Lipid nanoparticles and methods for sensitizing response to an immunotherapy

Abstract

Provided herein are versikine nucleic acid molecules comprising at least 90% sequence identity to SEQ ID NO: 1. Also provided herein are pharmaceutical compositions comprising any one of the versikine nucleic acid molecules described herein, and a lipid nanoparticle, wherein the versikine nucleic acid molecule is encapsulated in the lipid nanoparticle. Any of the versikine compositions described herein can be used for the treatment of cancer.

Description

[0001] LIPID NANOPARTICLES AND METHODS FOR SENSITIZING RESPONSE TO AN IMMUNOTHERAPY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 519,677, filed on August 15, 2023. The entire contents of the foregoing are incorporated herein byreference in their entireties.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grant No. CA252937 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] The large aggregating extracellular matrix proteoglycan versican (VC AN) is a central component of the embryonic provisional matrix, playing key non-redundant roles in development of the cardiovascular system and limbs. For example. Vcan-null mice die in utero by embryonic day 10.5 because of defects along the anterior-posterior cardiac axis. VCAN proteolysis by AD AMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteases at the Glu441-Ala442 bond (VI isoform enumeration) is an essential requirement that acts in part through the specific neoactivities of the released bioactive N- terminal fragment (matrikine) versikine, wherein disruption of the Glu441-Ala442 proteolytic site that generates versikine leads to developmental abnormalities. Furthermore, it has been shown that VCAN proteolysis at the versikine C-terminal Glu441-Ala442 bond correlates with CD8+ T cell infiltration in solid and hematopoietic human tumors.

[0008] SUMMARY

[0009] Provided herein are versikine nucleic acid molecules comprising at least 90% sequence identity to SEQ ID NO: 1. Also provided herein are versikine nucleic acid molecules comprising SEQ ID NO: 1. In some embodiments, the one or more nucleotides of the veriskine nucleic acid molecule comprises one or more chemically modified nucleotides. In some embodiments, the one or more chemically modified nucleotides comprises Nl- methyladenosine (ml A), N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil, Ml -pseudouracil, 5 -methoxy uridine (5moU), or N4- acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphorami dates, 2’-0-Me nucleoside analog, 2’F-RNA modification, 2’-deoxy-2'-fluoro-P-D-arabino nucleic acid (2’F- ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2’-O-(2-methoxyethyl)) modification. In some embodiments, the versikine nucleic acid molecule comprises 5-methoxyuridine (5moU) or pseudouridine.

[0010] Also provided herein are pharmaceutical compositions comprising: any one of the versikine nucleic acid molecules described herein, and a lipid nanoparticle. In some embodiments, the versikine nucleic acid molecule is encapsulated in the lipid nanoparticle. In some embodiments, one or more nucleotides of the versikine nucleic acid molecule comprises one or more chemically modified nucleotides. In some embodiments, the one or more chemically -modified nucleotides comprises N1 -methyladenosine (mlA), N6- methyladenosine (m6A). or adenosine to inosine (A-to-I) modification, pseudouracil, Ml- pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphoramidates, 2’-0-Me nucleoside analog, 2’F-RNA modification, 2’- deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F-ANA) modification. Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2’-O-(2-methoxyethyl)) modification. In some embodiments, the lipid nanoparticle comprises ALC-0315, 1,2 DSPC. cholesterol, ALC-0159, SM-102, DMG-PEG(2000), D-Lin-MC3-DMA, or any combinations thereof. In some embodiments, the lipid nanoparticle comprises SM-102, 1,2 DSPC, cholesterol, and DMG-PEG(2000) at a molar ratio of 50: 10:38.5: 1.5, respectively.

[0011] Also provided herein are isolated cells comprising any one of the versikine nucleic acid molecules described herein.

[0012] Also provided herein are methods for encapsulating any one of the versikine nucleic acid molecules described herein in a lipid nanoparticle, the method comprising: (a) generating the nucleic acid molecule by using a transcription vector, (b) mixing the nucleic acid molecule with a plurality of lipid nanoparticles, thereby forming lipid nanoparticles encapsulating the nucleic acid molecule. In some embodiments, the generating step (a) comprises using a transcription buffer comprising ATP, CTP, GTP, UTP, or a chemically modified nucleotide. In some embodiments, the chemically modified nucleotide comprises N1 -methyladenosine (ml A). N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil, Ml -pseudouracil, 5 -methoxy uridine (5moU), or N4- acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphorami dates, 2’-0-Me nucleoside analog. 2 F-RNA modification, 2’-deoxy-2’-fluoro-[3-D-arabino nucleic acid (2’F- ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2’-O-(2-methoxyethyl)) modification.

[0013] In some embodiments, a lipid nanoparticle of the plurality of lipid nanoparticles comprises ALC-0315, 1.2 DSPC, cholesterol, ALC-0159. SM-102. DMG-PEG(2000). D- Lin-MC3-DMA, or any combinations thereof. In some embodiments, the lipid nanoparticle comprises SM-102, 1,2 DSPC, cholesterol, and DMG-PEG(2000) at a molar ratio of 50:10:38.5:1.5, respectively. In some embodiments, a lipid nanoparticle of the plurality of lipid nanoparticles comprises phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), dimyristoyl phosphatidylglyerol (DMPG) and sphingomyelin (SM), l,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), l,2-dioleoyloxy-3- dimethylammoniumpropane (DODAP) and analogues, l,2-di-(9Z-octadecenoyl)-sn-glycero- 3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylcholine (PC) and dimyristoyl phosphatidylcholine (DMPC), 1 ,2-di-O- octadecenyl-3-trimethylammoniumpropane (DOTMA), 1 ,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), dioctadecyldi-methylammonium (DODA(Br)ZDDAB), dioctadecyldimethylammoniumchloride (DODAC). 1 ,2-dimyristoyloxypropyl-1.3- dimethylhydroxyethylammonium (DMRIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA) analogues, cholesterol, sphingomyelin (SM), or any combinations thereof.

[0014] Also provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof, the method comprising: administering any one of the versikine nucleic acid molecules, or any one of the pharmaceutical compositions described herein and an immunotherapeutic agent. In some embodiments, the immunotherapy comprises an anti- IL4 immunotherapy.

[0015] In some embodiments, the administering comprises delivery’ of the versikine nucleic acid molecule or pharmaceutical composition into a cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is an antigen-presenting cell. In some embodiments, the delivery of the nucleic acid molecule comprises a virus-based delivery. In some embodiments, the virus-based delivery is a lentivirus or an adenovirus.

[0016] In some embodiments, the subject has a tumor. In some embodiments, the tumor is a refractory tumor.

[0017] Also provided herein are methods for treating cancer in a subject in need thereof, the method comprising: administenng any one of the versikine nucleic acid molecules or any one of the pharmaceutical compositions described herein, thereby treating the cancer. In some embodiments, the subject is co-administered one or more agents that target the IL-4 pathway. In some embodiments, the one or more agents that target the IL-4 pathway comprise dupilumab or pasolizumab. In some embodiments, the one or more agents that target the IL-4 pathway is pitrakinra.

[0018] In some embodiments, the co-administration comprises administering the one or more agents that target the IL-4 pathway prior to administering the versikine nucleic acid molecule or the pharmaceutical composition. In some embodiments, the co-administration comprises administering the one or more agents that target the IL-4 pathway after administering the versikine nucleic acid molecule or the pharmaceutical composition. In some embodiments, the co-administration comprises administering the one or more agents that target the IL-4 pathway and the versikine nucleic acid molecule or the pharmaceutical composition at the same time.

[0019] In some embodiments, the subject is co-administered one or more agents that inhibit PD-1 and / or PD-L1. In some embodiments, one or more agents that inhibit PD-1 and / or PD- L1 comprise an antibody inhibitor. In some embodiments, the antibody inhibitor comprise cemiplimab, nivolumab, pembrolizumab, atezolizumab. avelumab, durvalumab, or any combination thereof. In some embodiments, the co-administration comprises administering the one or more agents that inhibit PD-1 and / or PD-L1 and the versikine nucleic acid molecule or the pharmaceutical composition prior to, after, and / or while simultaneously administering the versikine nucleic acid molecule or the pharmaceutical composition.

[0020] In some embodiments, the cancer is a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, lary ngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer. In some embodiments, the cancer is solid tumor. In some embodiments, the tumor is a refractory tumor.

[0021] In some embodiments, the administering of the versikine nucleic acid molecule or the pharmaceutical composition comprises, buccal, dermal, enteral, intra-arterial, intradermal, intragastric, intramedullar}', intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-tumoral, mucosal, nasal, oral, subcutaneous, or sublingual administration. In some embodiments, the administering comprises intra-tumoral or intravenous administration.

[0022] 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 invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0023] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 shows efficient translation of synthetic versikine mRNA from an in vitro transcription (IVT) vector. An in vitro transcription (IVT) vector was generated incorporating T7 start, 5’ and 3’ UTRs and a 1 10-nt poly A tail encoding HA-tagged versikine ORF. mRNA was synthesized incorporating 5-methoxyuridine and cap reagent. Western blotting with anti- HA tag antibody confirmed efficient translation of versikine migrating at the expected MW size (app. 75kDa). Lane 1, 293 cells transfected with luciferase synthetic mRNA. Lane 2, 293 cells transfected with versikine synthetic mRNA. Non-specific bands migrating slower than versikine indicate comparable protein loading in each lane.

