microcapsules.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-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPROM DEPOT™ (injectable microspheres made of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0256] In some embodiments, suitable surfactants include, but are not limited to, non-ionic agents such as polyoxyethylene sorbitans (e.g., TWEEN™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., SPAN™ 20, 30, 60, 80, or 85). In some embodiments, compositions that include a surfactant contain 0.05-5% of the surfactant. In some embodiments, compositions contain 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added if necessary.

[0257] In some embodiments, 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.

[0258] To prepare solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present disclosure, or a pharma-ceutically acceptable non-toxic salt thereof. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is evenly dispersed throughout the composition, thereby allowing the composition to be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above 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 offering the advantage of prolonged action. For example, the tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed released. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0259] Suitable emulsions can be prepared using commercially available fat emulsions, such as INTRALIPIDS™, LIPOSYN™, INFONUTROL™, LIPOFUNDIN™, and LIPIPHYSAN™. The active ingredient can be dissolved in a premixed emulsion composition or dissolved in oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) and water to form an emulsion. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to about 20%, e.g., about 5 to about 20%, of oil. The fat emulsion can contain fat droplets having a suitable size and can have a pH ranging from about 5.5 to about 8.0.

[0260] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0261] Compositions in pharma-ceutically acceptable solvents can be nebulized by using inert gas.Nebulized solutions can be inhaled directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions can be administered from a device that delivers the formulation in an appropriate manner.

[0262] VI. KITS FOR USE IN THERAPEUTICS The present disclosure also provides kits for use in immunotherapy for a disease or disorder, such as cancer (e.g., melanoma, lung cancer, colon cancer, or renal cell carcinoma), and / or treating or reducing the risk of a disease or disorder (e.g., cancer). In some embodiments, the kit comprises one or more containers comprising a composition described herein.

[0263] In some embodiments, the kit includes instructions for use according to any of the methods described herein.For example, the included instructions can include instructions for administering the pharmaceutical compositions described herein to treat, delay the onset, or alleviate the target disease.In some embodiments, the instructions include instructions for administering the compositions described herein to a subject at risk of the target disease / disorder (e.g., cancer).

[0264] In some embodiments, the instructions include dosage information, administration schedules, and routes of administration. In some embodiments, the containers are unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. In some embodiments, the instructions are written instructions on a label or package insert (e.g., a paper sheet included in the kit). In some embodiments, the instructions are machine-readable instructions (e.g., instructions carried on a magnetic or optical memory disk).

[0265] In some embodiments, the label or package insert indicates that the compositions disclosed herein are used for treating, delaying the onset, and / or ameliorating a cancer-related disease or disorder, such as those described herein. Instructions can be provided for practicing any of the methods described herein.

[0266] In some embodiments, the kit described herein is in suitable packaging. In some embodiments, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), or combinations thereof. In some embodiments, the packaging includes packaging for use in combination with a particular device, for example, an inhaler, a nasal administration device (e.g., an atomizer), or an injection device, such as a minipump. In some embodiments, the kit includes a sterile access port (e.g., the container can be an intravenous fluid bag or vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the container can also have a sterile access port (e.g., the container can be an intravenous fluid bag or vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, at least one active agent is a composition described herein.

[0267] In some embodiments, the kit further comprises additional components such as buffers and instructional information. In some embodiments, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described herein.

[0268] general technology 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. Molecular Cloning:A Laboratory Manual,second edition(Sambrook,et al.,1989)Cold Spring Harbor Press;Oligonucleotide Synthesis(MJGait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney,ed.1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGiffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons;Method of Enzymology(Academic Press,Inc.);Handbook of Experimental Immunology(DMWeir and CCBlackwell,eds.);Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds.,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,eds.,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,eds.,Harwood Academic Publishers,1995). Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. All publications cited herein (including those listed above and elsewhere in this disclosure) are incorporated by reference in their entirety. . EXAMPLES

[0269] Example 1: Construction of LNPs containing a VEE replicon The following materials and methods were used to construct the LNPs described herein.

[0270] Template preparation Template DNA was linearized using BspQI restriction enzyme. After linearization, the DNA was buffer exchanged through a filter at 6 diavolumes. This DNA was then subjected to IVT using T7 RNA polymerase and NTPs. After IVT, remaining DNA was digested using DNaseI. The mRNA was then buffer exchanged before liquid chromatography. After this, the mRNA was buffer exchanged again and then assayed for quality control.

[0271] Preparation of conventional BL-1 LNP formulations by T-junction The lipid materials were each weighed and dissolved in ethanol. The ethanol phase was prepared by mixing all the lipid materials according to the composition ratio of the following form: The aqueous phase was prepared by diluting the RNA molecule (e.g., mRNA containing VEE replicon or control mRNA without any VEE) with 20 mM citrate buffer (pH 4.0), 300 mM NaCl and water. The ethanol phase and aqueous phase of the LNP were mixed by T-shaped confluence mixing at a flow rate of 3:1 (aqueous phase:ethanol phase), followed immediately by in-line dilution with TBS buffer (pH 4.0) at a flow rate of 1:1 (LNP phase:PEG phase) by T-shaped confluence mixing to obtain the LNP. Table 2 below provides the relative amounts of each component included in the LNP composition. [Table 2]

[0272] Preparation of PEG-micelle-linked TT3 LNP formulations by T-junction The lipid materials were each weighed and dissolved in ethanol. The ethanol phase was prepared by mixing all lipid materials except DMG-PEG-2K according to the composition ratio of the following form: The aqueous phase was prepared by diluting the RNA molecule (e.g., mRNA containing VEE replicon or control mRNA without any VEE replicon) with 20 mM citrate buffer (pH 4.0), 300 mM NaCl, and water. The PEG micelle phase was prepared by adding the corresponding amount of DMG-PEG-2K to TBS buffer and mixing thoroughly by vortexing. Finally, the PEG micellarized TT3 LNP was obtained by first mixing the ethanol and aqueous phases of the LNPs at a flow rate of 3:1 (aqueous phase:ethanol phase) by T-mixing, followed immediately by in-line dilution with the PEG micelle phase at a flow rate of 1:1 (LNP phase:PEG phase) by T-mixing. Table 3 below provides the relative amounts (both exemplary amounts and weight ratios) of each component included in the LNP composition. [Table 3-1] [Table 3-2]

[0273] Buffer exchange and freeze / thaw of BL-1 / TT3 LNP The resulting BL-1 / TT3 LNPs were buffer exchanged and concentrated by tangential flow filtration. Then, a TBS stock solution containing 40% sucrose (W / V) was added to all prepared BL-1 / TT3 LNPs to bring the LNPs to a final solution of 10% sucrose. The final RNA concentration of the LNPs was measured by dissociating the LNPs with 2% TE+Triton and further detected by Quant-it RiboGreen assay. The LNPs were dispensed in aliquots of 50 μg / tube and frozen at -80°C. Before administration of the LNPs, the LNPs were thawed at room temperature.

[0274] Example 2: Analysis of liver expression To evaluate the ability of the LNPs constructed in Example 1 to target the liver, biodistribution was performed in mice. Briefly, animals were dosed with: (1) PBS (negative control), (2) LNPs encapsulating control mRNA without any VEE replicon (no RNA-VEE), or (3) LNPs encapsulating mRNA containing the VEE replicon (RNA-VEE).

[0275] As shown in Figure 1, in animals treated with No RNA-VEE, there was significant protein expression in the liver. In contrast, in animals treated with RNA-VEE, little detectable protein expression was observed in the liver. Protein expression in other tissues (e.g., spleen and lung) was comparable between animals in the No RNA-VEE and RNA-VEE groups, indicating the specificity of the effects observed in the liver.

[0276] The above results indicate that the LNPs described herein can selectively avoid and / or reduce expression in the liver, suggesting that the LNPs may allow for greater tolerability (e.g., reduced liver toxicity).

[0277] Example 3: Assessment of hepatotoxicity To further evaluate in vivo effects, the LNPs of the present disclosure are administered to animals (e.g., mice) as described in Example 2. Liver toxicity in animals is then evaluated in various ways. For example, in some embodiments, liver enzyme (ALT / AST) levels are measured, for example, in the blood of animals. In some embodiments, the general toxicity of animals is evaluated (e.g., activity, food intake, body score index, hydration status, or a combination thereof). In some embodiments, microscopic evaluation of liver is performed to detect any LNP-related effects on liver.