[0026] FIG. 2 shows a gel electrophoresis image of synthetic human versikine and control Thymus Cell Antigen 1.1 (Thy LI) mRNA.

[0027] FIG. 3 shows quantification and size analysis of control Thymus Cell Antigen 1.1 (Thy 1. 1) mRNA using Agilent® TapeStation® system. FIG. 4 shows quantification and size analysis of synthetic human versikine mRNA using Agilent® TapeStation® system.

[0028] FIG. 5 shows Western blot analysis of versikine expression in mammalian cells. Human Embryonic Kidney (HEK) 293T cells and murine colorectal carcinoma CT26 cells were transfected at approximately 40% confluency with 1 g / ml mRNA using Mirus TransYT® mRNA Transfection kit. Cell lysates were then harvested after 22 hours and prepared for Western blot analysis probing with antibodies (anti-hemagglutinin (HA) tag and secondary antibody conjugated to horseradish peroxidase (HRP)), which collectively recognize specifically the HA tag added to versikine.

[0029] FIGs. 6A-6F show the successful lipid nanoparticle (LNP)-mediated delivery' and translation of mRNA delivered through LNPs into mammalian cells. FIGs. 6A-6C show flow cytometry analysis of 293T human embryonic kidney (HEK) cells transfected at approximately 40% confluency with: no LNPs (control), 1 pg / ml of un-dialyzed LNPs encapsulating Thy 1.1 mRNA, and 1 pg / ml of dialyzed LNPs encapsulating Thy L I mRNA, respectively. FIGs. 6D-6F show flow cytometry analysis of CT26 murine colorectal carcinoma cells transfected at approximately 40% confluency with: no LNPs (control). 1 pg / ml of un-dialyzed LNPs encapsulating Thy 1.1 mRNA, and 1 pg / ml of dialyzed LNPs encapsulating Thy 1.1 mRNA, respectively. All cells were harvested after 23 hours and prepared for flow cytometry analysis.

[0030] DETAILED DESCRIPTION

[0031] Versican is an extracellular matrix proteoglycan with key roles in multiple facets of cancer development, ranging from proliferative signaling, evasion of growth-suppressor pathways, regulation of cell death, promotion of neoangiogenesis, and tissue invasion and metastasis. Multiple studies implicate versican and its bioactive proteolytic fragments (matrikines, e.g.. versikines) in the regulation of cancer inflammation and antitumor immune responses. The understanding of the dynamics of versican deposition and its proteolytic turnover holds potential for the development of novel immune biomarkers as w ell as approaches to promote efficacy of modem immunotherapies.

[0032] Lipid nanoparticles (LNPs) have emerged as promising vehicles to deliver a variety of therapeutic agents, wherein LNPs play a key role in effectively protecting and transporting mRNA to cells. Furthermore, with their ability to control the location and timing of drug delivery in the body, LNPs can be used to deliver treatments for a variety of diseases. The present disclosure describes versikine nucleic acid molecules comprising at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the versikine nucleic acid molecule comprises SEQ ID NO: 1. Also provided herein are pharmaceutical compositions that include any one of the versikine nucleic acid molecules described herein, and a lipid nanoparticle. Also provided herein are methods for encapsulating any one of the versikine nucleic acid molecules described herein in a lipid nanoparticle, the method including (a) generating the versikine nucleic acid molecule by using a transcription vector, (b) mixing the versikine nucleic acid molecule with a plurality of lipid nanoparticles, thereby forming lipid nanoparticles encapsulating the versikine nucleic acid molecule.

[0033] Also provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof that includes administering any one of the versikine nucleic acid molecules, or the pharmaceutical compositions described herein.

[0034] Particular implementations of the subject matter described in this specification can be implemented so as to realize one or more of the following advantages.

[0035] For example, the compositions and methods of the disclosure provide non-naturally occurring versikine nucleic acid molecules that work in synergy with the anti-IL4 pathway and can be used as an immunotherapy and / or a cancer therapy in patients in need thereof. In some embodiments, the compositions disclosed herein also provide versikine nucleic acid molecules encapsulated in lipid nanoparticles for efficient in vitro and in vivo delivery. In some embodiments, the versikine nucleic acid molecules and compositions of the disclosure can provide a signal to immune cells (e g., T cells) to activate in a tumor microenvironment, which may provide an anti-tumor immune response, may result in T cells directly attacking tumor cells and inducing their apoptosis, may result in T cells infiltrating a tumor mass and restricting tumor growth and metastasis, and may result in T cells counteracting a suppression of an immune response caused by the tumor microenvironment.

[0036] Various non-limiting aspects of these methods are described herein and can be used in any combination without limitation. Additional aspects of various components of the methods described herein are known in the art.

[0037] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about"’ in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0039] As used herein, “biological sample” can refer to a sample generally including cells and / or other biological material. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). Biological samples can be derived from a homogeneous culture or population of organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0040] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be a nucleic acid sample and / or protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.

[0041] As used herein, a “cell"’ can refer to either a prokaryotic or eukary otic cell, optionally- obtained from a subject or a commercially available source.

[0042] As used herein, “delivering,” “gene delivery,” “gene transfer,” “transducing” can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection / transfection. or various other proteinbased or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0043] In some embodiments, a polynucleotide can be inserted into a host cell by a gene delivery molecule. Examples of gene delivery molecules can include, but are not limited to, liposomes, micelle biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokary otic hosts, and may be used for gene therapy as well as for simple protein expression.

[0044] As used herein, the term "expression ' refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. In some embodiments, if the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular biological sample.

[0045] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules. mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5 -methylcytidine. C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C 5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8 -oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2 -thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0046] As used herein, “nucleic acid” is used to include any compound and / or substance that comprise a polymer of nucleotides. In some embodiments, a polymer of nucleotides is referred to as polynucleotides. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 ’-amino functionalization, and 2’-amino-a-LNA having a 2’-amino functionalization) or hybrids thereof. Naturally- occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

[0047] A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).

[0048] In some embodiments, the term "‘nucleic acid” refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA.

[0049] In some embodiments, a nucleic acid is a compound and / or substance that is, or can be incorporated into, a polynucleotide chain with a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid residue analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that one or more residues, and in some embodiments, are linked together other than by a phosphodiester. For example, in some embodiments, a nucleic acid includes one or more phosphorothioate and / or phosphoroamidite (e.g., 5'-N-phosphoramidite) linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5 -bromo uridine, C5- fluorouridine, C5 -iodouridine. C5-propynyl-uridine, C5 -propynyl-cytidine, C5- methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro). enzymatic synthesis in the absence of a complementary template, reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70. 75, 80, 85, 90, 95, 100, 1 10, 120. 130, 140, 150, 160, 170. 180, 190. 20. 225, 250, 275, 300, 325. 350, 375. 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0050] Modifications can be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)- mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Such families include amino acids with basic side chains (e.g., arginine, lysine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, and tryptophan), nonpolar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, and valine). beta-branched side chains (e.g., isoleucine, threonine, and valine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine).

[0051] As used herein, the term “nucleotides” and “nt” are used interchangeably herein to generally refer to biological molecules that comprise nucleic acids. Nucleotides can have moieties that contain the known purine and pyrimidine bases. Nucleotides may have other heterocyclic bases that have been modified. Such modifications include, e.g.. methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses, or other heterocycles. In some embodiments, nucleic acid modifications can also include a blocking modification comprising a 3’ end modification (e.g., a 3’ dideoxy C (3'ddC), 3’ddG, 3’ddA, 3?ddT, 3’ inverted dT, 3’ C3 spacer. 3’ amino, 3’ biotinylation, or 3’ phosphorylation). The terms “polynucleotides,” “nucleic acid,” and “oligonucleotides” can be used interchangeably, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozy mes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise non-naturally occurring sequences. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0052] As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility' to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0053] As used herein, the term '‘treatment” (also '‘treat” or “treating”) refers to administration of a therapy or composition that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0054] Versikine nucleic acid molecules

[0055] Provided herein are versikine nucleic acid molecules comprising at least 90% sequence identity to SEQ ID NO: 1 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity). In some embodiments, the versikine nucleic acid molecule comprises SEQ ID NO: 1.

[0056] SEQ ID NO: 1 - Versikine mRNA

[0057] AUGUUCAUAAAUAUAAAGAGCAUCUUAUGGAUGUGUUCAACCUUAAUAGUAA CCCAUGCGCUACAUAAAGUCAAAGUGGGAAAAAGCCCACCGGUGAGGGGCUCC CUCUCUGGAAAAGUCAGCCUACCUUGUCAUUUUUCAACGAUGCCUACUUUGCC ACCCAGUUACAACACCAGUGAAUUUCUCCGCAUCAAAUGGUCUAAGAUUGAAG UGGACAAAAAUGGAAAAGAUUUGAAAGAGACUACUGUCCUUGUGGCCCAAAA UGGAAAUAUCAAGAUUGGUCAGGACUACAAAGGGAGAGUGUCUGUGCCCACA CAUCCCGAGGCUGUGGGCGAUGCCUCCCUCACUGUGGUCAAGCUGCUGGCAAG UGAUGCGGGUCUUUACCGCUGUGACGUCAUGUACGGGAUUGAAGACACACAA GACACGGUGUCACUGACUGUGGAUGGGGUUGUGUUUCACUACAGGGCGGCAA CCAGCAGGUACACACUGAAUUUUGAGGCUGCUCAGAAGGCUUGUUUGGACGU UGGGGCAGUCAUAGCAACUCCAGAGCAGCUCUUUGCUGCCUAUGAAGAUGGA UUUGAGCAGUGUGACGCAGGCUGGCUGGCUGAUCAGACUGUCAGAUAUCCCA UCCGGGCUCCCAGAGUAGGCUGUUAUGGAGAUAAGAUGGGAAAGGCAGGAGU CAGGACUUAUGGAUUCCGUUCUCCCCAGGAAACUUACGAUGUGUAUUGUUAU GUGGAUCAUCUGGAUGGUGAUGUGUUCCACCUCACUGUCCCCAGUAAAUUCAC CUUCGAGGAGGCUGCAAAAGAGUGUGAAAACCAGGAUGCCAGGCUGGCAACA GUGGGGGAACUCCAGGCGGCAUGGAGGAACGGCUUUGACCAGUGCGAUUACG GGUGGCUGUCGGAUGCCAGCGUGCGCCACCCUGUGACUGUGGCCAGGGCCCAG UGUGGAGGUGGUCUACUUGGGGUGAGAACCCUGUAUCGUUUUGAGAACCAGA CAGGCUUCCCUCCCCCUGAUAGCAGAUUUGAUGCCUACUGCUUUAAACGUCGA AUGAGUGAUUUGAGUGUAAUUGGUCAUCCAAUAGAUUCAGAAUCUAAAGAAG AUGAACCUUGUAGUGAAGAAACAGAUCCAGUGCAUGAUCUAAUGGCUGAAAU UUUACCUGAAUUCCCUGACAUAAUUGAAAUAGACCUAUACCACAGUGAAGAA AAUGAAGAAGAAGAAGAAGAGUGUGCAAAUGCUACUGAUGUGACAACCACCC CAUCUGUGCAGUACAUAAAUGGGAAGCAUCUCGUUACCACUGUGCCCAAGGAC CCAGAAGCUGCAGAA

[0058] In some embodiments, one or more nucleotides of the versikine nucleic acid molecule comprises one or more chemically modified nucleotides. As used herein, “chemically modified nucleotides” refer to nucleotides that have chemical modifications to the phosphate backbone, sugar, and bases of the nucleotide (e.g., DNA or RNA). In some embodiments, a chemical modification can include phophorothioate linkages, Locked Nucleic Acids (LNAs), or 2’-O-methyl modifications. In some embodiments, a nucleotide chemical modification is a N1 -methyladenosine (ml A), N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification. In some embodiments, a nucleotide chemical modification is a pseudouracil, Ml -pseudouracil, 5 -methoxy uridine (5moU), or N4-acetylcytidine. In some embodiments, a nucleotide chemical modification is a I,2'-O-dimethyladenosine (mlAm), l,2'-O- dimethylguanosine (mlGm). l,2’-O-dimethylinosine (mllm), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (mlacp3Y), 1 -methyladenosine (mlA), 1 -methylguanosine (mlG), 1-methylinosine (mil), 1 -methylpseudouridine (mlY), 2,8-dimethyladenosine (m2, 8 A), 2-geranylthiouridine (ges2U), 2-lysidine (k2C), 2-methyladenosine (m2 A). 2- methylthiomethylenethio-N6-isopentenyl-adenosine (msms2i6A), 2-methylthio-cyclic-N6- threonylcarbamoyladenosine (ms2ct6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)- adenosine (ms2io6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine (ms2hn6A), 2- methylthio-N6-isopentenyladenosine (ms2i6A). 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A). 2-selenouridine (se2U), 2-thio-2’-O-methyluridine (s2Um), 2-thiocytidine (s2C), 2-thiouridine (s2U), 2’-O- methyladenosine (Am), 2’-O-methylcytidine (Cm), 2’-O-methylguanosine (Gm), 2’-0- methylinosine (Im), 2'-O-methylpseudouridine (Ym). 2’-O-methyluridine (Um), 2’-0- methyluridine 5-oxyacetic acid methyl ester (mcmo5Um), 2’-0-ribosyladenosine (phosphate) (Ar(p)), 2’-O-ribosylguanosine (phosphate) (Gr(p)), 3,2’-O-dimethyluridine (m3Um), 3-(3- amino-3-carboxypropyl)-5,6-dihydrouridine (acp3D), 3-(3-amino-3- carboxypropyl)pseudouridine (acp3Y), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 3- methylcytidine (m3C). 3-methylpseudouridine (m3Y), 3-methyluridine (m3U). 4- demethylwyosine (imG-14), 4-thiouridine (s4U), 5,2’-O-dimethylcytidine (m5Cm), 5,2’-O- dimethyluridine (m5Um), 5-(carboxyhydroxymethyl)-2’-O-methyluridine methyl ester (mchm5Um), 5-(carboxyhydroxymethyl)uridine methyl ester (mchm5U), 5- (isopentenylaminomethyl)-2-thiouridine (inm5s2U), 5-(isopentenylaminomethyl)-2'-O- methyluridine (inm5Um), 5-(isopentenylaminomethyl)uridine (inm5U), 5-aminomethyl-2- geranylthiouridine (nm5ges2U), 5-aminomethyl-2-selenouridine (nm5se2U), 5-aminomethyl- 2-thiouridine (nm5s2U), 5 -aminomethyluridine (nm5U), 5-carbamoylhydroxymethyluridine (nchm5U), 5-carbamoylmethyl-2-thiouridine (ncm5s2U), 5-carbamoylmethyl-2'-O- methyluridine (ncm5Um), 5 -carbamoylmethyluridine (ncm5U), 5- carboxyhydroxymethyluridine (chm5U), 5-carboxymethyl-2-thiouridine (cm5s2U), 5- carboxymethylaminomethyl-2-geranylthiouridine (cmnm5ges2U), 5- carboxymethy laminomethyl-2-sel enouridine (cmnm5 se2U), 5 -carboxy methylaminomethy 1-2- thiouridine (cmnm5s2U), 5-carboxymethylaminomethyl-2’-O-methyluridine (cmnm5Um), 5- carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethyluridine (cm5U), 5- cyanomethyluridine (cnm5U), 5-formyl-2’-O-methylcytidine (f5Cm), 5-formylcytidine (f5C), 5 -hydroxy cytidine (ho5C), 5-hydroxymethylcytidine (hm5C), 5 -hydroxy uridine (ho5U), 5- methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyl-2’-O- methyluridine (mcm5Um), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxyuridine (mo5U), 5-methyl-2-thiouridine (m5s2U), 5-methylaminomethyl-2-geranylthiouridine (mnm5ges2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-methylaminomethyl- 2-thiouridine (mnm5s2U), 5 -methylaminomethyluridine (mnm5U), 5-methylcytidine (m5C), 5-methyldihydrouridine (m5D), 5 -methyluridine (m5U), 5 -taurinomethy 1-2 -thiouridine (tm5s2U), 5-taurinomethyluridine (tm5U), 7-aminocarboxypropyl-demethylwyosine (yW- 86), 7-aminocarboxypropylwyosine (yW-72), 7-aminocarboxypropylwyosine methyl ester (yW-58). 7-aminomethyl-7-deazaguanosine (preQltRNA), 7-cyano-7-deazaguanosine (preQOtRNA), 7-methylguanosine (m7G), 8-methyladenosine (m8A), N2,2’-O- dimethylguanosine (m2Gm), N2,7,2’-O-trimethylguanosine (m2,7Gm), N2,7- dimethylguanosine (m2,7G), N2,N2,2’-O-trimethylguanosine (m2,2Gm), N2,N2,7- trimethylguanosine (m2,2,7G), N2,N2-dimethylguanosine (m2.2G), N2-methylguanosine (m2G). N4,2?-O-dimethylcytidine (m4Cm). N4.N4.2’-O-trimethylcytidine (m4,4Cm), N4,N4-dimethylcytidine (m4,4C), N4-acety 1-2 ’-O-methylcyti dine (ac4Cm), N4- acetylcytidine (ac4C), N4-methylcytidine (m4C), N6,2’-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m6,6Am), N6.N6-dimethyladenosine (m6,6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), N6-acetyladenosine (ac6A). N6-formyladenosine (f6A), N6-glycinylcarbamoyladenosine (g6A), N6-hydroxymethyladenosine (hm6A), N6- hydroxynorvalylcarbamoyladenosine (hn6A), N6-isopentenyladenosine (i6A), N6-methyl- N6-threonylcarbamoyladenosine (m6t6A), N6-methyladenosine (m6A), N6- threonylcarbamoyladenosine (t6A), Agmatidine (C+). Archaeosine (G+). cyclic N6- threonylcarbamoyladenosine (ct6A). dihydrouridine (D), epoxy queuosine (oQtRNA), galactosyl-queuosine (galQtRNA), glutamyl -queuosine (gluQtRNA), hydroxy-N6- threonylcarbamoyladenosine (ht6A), hydrox wybutosine (OHyW), inosine (I), isowyosinc (imG2), mannosyl-queuosine (manQtRNA). methylated undermodified hydroxy wybutosine (OHy Wy), methylwyosine (mimG), peroxywybutosine (o2yW), pseudouridine (Y), queuosine (QtRNA), undermodified hydroxywybutosine (OHyWx), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), wybutosine (y W), or wyosine (imG) modification. In some embodiments, the one or more chemically modified nucleotides comprises N1 -methyladenosine (mlA), N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil. Ml -pseudouracil, 5-methoxyuridine (5moU), or N4- acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphorami dates, 2’-0-Me nucleoside analog. 2 F-RNA modification, 2’-deoxy-2’-fluoro-[3-D-arabino nucleic acid (2’F- ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2’-O-(2-methoxyethyl)) modification.