[0278] Example 4: Construction of additional lipid nanoparticles To further evaluate the VEE replicons described herein, additional LNPs containing different lipids (e.g., ionizable lipids, cationic lipids, or lipid-like substances, e.g., lipidoids) described herein were constructed as described in Example 1. In total, nine different LNPs were constructed (referred to herein as LNP1 through LNP9) and used to encapsulate mRNAs containing a VEE replicon (as described herein) or a control mRNA that does not contain any VEE replicon. The ability of these LNPs to induce payload expression was evaluated as described in the Examples below.

[0279] Example 5: Analysis of hepatic expression by lipid nanoparticles encapsulating RNA containing the VEE replicon 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) mice were administered a single intravenous dose of LNP1 or LNP2. Both LNP1 and LNP2 contained 20 μg of the VEE replicon containing a nucleic acid sequence encoding nuclear localization signal (NLS)-Cre. Control animals were administered phosphate-buffered saline (PBS). The Cre gene encodes the Cre protein, a site-specific DNA recombinase that can catalyze the recombination of DNA between specific sites in a DNA molecule. These sites contain specific binding sites for Cre, known as loxP sequences, surrounding a directional core sequence where recombination can occur. Cre induces tdTomato fluorescence, which indicates Cre-mediated recombination in liver tissue. Approximately 72 hours after LNP administration, tdTomato fluorescent expression was measured in the liver of mice using fluorescent IVIS imaging.

[0280] As shown in Figures 2A and 2B, the fluorescent signals observed in the livers of mice administered either LNP1 or LNP2 were not significantly different from those observed in control animals. Furthermore, there was no obvious difference in fluorescent expression in the liver between animals administered LNP1 and LNP2. These results strongly indicate the ability of the VEE replicon described herein to selectively avoid and / or reduce expression in the liver. The results further suggest that the liver avoidance property of the VEE replicon is independent of any difference between at least LNP1 and LNP2.

[0281] Example 6: Analysis of hepatic expression by lipid nanoparticles encapsulating VEE replicon-free RNA To confirm that the LNPs described herein can indeed target the liver, B6.Cg Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J(Ai reporter) mice were intravenously administered a single dose of LNP3, LNP4, LNP5, or LNP6. Each LNP was loaded with 20 μg of mRNA encoding nuclear localization signal (NLS)-Cre (no RNA-VEE) that does not contain any VEE replicon. Control animals were administered PBS. Approximately 72 hours after LNP administration, tdTomato fluorescent expression in the liver of mice was measured again using fluorescent IVIS imaging. As described in Example 4, td-Tomato fluorescent expression in the liver was expected to indicate Cre-mediated recombination in that tissue.

[0282] As shown in Figures 3A and 3B, in control animals, no detectable fluorescent signal was observed in the liver of the control mice. In contrast, a strong fluorescent signal was observed in the liver of the mice administered one of the tested LNPs (each encapsulating no RNA-VEE).

[0283] For further analysis, LNP7 (also encapsulating mRNA without any VEE replicon, the mRNA encoding NLS-Cre; RNA-VEE) was administered intravenously to tdTomato reporter mice. For comparison, some animals received LNP3 (i.e., encapsulating mRNA encoding NLS-Cre, without any VEE replicon) used above. Control animals received PBS only. Again, td-Tomato fluorescence was quantified using fluorescent IVIS imaging approximately 72 hours after LNP administration.

[0284] As shown in Figures 4A and 4B, significant fluorescent signals were observed in the livers of animals administered LNP7 compared to control animals, and the fluorescent signals were comparable to those observed in mice administered LNP3.

[0285] The results provided in this example relatively demonstrate that the LNPs described herein can indeed target the liver, and suggest that the liver avoidance and / or reduced liver expression observed in the previous examples is restricted to the VEE replicon itself.

[0286] Example 7: Analysis of firefly luciferase expression in the liver after administration of lipid nanoparticles encapsulating RNA containing the VEE replicon or RNA without any VEE replicon To confirm the above results in a different fluorescent model, C57BL / 6J mice were administered a single dose of one of the following LNPs: LNP8, LNP9, and LNP2. LNP9 and LNP2 were administered intravenously to the animals. Animals were administered LNP8 intravenously or intratumorally. Each of the LNPs (i.e., LNP8, LNP9, and LNP2) contained a VEE replicon and was encapsulated with 40 μg of mRNA encoding the firefly luciferase gene FLuc. The gene encodes an enzyme that catalyzes the oxygenation of d-luciferin to oxyluciferin, a reaction that produces visible light (530 nm to 640 nm). Control animals were administered PBS. Approximately 24 hours after administration, luciferase expression in the liver was measured using bioluminescence IVIS imaging.