[0059] In some embodiments, the versikine nucleic acid molecule is combined, fused to, conjugated, or otherwise linked to other nucleic acid molecules. In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes an immunomodulator protein. In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes XCL1, CCL5. GM-CSF, or FLT3LG. In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that comprises at least 90% sequence identity to SEQ ID NO: 2 (e.g., having at least 91% sequence identity7, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), SEQ ID NO: 3 (e.g., having at least 91% sequence identity, at least 92% sequence identity7, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), SEQ ID NO: 4 (e.g., having at least 91% sequence identity7, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity7, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), or SEQ ID NO: 5 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity7, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity7, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity ).

[0060] In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the immunomodulator protein XCL1 having an amino acid sequence that comprises at least 90% sequence identity7of SEQ ID NO: 12 (e.g., having at least 91% sequence identity, at least 92% sequence identity7, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity ). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the immunomodulator protein CCL5 having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 13 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity7, at least 99% sequence identity ). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the immunomodulator protein GM-CSF having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 14 (e.g., having at least 91% sequence identity, at least 92% sequence identity7, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity ). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the immunomodulator protein FLT3LG having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 15 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity', at least 99% sequence identity).

[0061] In some embodiments, a versikine nucleic acid molecule is otherwise linked to a secretory signal. In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes a secretory peptide. In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes human OSM, human immunoglobulin, human chymotry psinogen, human try psinogen 2, human IL-2, or human insulin. In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that comprises at least 90% sequence identity to SEQ ID NO: 6 (e g., haying at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity', at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), SEQ ID NO: 7 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity', at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity', at least 98% sequence identity, at least 99% sequence identity), SEQ ID NO: 8 (e g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity ), SEQ ID NO: 9 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), SEQ ID NO: 10 (e g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity', at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity), or SEQ ID NO: 11 (e.g., having at least 91% sequence identity, at least 92% sequence identity’, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity).

[0062] In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the human OSM having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 16 (e.g., having at least 91% sequence identity’, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity', at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity', at least 98% sequence identity, at least 99% sequence identity). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the human immunoglobulin having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 17 (e.g., having at least 91% sequence identity', at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the human chymotrypsinogen having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 18 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity’, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity', at least 98% sequence identity, at least 99% sequence identity). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the human try psinogen 2 having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 19 (e.g., having at least 91% sequence identity', at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity', at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the human IL-2 having an ammo acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 20 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity', at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity). In some embodiments, the versikine nucleic acid molecule is otherwise linked to a nucleic acid molecule that encodes the human insulin having an amino acid sequence that comprises at least 90% sequence identity of SEQ ID NO: 21 (e.g., having at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity', at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity).

[0063] SEQ ID NO: 2 - Human XCL1 nucleic acid sequence

[0064] GCGGGACCTC AGCCATGAGA CTTCTCATCC TGGCCCTCCT TGGCATCTGC TCTCTCACTG CATACATTGT GGAAGGTGTA GGGAGTGAAG TCTCAGATAA GAGGACCTGT GTGAGCCTCA CTACCCAGCG ACTGCCGGTT AGCAGAATCA AGACCTACAC CATCACGGAA GGCTCCTTGA GAGCAGTAAT TTTTATTACC AAACGTGGCC TAAAAGTCTG TGCTGATCCA CAAGCCACGT GGGTGAGAGA CGTGGTCAGG AGCATGGACA GGAAATCCAA CACCAGAAAT AACATGATCC AGACCAAGCC AACAGGAACC CAGCAATCGA CCAATACAGC TGTGACCCTG ACTGGCTAGT AGTCTCTGGC ACCCTGTCCG TCTCCAGCCA GCCAGCTCAT TTCACTTTAC ACCCTCATGG ACTGAGTTTA TACTCACCTT TTATGAAAGC ACTGCATGAA TAAAATTATT CCTTTGTATT TTTACTTTTA AATGTCTTCT GTATTCACTT ATATGTTCTA ATTAATAAAT TATTTATTAT TAAGAAT

[0065] SEQ ID NO: 3 - Human CCL5 nucleic acid sequence

[0066] CCTCCGACAG CCTCTCCACA GGTACCATGA AGGTCTCCGC GGCACGCCTC GCTGTCATCC TCATTGCTAC TGCCCTCTGC GCTCCTGCAT CTGCCTCCCC ATATTCCTCG GACACCACAC CCTGCTGCTT TGCCTACATT GCCCGCCCAC TGCCCCGTGC CCACATCAAG GAGTATTTCT ACACCAGTGG CAAGTGCTCC AACCCAGCAG TCGTCTTTGT CACCCGAAAG AACCGCCAAG TGTGTGCCAA CCCAGAGAAG AAATGGGTTC GGGAGTACAT CAACTCTTTG GAGATGAGCT AGGATGGAGA GTCCTTGAAC CTGAACTTAC ACAAATTTGC CTGTTTCTGC TTGCTCTTGT CCTAGCTTGG GAGGCTTCCC CTCACTATCC TACCCCACCC GCTCCTTGAA GGGCCCAGAT TCTGACCACG ACGAGCAGCA GTTACAAAAA CCTTCCCCAG GCTGGACGTG GTGGCTCAGC CTTGTAATCC CAGCACTTTG GGAGGCCAAG GTGGGTGGAT CACTTGAGGT CAGGAGTTCG AGACAGCCTG GCCAACATGA TGAAACCCCA TGTGTACTAA AAATACAAAA AATTAGCCGG GCGTGGTAGC GGGCGCCTGT AGTCCCAGCT ACTCGGGAGG CTGAGGCAGG AGAATGGCGT GAACCCGGGA GCGGAGCTTG CAGTGAGCCG AGATCGCGCC ACTGCACTCC AGCCTGGGCG ACAGAGCGAG ACTCCGTCTC AAAAAAAAAA AAAAAAAAAA AAAAAATACA AAAATTAGCC GCGTGGTGGC CCACGCCTGT AATCCCAGCT ACTCGGGAGG CTAAGGCAGG AAAATTGTTT GAACCCAGGA GGTGGAGGCT GCAGTGAGCT GAGATTGTGC CACTTCACTC CAGCCTGGGT GACAAAGTGA GACTCCGTCA CAACAACAAC AACAAAAAGC TTCCCCAACT AAAGCCTAGA AGAGCTTCTG AGGCGCTGCT TTGTCAAAAG GAAGTCTCTA GGTTCTGAGC TCTGGCTTTG CCTTGGCTTT GCAAGGGCTC TGTGACAAGG AAGGAAGTCA GCATGCCTCT AGAGGCAAGG AAGGGAGGAA CACTGCACTC TTAAGCTTCC GCCGTCTCAA CCCCTCACAG GAGCTTACTG GCAAACATGA AAAATCGGGG

[0067] SEQ ID NO: 4 - Human GM-CSF nucleic acid sequence

[0068] AAAGTTCTCT GGAGGATGTG GCTGCAGAGC CTGCTGCTCT TGGGCACTGT GGCCTGCAGC ATCTCTGCAC CCGCCCGCTC GCCCAGCCCC AGCACACAGC CCTGGGAGCA TGTGAATGCC ATCCAGGAGG CCCGGCGTCT CCTGAACCTG AGTAGAGACA CTGCTGCTGA GATGAATGAA ACAGTAGAAG TCATCTCAGA AATGTTTGAC CTCCAGGAGC CGACCTGCCT ACAGACCCGC CTGGAGCTGT ACAAGCAGGG CCTGCGGGGC AGCCTCACCA AGCTCAAGGG CCCCTTGACC ATGATGGCCA GCCACTACAA ACAGCACTGC CCTCCAACCC CGGAAACTTC CTGTGCAACC CAGATTATCA CCTTTGAAAG TTTCAAAGAG AACCTGAAGG ACTTTCTGCT TGTCATCCCC TTTGACTGCT GGGAGCCAGT CCAGGAGTGA GACCGGCCAG ATGAGGCTGG CCAAGCCGGG GAGCTGCTCT CTCATGAAAC AAGAG

[0069] SEQ ID NO: 5 - Human FLT3LG nucleic acid sequence

[0070] TGGAAAGGGC TGTCACCCGG CTTGGCCCCT TCCACACCCA ACTGGGGCAA GCCTGACCCG GCGACAGGAG GCATGAGGGG CCCCCGGCCG AAATGACAGT GCTGGCGCCA GCCTGGAGCC CAACAACCTA TCTCCTCCTG CTGCTGCTGC TGAGCTCGGG ACTCAGTGGG ACCCAGGACT GCTCCTTCCA ACACAGCCCC ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGCGCT CTGGCGGCTG GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG CAGTGTCAGC CCGACTCCTC AACCCTGCCA CCCCCATGGA GTCCCCGGCC CCTGGAGGCC ACAGCCCCGA CAGCCCCGCA GCCCCCTCTG CTCCTCCTAC TGCTGCTGCC CGTGGGCCTC CTGCTGCTGG CCGCTGCCTG GTGCCTGCAC TGGCAGAGGA CGCGGCGGAG GACACCCCGC CCTGGGGAGC AGGTGCCCCC CGTCCCCAGT CCCCAGGACC TGCTGCTTGT GGAGCACTGA CCTGGCCAAG GCCTCATCCT GGGGAGGATA CGTAGGCACA CAGAGGGGAG TCACCAGCC