[0287] As previously observed in td-Tomato reporter mice, luciferase expression in the liver of C57BL / 6J mice treated with LNP2 was not significantly different compared to control animals. Similar results were observed in C57BL / 6J mice treated with either LNP8 or LNP9. Furthermore, the route of administration (at least intravenous and intratumoral) did not appear to have any effect, as no significant differences in luciferase expression were observed in animals treated with LNP8 intravenously or intratumorally.

[0288] Again, to confirm that the lipid nanoparticles themselves could indeed target the liver, LNP2, LNP8, and LNP9 were loaded with 10 μg of firefly luciferase-encoding mRNA, this time without any VEE replicon. A single dose of LNP was then administered intravenously to C57BL / 6J mice, and luciferase expression in the liver was measured approximately 24 hours later using bioluminescence IVIS imaging.

[0289] As shown in Figures 6A and 6B, the bioluminescence signals in the liver of the LNP groups, including LNP8, LNP9, and LNP2, were significantly higher than that of the control, indicating the specific expression of FLuc-mod RNA in the liver of each LNP tested.

[0290] These results confirm that the LNP described herein can target the liver.These results also help to confirm the liver avoidance properties of the VEE replicon described herein.

[0291] Example 8: Comparison of liver-specific delivery of lipid nanoparticles encapsulating either RNA containing a VEE replicon or RNA without any VEE replicon To provide a direct comparison between mRNA without any VEE replicon (RNA-VEE-free) and mRNA with VEE replicon (RNA-VEE-free) in terms of liver specificity, C57BL / 6J mice were intravenously administered a single dose of LNP8 encapsulated with either (1) no RNA-VEE-encoding firefly luciferase or (2) RNA-VEE-encoding firefly luciferase at 2 mg / kg. Luciferase expression in the liver of the animals was then measured approximately 48 hours after administration using bioluminescence IVIS imaging.

[0292] As shown in Figures 7A and 7B, the bioluminescence signal in the liver of the group without RNA-VEE was significantly higher than that of the RNA-VEE replicon group. This result further supports that RNA-VEE prevents expression in the liver, whereas expression in the liver is possible without RNA-VEE. Furthermore, this result further indicates that the liver avoidance property of the VEE replicon described herein is independent of the lipid nanoparticles used.

[0293] Example 9: Lipid nanoparticles containing RNA containing a VEE replicon and ionized lipids To evaluate the liver evasion properties of the VEE replicon described herein, lipid nanoparticles containing ionizable lipids are constructed to produce lipid nanoparticles that are then used to encapsulate the VEE replicon and mRNA encoding a payload (RNA-VEE). The ionizable lipids are selected from the following: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5). , di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), Nl-[2(didodecylamino)ethyl]-N1,N4,N4-tridodecyl 1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25) ), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Di Oleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA),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)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), or any combination thereof.

[0294] The LNPs (i.e., comprising ionized lipids and RNA-VEE) are administered in vivo and then expression of the encoded payload in the liver is assessed (e.g., as described in Examples 4-7). In some embodiments, hepatotoxicity is also assessed in animals (e.g., as described in Example 3).

[0295] Example 10: Lipid nanoparticles containing RNA containing a VEE replicon and cationic lipids To evaluate the liver evasion properties of the VEE replicon described herein, lipid nanoparticles containing cationic lipids were constructed to produce lipid nanoparticles that were then used to encapsulate mRNA containing the VEE replicon and encoding a payload (RNA-VEE).The cationic lipid is selected from the following: l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), lipofectamine, N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolium chloride (DOTEVI), 2,3-dioleyloxy-N-[2(sperminecarboxamide )ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-(l,2-dioleoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), N,N- Dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-dilauroyl-sn-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), l,2-di-(9Z-tetradecenoyl)-sn-glycero-3-ethylphosphocholine (14:1 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), or any combination thereof.

[0296] The LNP (i.e., comprising a cationic lipid and an RNA-VEE) is administered in vivo and then expression of the encoded payload in the liver is assessed (e.g., as described in Examples 4-7). In some embodiments, hepatotoxicity is also assessed in animals (e.g., as described in Example 3).

[0297] Example 10: Lipid nanoparticles containing RNA and lipid-like materials containing a VEE replicon To evaluate the liver evasion properties of the VEE replicon described herein, lipid nanoparticles containing lipid mimetics are constructed to generate lipid-like nanoparticles (LLNs) that are then used to encapsulate the VEE replicon and mRNA encoding a payload (RNA-VEE). The lipid mimetics are 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-hydroxyoctadeca9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane 2 ,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(butane-4,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin), 12-dienoate) (OF-C4-Deg-Lin), N1,N3,N5-tris(3-(didodecylamino)propyl)benzene 1,3,5-tricarboxamide (TT3), hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-(((benzene-1,3,5-tricarbonyl)ris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate (FTT5), PL-1 [Nature Communications,12-7264(2021) and incorporated herein by reference], 98N12-5 [disclosed in Molecular Therapy vol.17 no.5 May 2009 and incorporated herein by reference], ethyl 5,5-di((Z)-heptadec-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.

[0298] The LLP (i.e., comprising a lipid mimetic and an RNA-VEE) is administered in vivo and then expression of the encoded payload in the liver is assessed (e.g., as described in Examples 4-7). In some embodiments, hepatotoxicity is also assessed in animals (e.g., as described in Example 3).

[0299] Example 12: Lipid nanoparticles containing RNA containing a VEE replicon and polymeric material To evaluate the liver evasion properties of the VEE replicon described herein, lipid nanoparticles containing polymeric materials are constructed to produce polymeric nanoparticles (PNPs), which are then used to encapsulate the VEE replicon and mRNA encoding a payload (RNA-VEE). The polymeric materials are 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).

[0300] The PNPs (i.e., comprising a polymeric material and an RNA VEE) are administered in vivo and then expression of the encoded payload in the liver is assessed (e.g., as described in Examples 4-7). In some embodiments, hepatotoxicity in animals is also assessed (e.g., as described in Example 3). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13]

[0301] It is understood that the Detailed Description section is intended to be used to interpret the claims, and not the Summary and Abstract sections. The Summary and Abstract sections may set forth one or more exemplary aspects of the disclosure contemplated by the inventor(s), but are not all inclusive and are in no way intended to limit the scope of the disclosure and the appended claims.

[0302] The present disclosure has been described above using functional building blocks illustrating the implementation of certain functions and their relationships. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of description. Alternative boundaries may be defined as long as the certain functions and their relationships are appropriately implemented.

[0303] The foregoing description of the specific embodiments fully discloses the general nature of the present disclosure, such that those skilled in the art can easily modify and / or adapt such specific embodiments to various applications without undue experimentation and without departing from the general outline of the present disclosure by applying the knowledge of those skilled in the art. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the embodiments of the present disclosure, based on the teaching and guidance provided herein. It is to be understood that the phraseology or terminology used herein is for the purpose of description, not of limitation, and that the terminology or terminology used herein will be interpreted by those skilled in the art in light of the teachings and guidance provided herein.

[0304] 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. (i) Reduce or avoid the expression of heterologous proteins in the target liver, and / or (ii) Selectively express a heterologous protein in the target non-liver tissue. A pharmaceutical composition for the purpose of A pharmaceutical composition comprising (i) one or more lipids, and (ii) lipid nanoparticles containing a replicon derived from the Venezuelan encephalitis (VEE) virus ("VEE replicon"), wherein the VEE replicon contains a nucleic acid sequence encoding the heterologous protein.

2. The pharmaceutical composition according to claim 1, wherein reducing the expression means, compared to a reference subject (for example, a subject administered the corresponding lipid nanoparticles but whose replicon is not a VEE replicon), (i) reducing the amount of the heterologous protein expressed in the liver, (ii) reducing the duration of expression of the heterologous protein in the liver, or (iii) both (i) and (ii).

3. The pharmaceutical composition according to claim 2, wherein, after administration of the pharmaceutical composition, the amount of the 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 that of the reference subject, and / or 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 that of the reference subject.