[0071] SEQ ID NO: 6 - Human OSM nucleic acid sequence

[0072] ATGGGCGTGCTGCTGACCCAGCGCACCCTGCTGAGCCTGGTGCTGGCGCTGCTGT TTCCGAGCATGGCGAGCATG

[0073] SEQ ID NO: 7 - Human immunoglobulin nucleic acid sequence

[0074] ATGGATATGCGCGTGCCGGCGCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGCGCG GCGCGCGCTGC

[0075] SEQ ID NO: 8 - Human chymotrypsinogen nucleic acid sequence

[0076] ATGGCGTTTCTGTGGCTGCTGAGCTGCTGGGCGCTGCTGGGCACCACCTTTGGC

[0077] SEQ ID NO: 9 - Human trypsinogen 2 nucleic acid sequence

[0078] ATGAACCTGCTGCTGATTCTGACCTTTGTGGCGGCGGCGGTGGCG

[0079] SEQ ID NO: 10 - Human IL-2 nucleic acid sequence

[0080] ATGTATCGCATGCAGCTGCTGAGCTGCATTGCGCTGAGCCTGGCGCTGGTGACCA ACAGC

[0081] SEQ ID NO: 11 - Human insulin nucleic acid sequence

[0082] ATGGCGCTGTGGATGCGCCTGCTGCCGCTGCTGGCGCTGCTGGCGCTGTGGGGCC CGGATCCGGCGGCGGCG

[0083] SEQ ID NO: 12 - Human XCL1 amino acid sequence

[0084] MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIF ITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG

[0085] SEQ ID NO: 13 - Human CCL5 amino acid sequence MKVSAAALAVILIATALCAPASAFPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSN

[0086] PAVVFVTRKNRQVCANPEKKWVREYINSLEMS

[0087] SEQ ID NO: 14 - Human GM-CSF amino acid sequence

[0088] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLN

[0089] LSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASH YKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE

[0090] SEQ ID NO: 15 - Human FLT3LG amino acid sequence

[0091] MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVK

[0092] 1RELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE

[0093] RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQC QP

[0094] DSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPG EQVPPVPSPQDLLLVEH

[0095] SEQ ID NO: 16 - Human OSM amino acid sequence

[0096] MGVLLTQRTLLSLVLALLFPSMASM

[0097] SEQ ID NO: 17 - Human immunoglobulin amino acid sequence

[0098] MDMRVPAQLLGLLLLWLRGARC

[0099] SEQ ID NO: 18 - Human chymotrypsinogen amino acid sequence

[0100] MAFLWLLSCWALLGTTFG

[0101] SEQ ID NO: 19 - Human trypsinogen 2 amino acid sequence

[0102] MNLLLILTFVAAAVA

[0103] SEQ ID NO: 20 - Human IL-2 amino acid sequence

[0104] MYRMQLLSCIALSLALVTNS

[0105] SEQ ID NO: 21 - Human insulin amino acid sequence

[0106] MALWMRLLPLLALLALWGPDPAAA

[0107] Pharmaceutical compositions - Versikine and lipid nanoparticles

[0108] Provided herein are pharmaceutical compositions that include any one of the versikine nucleic acid molecules described herein, optionally linked to any of the nucleic acid sequences disclosed, and a lipid nanoparticle. In some embodiments, the versikine nucleic acid molecule is encapsulated in the lipid nanoparticle.

[0109] As used herein, the term '‘pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, a pharmaceutical composition is suitable for administration to a human or animal subject, e.g.. via a particular route of administration (e.g., parenteral). In some embodiments, an active agent is present in a pharmaceutical composition in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0110] As used herein, a “lipid nanoparticle” refers to a nanoparticle composed of lipids, wherein a lipid nanoparticle comprises a solid lipid core matrix that can solubilize lipophilic molecules. Lipid nanoparticle (LNP) synthesis comprises (i) lipid component preparation, which involves the combination of different types of lipids to achieve the desired lipid composition; and (ii) aqueous phase preparation, which includes the substance to be encapsulated by the LNP. In some embodiments, a lipid nanoparticle can include a liposome, solid lipid nanoparticle, nanostructured lipid carrier, or cationic lipid-nucleic acid complex.

[0111] In some embodiments, lipid nanoparticles can be used as a pharmaceutical drug delivery system. In some embodiments, LNPs can be used as a delivery vehicle for nucleic acids (e.g.. siRNA. mRNA). As used herein, the term “encapsulation” refers to the process of confining an individual mRNA molecule within a nanoparticle. In some embodiments, a lipid nanoparticle can encapsulate a versikine nucleic acid. In some embodiments, a lipid nanoparticle can encapsulate a versikine mRNA.

[0112] In some embodiments, one or more nucleotides of the versikine nucleic acid molecule comprises one or more chemically modified nucleotides. In some embodiments, the one or more chemically -modified nucleotides comprises N1 -methyladenosine (ml A), N6- methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil, Ml- pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification. Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphoramidates, 2’-0-Me nucleoside analog, 2’F-RNA modification, 2’- deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F-ANA) modification, Locked nucleic acid (LNA). unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2’-O-(2-melhoxy ethyl)) modification. In some embodiments, a lipid nanoparticle comprises a lipid. In some embodiments, the lipid nanoparticle comprises ALC-0315. 1,2 DSPC, cholesterol. ALC-0159, SM-102, DMG-PEG(2000), D-Lin-MC3-DMA, or any combinations thereof. In some embodiments, a lipid nanoparticle comprises phosphatidyl serine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), dimyristoyl phosphatidylglyerol (DMPG) and sphingomyelin (SM), l,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). l,2-dioleoyloxy-3- dimethylammoniumpropane (DODAP) and analogues, l,2-di-(9Z-octadecenoyl)-sn-glycero- 3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylcholine (PC) and dimyristoyl phosphatidylcholine (DMPC), 1,2-di-O- octadecenyl-3-trimethylammoniumpropane (DOTMA). l,2-dioleyloxy-N,N- dimethyl aminopropane (DODMA), dioctadecyldi-methylammonium (DODA(Br) / DDAB), dioctadecyldimethylammoniumchloride (DODAC), 1 ,2-dimyristoyloxypropyl-l ,3- dimethylhydroxy ethylammonium (DMRIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N.N-dimethyl-l-propanamium trifluoroacetate (DOSPA) analogues, cholesterol, sphingomyelin (SM), or any combinations thereof.

[0113] Also provided herein are pharmaceutical compositions that include any of the lipid nanoparticle compositions of the disclosure and / or the versikine nucleic acid molecules in combination with at least one pharmaceutically acceptable carrier or diluent. The pharmaceutical compositions can be formulated in any manner known in the art.

[0114] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e g., sterile water or saline), a fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants such as ascorbic acid or sodium bisulfate, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g.. sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating such as lecithin, or a surfactant. Absorption of the lipid nanoparticle compositions can be prolonged by including an agent that delays absorption (e.g.. aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid). Compositions containing one or more of any of the lipid nanoparticles and / or the versikine nucleic acid molecules described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).

[0115] Toxicity' and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0116] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of the lipid nanoparticles (e.g.. any of the lipid nanoparticles described herein) and / or the versikine nucleic acid molecules will be, for example, an amount that decreases cancer cell invasion or metastasis in a subject having cancer, treats a metastatic cancer in a subject, decreases or stabilizes metastatic tumor size in in a subject, decreases the rate of metastatic tumor growth in a subject, decreases the severity, frequency, and / or duration of one or more symptoms of a metastatic cancer in a subject (e.g., a human), or decreases the number of symptoms of a metastatic cancer in a subject (e.g., as compared to a control subject having the same disease but not receiving treatment or a different treatment, or the same subject prior to treatment).

[0117] The effectiveness and dosing of any of the lipid nanoparticle compositions and / or the versikine nucleic acid molecules described herein can be determined by a health care professional using methods know n in the art, as well as by the observation of one or more symptoms of a metastatic cancer in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0118] Exemplary doses include milligram or microgram amounts of any of the lipid nanoparticle compositions and / or the versikine nucleic acid molecules described herein per kilogram of the subject’s weight. While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including the lipid nanoparticle compositions and / or the versikine nucleic acid molecules described herein, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the lipid nanoparticle compositions and / or the versikine nucleic acid molecules in vivo.

[0119] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0120] Methods for encapsulating a versikine nucleic acid molecule

[0121] Provided herein are methods for encapsulating any one of the versikine nucleic acid molecules described herein in a lipid nanoparticle, including, but not limited to ultrasonication, solvent emulsification evaporation, solvent emulsification-diffusion, membrane contactor, double emulsification, supercritical fluid extraction, spray drying, or other methods for making lipid nanoparticles known in the art. In one non-limiting example, the method for encapsulating a versikine nucleic acid molecule includes (a) generating the versikine nucleic acid molecule by using a transcription vector, (b) mixing the versikine nucleic acid molecule with a plurality of lipid nanoparticles, thereby forming lipid nanoparticles encapsulating the versikine nucleic acid molecule.

[0122] Versikine mRNA

[0123] In some embodiments, a lipid nanoparticle is used to encapsulate a versikine nucleic acid molecule. In some embodiments, the lipid nanoparticle is used to encapsulate a versikine mRNA. In some embodiments, mRNAs may be synthesized according to any of a variety of known methods. For example, a versikine mRNAs can be synthesized via in vitro transcription (IVT). For example, in some embodiments, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase 1, pyrophosphatase, and / or RNAse inhibitor. In some embodiments, the in vitro synthesized mRNA may be purified before formulation and encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0124] In some embodiments, the generating step (a) comprises using a transcription buffer comprising ATP, CTP, GTP, UTP, or a chemically modified nucleotide. In some embodiments, the chemically modified nucleotide comprises N1 -methyladenosine (mlA), N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil, Ml- pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine. phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphoramidates, 2’-0-Me nucleoside analog, 2’F-RNA modification. 2’- deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F-ANA) modification. Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2'-O-(2-methoxyethyl)) modification.

[0125] In some embodiments, the versikine nucleic acid molecule is conjugated or otherwise operably linked to one or more other nucleic acid sequences of the disclosure before the resulting nucleic acid is introduced to a lipid nanoparticle.

[0126] Lipid nanoparticles (LNPs)

[0127] In some embodiments, the plurality of lipid nanoparticles (LNPs) can include a mixture of lipids suitable to form lipid nanoparticles and / or liposomes for encapsulation of mRNA.

[0128] Methods of Manufacturing LNPs

[0129] In some embodiments, the plurality of nanoparticles are manufactured by using a microfluidic device. In some embodiments, this microfluidic method of LNP manufacturing allows for controllable and tunable nanoparticle production. In some embodiments, the microfluidic device includes a microfluidic channel with micro-mixing structures for nanoparticle synthesis. The microfluidic device leverages precise manipulation of fluids (e.g., an aqueous phase such as the mRNA solution and a solvent phase such as a lipid mixture or a lipid solution) at the microscale, achieving fast, controlled, and highly efficient mixing processes of different solution phases.

[0130] In some embodiments, the microfluidic device is scalable and includes a first subunit and a second subunit that are interchangeable and removably coupled to the microfluidic device. In some embodiments, the first subunit can be used to synthesize LNPs at a small scale. For example, in some embodiments, the first subunit is configured to yield about 0. 1 ml to about 0.5 ml of LNPs per sample. In some implementations, the first subunit is configured to yield about 0. 1 ml to about 0.2 ml, about 0. 1 ml to about 0.3 ml, about 0.1 ml to about 0.4 ml, about 0.2 ml to about 0.3 ml, about 0.2 ml to about 0.4 ml, about 0.2 ml to about 0.5 ml. about 0.3 ml to about 0.4 ml, about 0.3 ml to about 0.5 ml, or about 0.4 ml to about 0.5 ml of LNPs per sample. In some embodiments, the first subunit is configured to yield about 0. 1 ml to about 2 ml of LNPs per sample. In some embodiments, the first subunit is configured to yield about 1 to about 4 LNP samples per run.

[0131] In some embodiments, the second subunit can be used to synthesize LNPs at a larger scale when compared to the first subunit’s yields. For example, in some embodiments, the second subunit is configured to yield about 1 ml to about 12 ml of LNPs per sample. In some embodiments, the second subunit is configured to yield about 1 ml to about 2 ml, about 1 ml to about 3 ml, about 1 ml to about 4 ml, about 1 ml to about 5 ml. about 1 ml to about 6 ml, about 1 ml to about 7 ml, about 1 ml to about 8 ml, about 1 ml to about 9 ml. about 1 ml to about 10 ml, about 1 ml to about 1 1 ml, or about 1 ml to about 12 ml of LNPs per sample. In some embodiments, the disclosed microfluidic methods of manufacturing are scalable and can generate a higher yield of LNP product than the first and second subunits. For example, disclosed microfluidic methods of manufacturing can yield about 2 ml to about 200 ml of LNPs per run, about 50 ml to about 1 L of LNPs per run, about 50 ml to about 1 L of LNPs per run, at least about 10 L of LNPs per run, or at least about 20 L of LNPs per run.

[0132] In some embodiments, a suitable lipid solution is ethanol-based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In some embodiments, a suitable lipid solution is isopropyl alcohol-based. In some embodiments, a suitable lipid solution is dimethylsulfoxide-based. In some embodiments, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.

[0133] In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of about 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg / ml, 0.5-90 mg / ml, 1.0-80 mg / ml. 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml. 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml. or 10 mg / ml.

[0134] In another example, a suitable lipid solution may contain a mixture of desired lipids at atotal concentration of about 10 mM. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of about 5 mM to about 15 mM (e.g., about 5 mM to about 6 mM, about 5 mM to about 7 mM, about 5 mM to about 8 mM, about 5 mM to about 9 mM. about 5 mM to about 10 mM, about 6 mM to about 7 mM, about 6 mM to about 8 mM, about 6 mM to about 9 mM, about 6 mM to about 10 mM, about 7 mM to about 8 mM, about 7 mM to about 9 mM, about 7 mM to about 10 mM, about 8 mM to about 9 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 10 mM to about 11 mM, about 10 mM to about 12 mM, about 10 mM to about 13 mM. about 10 mM to about 14 mM, or about 10 mM to about 15 mM.

[0135] In some embodiments, any desired lipids may be mixed at any ratios suitable for encapsulating mRNAs. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g.. non-cationic lipids and / or cholesterol) and / or PEGylated lipids. In some embodiments, a helper lipid is a class of lipid molecules that may increase particle stability and fluidity of LNPs. Non-limiting examples of helper lipids include dioleoy lphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), cholesterol, 1.2-Dimyristoyl-sn-glycero-3- phosphocholine (DMPC), and sphingomyelin. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol) and one or more PEGylated lipids. In some embodiments, the LNP compositions of the disclosure include an ionizable cationic lipid, a helper lipid, cholesterol, and a PEGylated lipid at a molar ratio of 50: 10:38.5: 1.5, respectively. In some embodiments, the LNP compositions of the disclosure include heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), 1,2- distearoyl-sn-glycero-3-phosphatidylcholine (1,2-DSPC). cholesterol, and 1 ,2-dimyristoyl- rac-glycero-3-methoxypoly ethylene glycol-2000 (DMG-PEG 2000) at a molar ratio of 50: 10:38.5:1.5, respectively. In some embodiments, the LNP compositions of the disclosure include an ionizable cationic lipid comprising at least 50 mole percent (mol %). In some embodiments, the LNP compositions of the disclosure include a helper lipid (e g., a non- catiomc lipid) comprising at least 10 mol %. In some embodiments, the LNP compositions of the disclosure include cholesterol comprising at least 38.5 mol %. In some embodiments, the LNP compositions of the disclosure include a PEGylated lipid comprising at least 1.5 mol %. In some embodiments, the LNP compositions of the disclosure include an ionizable cationic lipid comprising at least 50 mol % and a helper lipid comprising at least 10 mol %. In some embodiments, the LNP compositions of the disclosure include an ionizable cationic lipid comprising at least 50 mol % and cholesterol comprising at least 38.5 mol %. In some embodiments, the LNP compositions of the disclosure include an ionizable cationic lipid comprising at least 50 mol % and a PEGylated lipid comprising at least 1.5 mol %. In some embodiments, the LNP compositions of the disclosure include a helper lipid comprising at least 50 mol % and cholesterol comprising at least 38.5 mol %. In some embodiments, the LNP compositions of the disclosure include a helper lipid comprising at least 50 mol % and a PEGylated lipid comprising at least 1.5 mol %. In some embodiments, the LNP compositions of the disclosure include cholesterol comprising at least 38.5 mol % and a PEGylated lipid comprising at least 1.5 mol %.

[0136] In some embodiments, a lipid nanoparticle of the plurality of lipid nanoparticles comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4- hydroxybutyl)hexan-l-aminium (ALC-0315), 1,2-DSPC, cholesterol, methoxypoly ethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159), SM-102, DMG-PEG(2000), 4-(dimethylammo)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12- octadecadien-l-yl-10,13-nonadecadien-l-yl ester (D-Lin-MC3-DMA), or any combinations thereof. In some embodiments, a lipid nanoparticle of the plurality of lipid nanoparticles comprises phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), dimyristoyl phosphatidylglycerol (DMPG) and sphingomyelin (SM), l,2-dioleoyl-3- trimethylammoniumpropane (DOTAP), 1.2-dioleoyloxy-3-dimethylammoniumpropane (DODAP) and analogues, l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylcholine (PC) and dimyristoyl phosphatidylcholine (DMPC). l,2-di-O-octadecenyl-3- trimethylammoniumpropane (DOTMA), 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), dioctadecyldi-methylammonium (DODA(Br) / DDAB), dioctadecyldimethylammoniumchloride (DODAC), 1 ,2-dimyristoyloxypropyl-l ,3- dimethylhydroxy ethylammonium (DMRIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N.N-dimethyl-l-propanamium trifluoroacetate (DOSPA) analogues, cholesterol, sphingomyelin (SM), or any combinations thereof.