4. The pharmaceutical composition according to claim 3, wherein, after administration of the pharmaceutical composition, the liver does not express the heterologous protein.

5. The pharmaceutical composition according to claim 1, wherein, after administration of the pharmaceutical composition, the heterologous protein is expressed in the non-liver tissue of the target, and the non-liver tissue is selected from the spleen, lungs, tumor, or a combination thereof.

6. After administration of the aforementioned pharmaceutical composition, (a) 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 that observed in the reference non-liver tissue. (b) The duration of expression of the heterologous protein 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%, compared to that observed in the reference non-liver tissue. (c) The amount of the heterologous protein expressed in the non-liver 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 greater than the corresponding amount observed in the liver of the subject. (d) The duration of expression of the heterologous protein in the non-liver 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 longer than the corresponding duration observed in the liver of the subject, or (e) Any two or more combinations of (a) to (d), The pharmaceutical composition according to claim 5.

7. The pharmaceutical composition according to claim 1, wherein the VEE replicon has the nucleotide sequence described in Sequence ID No.

187.

8. The pharmaceutical composition according to claim 1, wherein the heterologous protein comprises a cytokine, an antibody or an antigen-binding fragment thereof, a chimeric antigen receptor, or a combination thereof.

9. The pharmaceutical composition according to claim 8, wherein the cytokine comprises the interleukin (IL)-12 protein.

10. The nucleic acid sequence of the VEE replicon is (i) Encoding the IL-12β subunit, with respect to the sequences described in SEQ ID NO: 65, 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: 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, at least about 75%, at least about 76%, at least about 77%, at least about 78 nucleotide sequences having sequence identity of %, 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%, (ii) IL-12α The subunit is coded, and for the sequences described in SEQ ID NO: 115, 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: 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, at least approximately 77%, at least approximately 78%, nucleotide sequences that are identical by 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 at least 100%. A pharmaceutical composition according to claim 9, comprising:

11. The pharmaceutical composition according to claim 1, wherein the one or more lipids include ionized lipids, cationic lipids, lipidoids, phospholipids, sterols, or a combination thereof.

12. The pharmaceutical composition according to claim 11, wherein the one or more lipids include N1,N3,N5-tris(3-(didodecylamino)propyl)benzene 1,3,5-tricarboxamide (TT3), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (C14-PEG2000)], or a combination thereof.

13. The pharmaceutical composition according to claim 12, wherein the C14-PEG2000 is embedded in lipid nanoparticles and / or the C14-PEG2000 is not embedded in lipid nanoparticles.

14. The pharmaceutical composition according to claim 1, wherein the lipid nanoparticles have a diameter of about 30 to 500 nm.

15. The pharmaceutical composition according to claim 1, wherein the one or more lipids and the VEE replicon have a mass ratio of about 1:2 to about 15:

1.

16. The pharmaceutical composition according to claim 1, wherein the VEE replicon further comprises a regulatory element, optionally comprising at least one translation enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or its seed, a 3' tailing region of a linked nucleoside, an AU-rich element (ARE), a post-transcriptional regulatory modulator, a 5' UTR, a 3' UTR, or a combination thereof.

17. The VEE replicon comprises at least one modified nucleoside, and optionally the at least one modified nucleoside is 6-azacytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iod-uridine, N1-methyl-pseudridine, 5,6-dihydrouridine, α-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, pseudouridine, inosine, α-thio- The pharmaceutical composition according to claim 1, comprising anosine, 8-oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolocytidine, 5-methylcytidine, N4-acetylcytidine, 5-methyluridine, 5-iodocytidine, or a combination thereof.

18. The pharmaceutical composition according to claim 1, wherein the lipid nanoparticles are administered to the subject via intratumor, intrathecal cavity, intramuscular, intravenous, subcutaneous, inhalation, intradermal, lymphatic vessel, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration.

19. The pharmaceutical composition according to claim 1, which is used in combination with at least one additional therapeutic agent, wherein the at least one additional therapeutic agent optionally comprises a chemotherapeutic agent, targeted anticancer therapy, oncolytic agent, cytotoxic agent, immunotherapy, cytokine, surgical procedure, radiotherapy, activator of a costimulatory molecule, immune checkpoint inhibitor, vaccine, cellular immunotherapy, or any combination thereof.

20. The pharmaceutical composition according to claim 19, 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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