[0137] In some embodiments, versikine mRNA is encapsulated using the following lipid formulations with substitutes SM-102: 1,2-DSPC: cholesterol: DMG-PEG(2000). In some embodiments, versikine mRNA is encapsulated using the following lipid formulations with substitutes including, but not limited to, ALC-0315: 1,2 DSPC: cholesterol: ALC-0159; and D-Lin-MC3-DMA: 1,2 DSPC: cholesterol: DMG-PEG(2000).

[0138] In some embodiments, one or more of the lipids can be in cis. In some embodiments, one or more of the lipids can be in trans. In some embodiments, a lipid nanoparticle can comprise a mixture of cis and trans lipids. In some embodiments, a pharmaceutical composition comprising multiple lipid nanoparticles can comprise a mixture of cis and trans lipid nanoparticles.

[0139] LNP Characterization

[0140] In some embodiments, the hpid nanoparticles of the disclosure have a diameter between about 40 nanometers (nm) to about 200 nm (e g., between about 40 nm to about 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 40 nm to about 90 nm. about 40 nm to about 100 nm, about 40 nm to about 110 nm, about 40 nm to about 120 nm, about 40 nm to about 130 nm, about 40 nm to about 140 nm, about 40 nm to about 150 nm, about 40 nm to about 160 nm, about 40 nm to about 170 nm, about 40 nm to about 180 nm, or about 40 nm to about 190 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm. about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, or about 80 nm to about 90 nm). In some embodiments, the lipid nanoparticles of the disclosure have a diameter between about 50 nm and 90 nm.

[0141] In some embodiments, the lipid nanoparticles provided herein have a poly dispersityindex (PDI) of about 0.05 to about 0.25. The PDI is essentially a representation of the distribution of size populations within a given LNP sample. The numerical value of PDI ranges from 0.0 (for a perfectly uniform sample with respect to the particle size) to 1.0 (for a highly polydisperse sample with multiple particle size populations). In some embodiments, the lipid nanoparticles provided herein can have a PDI of about 0.050 to 0.100, about 0.100 to 0.110, about 0.110 to 0.120, about 0.120 to 0.130, about 0.130 to 0.140, about 0.140 to 0.150, about 0.150 to 0.160, about 0.160 to 0.170, about 0.170 to 0.180, about 0.180 to 0.190, about 0.190 to 0.200. about 0.200 to 0.210, about 0.210 to 0.220, about 0.230 to 0.240, or about 0.240 to 0.250.

[0142] In some embodiments, the lipid nanoparticles disclosed herein have a zeta potential ranging from about -10 millivolts (mV) to about 10 mV. The zeta potential describes the surface charge of the LNPs while suspended, e.g., in a buffer solution. In some embodiments, the lipid nanoparticles disclosed herein have a zeta potential ranging from about -10 mV to about -5 mV, -10 mV to about 0 mV, -5 mV to about 0 mV, 0 mV to about 5 mV, 0 mV to about 10 mV, or 5 mV to about 10 mV.

[0143] In some embodiments, the lipid nanoparticle compositions disclosed herein are stable for at least about 1 day to about 6 months or more. The term '’stable" or “stability,” as used herein, indicates a lack of change in any of the physical properties of a same sample of the lipid nanoparticles or compositions as measured and compared from the day when they were prepared to the day after being in storage. In some embodiments, the lipid nanoparticle compositions disclosed herein are stable for at least about 1 day to about 5 days, for about 5 days to 10 days, about 10 days to about 15 days, for about 15 days to 30 days, about 30 days to about 40 days, for about 40 days to 50 days, about 50 days to about 60 days, about 3 months to about 4 months, about 4 months to about 5 months, about 5 months to about 6 months, or more.

[0144] Methods for sensitizing a response to an immunotherapy

[0145] Provided herein are methods for sensitizing a response to an immunotherapy in a subject in need thereof that include administering any one of the versikine nucleic acid molecules or the pharmaceutical compositions described herein. In some embodiments, the immunotherapy comprises an anti-IL4 immunotherapy.

[0146] Immunotherapy

[0147] As used herein, "‘immunotherapy” refers to a treatment of disease (e.g., cancer) by activating or suppressing the immune system. For example, cancer immunotherapy uses the immune system and its components to mount an anti-tumor response through immune activation. In some embodiments, an immunotherapy can include an immune checkpoint inhibitor, an oncolytic virus therapy, a cell-based therapy, a CAR-T cell therapy, or a cancer vaccine. In some embodiments, an immunotherapy can include immune checkpoint blockade (ICB), wherein an immune checkpoint inhibitor is administered. Immune checkpoint blockade (ICB) is a treatment that uses immune checkpoint inhibitors to address a disease (e.g., cancer), wherein the immune checkpoint inhibitor blocks a checkpoint protein from binding to its partner protein or receptor.

[0148] In some embodiments, the immunotherapy includes an anti-IL4 immunotherapy. Interleukin (IL)-4 is a cytokine in tumor immunology7, wherein IL-4 exhibited potent antitumor ability. It has been shown that administration of a neutralizing antibody against IL-4 enhances anti-tumor immunity and delays tumor progression. Furthermore. IL-4 blockade alters inflammation in the tumor microenvironment, reducing the generation of both immunosuppressive M2 macrophages and myeloid-derived suppressor cells, and enhancing tumor-specific cytotoxic T lymphocytes. IL-4 affects anti-tumor immunity and constitutes a therapeutic target to reduce immune suppression in the tumor microenvironment, thus enhancing the efficacy of cancer therapy.

[0149] As used herein, the term '‘sensitizing” or “improve sensitivity” can refer to increasing response rates to a specific treatment (e.g., immunotherapy). In some embodiments, improving sensitivity to ICB treatment can refer to inhibiting negative regulatory immune checkpoints or stimulating activating immune checkpoints.

[0150] Administration of Versikine nucleic acid molecule

[0151] In some embodiments, a method described herein includes administering any one of the versikine nucleic acid molecules, lipid nanoparticle compositions, or the pharmaceutical compositions described herein.

[0152] As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g.. by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), intratumoral, intralesional, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0153] In some embodiments, the administering comprises delivery’ of the versikine nucleic acid molecule, the liposomal compositions encapsulating the versikine nucleic acid molecule, or the pharmaceutical composition into a cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is an antigen-presenting cell (e.g., macrophage, B cell, and / or dendritic cell). In some embodiments, the cell is a non-hematopoietic antigen- presenting cell (e.g.. antigen-presenting fibroblast, lymph node stromal cell, or endothelial cell). In some embodiments, the cell is a cancer cell. In some embodiments, the versikine nucleic acid molecule or pharmaceutical composition can be delivered into a cell by a variety’ of well-known techniques. In some embodiments, the delivery method can include a vector- mediated gene transfer (e.g.. viral infection / transfection, or various other protein-based or lipid-based gene delivery' complexes). In some embodiments, the delivery method can include methods facilitating the delivery’ of "naked" polynucleotides (e.g., electroporation, “gene gun’' delivery and various other techniques used for the introduction of polynucleotides). In some embodiments, the delivering comprises transfection, electroporation, or a virus-based delivery. In some embodiments, the delivery of the nucleic acid molecule comprises a virus-based delivery. In some embodiments, the virus-based delivery’ comprises an adeno-associated virus or a lentivirus. In some embodiments, the virusbased delivery comprises a plant-virus.

[0154] Method of treating a cancer in a subject

[0155] Provided herein are also methods for treating cancer in a subject in need thereof, the method including administering any one of the versikine nucleic acid molecules or the pharmaceutical compositions described herein, thereby treating the cancer. As used herein, the terms ‘‘cancer’", “tumor’", and “carcinoma"’ refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a metastatic solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary7peritoneal cancer, salivary7gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer. In some embodiments, hematopoietic cancers can include leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, precancerous pathology such as myelodysplastic syndromes, acquired aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria (PNH) and 5q- syndrome and the like. In some embodiments, a relevant cancer may be colorectal cancer.

[0156] In some embodiments, the versikine nucleic acid molecules, the liposomal compositions, or the pharmaceutical compositions described herein can be administered alone or with an immunotherapy agent and / or a pharmaceutical agent. In some embodiments, the versikine nucleic acid molecules, the liposomal compositions, or the pharmaceutical compositions described herein can be co-administered to the subject with an immunotherapy agent. In some embodiments, the subject is co-administered one or more immunotherapy agents that target the IL-4 pathway. In some embodiments, the subject is co-administered one or more immunotherapy agents that inhibit programmed death 1 (PD-1) and / or programmed death-ligand 1 (PD-L1). In some embodiments, the versikine nucleic acid molecules, the liposomal compositions, or the pharmaceutical compositions and the immunotherapy agent can be administered simultaneously. In some embodiments, the versikine nucleic acid molecules, the liposomal compositions, or the pharmaceutical compositions can be administered before the immunotherapy agent. In some embodiments, the versikine nucleic acid molecules, the liposomal compositions, or the pharmaceutical compositions can be administered after the immunotherapy agent. In some embodiments, the co-administration comprises administering the one or more immunotherapy agents prior to administering the versikine nucleic acid molecule, the liposomal compositions, or the pharmaceutical composition. In some embodiments, the co-administration comprises administering the one or more immunotherapy agents after administering the versikine nucleic acid molecule, the liposomal compositions, or the pharmaceutical composition. In some embodiments, the co- administration comprises administering the one or more immunotherapy agents and the versikine nucleic acid molecule, the liposomal compositions, or the pharmaceutical composition at the same time.

[0157] In some embodiments, the one or more immunotherapy agents that target the IL-4 pathway can comprise antibody inhibitors of IL-4 (e.g., Dupilumab, pasolizumab), or an IL-4 receptor antagonist (e.g.. pitrakinra). In some embodiments, the one or more immunotherapy agents comprise antibody inhibitors of PD-1 and / or PD-L1. In some embodiments, the one or more immunotherapy agents comprise cemiplimab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, or any combination thereof. In some embodiments, the one or more immunotherapy agents comprise nucleic acid molecules that encode one or more inhibitors of PD-1 and / or PD-L1. For example, in some embodiments, the one or more immunotherapy agents comprise mRNA encoding for one or more inhibitors of PD-1 and / or PD-L1. In some embodiments, the nucleic acid molecules encoding for one or more inhibitors of PD-1 and / or PD-L1 can also be encapsulated within the same lipid nanoparticles as the versikine nucleic acid molecules. In some embodiments, the nucleic acid molecules encoding for one or more inhibitors of PD-1 and / or PD-L1 are encapsulated in lipid nanoparticles that are separate from the lipid nanoparticles used to encapsulate the versikine nucleic acid molecules. In some embodiments, the nucleic acid molecules encoding for one or more inhibitors of PD-1 and / or PD-L1 can be delivered into a cell with a delivery method that is different than lipid nanoparticles. In some embodiments, the delivery method to deliver the nucleic acid molecules encoding for one or more inhibitors of PD-1 and / or PD-L1 can include one or more of vector-mediated gene transfer, electroporation, “gene gun” delivery, transfection, electroporation, and virus-based delivery.

[0158] In some embodiments, administering any one of the versikine nucleic acid molecules or the pharmaceutical compositions described herein can treat a cancer. In some embodiments, the cancer is a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary' gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer. In some embodiments, the cancer is a refractory tumor.

[0159] In some embodiments, the administering of the versikine nucleic acid molecule or the pharmaceutical composition comprises bronchial (e.g., by bronchial instillation), buccal, dermal, enteral, intra-arterial, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intracistemal, intratumoral, intralesional. within a specific organ (e.g.. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e g., by intratracheal instillation), vaginal, or vitreal administration.

[0160] EXAMPLES

[0161] Certain embodiments of the present disclosure are further described in the following examples, which do not limit the scope of any embodiments described in the claims.

[0162] Example 1 - Versikine mRNA Synthesis

[0163] The versikine open-reading-frame (or fusions thereol) w as cloned in a vaccine- appropriate in vitro transcription vector encompassing a T7 promoter, 5’ and 3’ UTR and 110-long poly-A tail. Sequence was confirmed after cloning. The vector was then linearized using restriction enzyme digestion.

[0164] In vitro transcription w as carried out in a reaction mix consisting of: lOx Transcription buffer (2 pl) lOx ATP (or chemically modified nucleotides*) (2 pl) lOx CTP (or chemically modified nucleotides*) (2 pl) lOx GTP (or chemically modified nucleotides*) (2 pl) 5-Methoxy-UTP (100 mM) (or chemically modified nucleotides*) (2 pl) CleanCap® Reagent AG (1.6 pl)

[0165] Template DNA (1 pl) lOx Enzyme Mix (2 pl) Water Total (20 pl)

[0166] Chemically modified nucleotides can include, but are not limited to N1 - methyladenosine (ml A). N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil. Ml -pseudouracil, 5 -methoxy uridine (5moU), or N4- acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification. Phosphonoacetate (PACE) linkage modification, Morpholino phosphorami dates. 2’-O-Me nucleoside analog, 2’F-RNA modification, 2’-deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F- ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) (or “seconucleoside”) modification, 2’-0-M0E (2’-O-(2-methoxyethyl)) modification.

[0167] RNA synthesis was allowed to proceed at 37 °C for 2 hours. Briefly, 4 pl of DNase I (Takara IVTpro™ T7 mRNA Synthesis Kit. Cat #6144) was added to the reaction, mixed briefly, and incubated and allowed to react for 15 mins, at 37 °C. RNA was then recovered using LiCl precipitation. mRNA purification was performed by LiCl precipitation. Lithium Chloride Precipitation Solution (Takara IVTpro™ T7 mRNA Synthesis Kit, Cat #6144) was thawed at room temperature before use. The solution was mixed well, and if a precipitate was observed, the solution was warmed at 37 °C. If the precipitate persisted after the solution was armed, it was used as is. The precipitate did not affect the subsequent steps. 30 pl of Nuclease-Free Water (Takara IVTpro™ T7 mRNA Synthesis Kit. Cat #6144) and 30 pl of Lithium Chloride Precipitation Solution (Takara IVTpro™ T7 mRNA Synthesis Kit, Cat #6144) were added to the IVT reaction solution (about 24 pl) treated with DNase I to stop the reaction. The solution was mixed well, then cooled at -20°C for at least 30 minutes. The solution was then centrifuged at 4 °C for 15 minutes at maximum speed. The supernatant was carefully removed and the pellet was washed with 1 ml of 70% ethanol. The sample was centrifuged again at 4 °C for 15 minutes at maximum speed. The supernatant was carefully removed. The pellet was allowed to air-dry and then dissolved in 100 pl of Nuclease-Free Water (Takara IVTpro™ T7 mRNA Synthesis Kit, Cat #6144). The mRNA was stored at -80°C until further use. About 150 to 200 pg of mRNA was generated per reaction containing 1 pg of template DNA. mRNA length and purity was confirmed using an Agilent® TapeStation® or an Agilent® Bioanalyzer® device.

[0168] Example 2 - LNP Encapsulation of Versikine mRNA

[0169] LNP encapsulation of mRNA was performed using Preci Genome’s NanoGenerator™ microfluidic system (a pressure controller with two attachable subunits: NanoGenerator™ Flex-S platform for smaller-scale syntheses and larger-scale NanoGenerator1MFlex-M platform). mRNA-LNPs contain two core phases: aqueous (mRNA in acidification buffer) and solvent / organic (various lipids dissolved in ethanol). They are often diluted with buffer (e.g., phosphate buffer saline (PBS)), or concentrated, to reach the ideal concentration for experiments.

[0170] Three steps of LNP production are described below: pre-synthesis with Precigenome’s system (making the aqueous and solvent / organic phase), synthesis (running the microfluidic machine), and post-synthesis (processing and quality control).

[0171] A) Pre-Synthesis of Versikine mRNA-LNPs

[0172] Cayman Chemical's LipidLaunch™ LNP- 102 Exploration Kit (Item No. 35425) was used to formulate the solvent phase with the ionizable cationic lipid SM-102 and component lipids 1,2-DSPC, cholesterol, and DMG-PEG(2000).

[0173] First, stock solutions of each of the lipid components were made as follows. Absolute ethanol was added to each of the provided solids 1,2-DSPC, cholesterol, and DMG- PEG(2000), such that their concentrations were 25 mg / mL, 5 mg / mL, and 1 mg / mL, respectively. SM-102 was supplied by the kit already suspended in ethanol at a concentration of 100 mg / mL.

[0174] Next, 35 pL of the SM-102 stock, 32 pL of the 1,2-DSPC stock, 296 pL of the cholesterol stock, 378 pL of the DMG-PEG(2000) stock, and 259 pL of absolute ethanol were combined to create 1 mL of a 10 mM total lipid solvent phase, with the following lipid molar ratios:

[0175] SM-102: 50

[0176] 1,2-DSPC: 10

[0177] Cholesterol: 38.5

[0178] DMG-PEG2000: 1.5 This production of the lipid solvent phase was scaled as necessary'.

[0179] Next, to make the aqueous phase, versikine mRNA was diluted in either 50 mM sodium acetate or sodium citrate, at pH 4, to a concentration of about 0.0893 mg / mL. B) Synthesis of Versikine mRNA-LNPs

[0180] Microfluidic devices were used to combine the aqueous and solvent phases of mRNA- LNPs in a rapid and consistent manner. Preci Genome’s microfluidic devices (including the NanoGenerator™ Flex-S platform and NanoGenerator™ Flex-M platform) both used the same mixing chip with three linked wells: a first well for loading of the aqueous phase, a second well for loading of the solvent phase, and a third well for collection of the mRNA- LNPs. mRNA-LNPs were synthesized with a 3: 1 aqueous to solvent ratio by volume while using both the NanoGenerator™ Flex-S platform and NanoGenerator™ Flex-M platform. i) NanoGenerator™ Flex-S platform

[0181] Between 200-500 pL of mRNA-LNPs were generated using a 3: 1 aqueous to solvent ratio and the NanoGenerator™ Flex-S system. This production was diluted with 25-500 pL of phosphate-buffered saline (PBS) to reach a final production volume of 225-1000 pL.

[0182] First, it was confirmed that the tubes connecting the Flex-S flow unit to the NanoGenerator™ were attached to the desired channel where the product was to be deposited in. Tube attachments for each channel are listed below. (Al / Sl is found on the Flex-S unit, Ch# is found on the generator itself.)

[0183] Channel 1 : aqueous (green) tube on Al / Ch#L solvent (yellow) tube on Sl / Ch#3

[0184] Channel 2: green on A2 / Ch#2, yellow on S2 / Ch#4

[0185] Channel 3: green on A3 / Ch#5, yellow on S3 / Ch#7

[0186] Channel 4: green on A4 / Ch#6, yellow on S4 / Ch#8

[0187] Next, the mixing chip was added to the NanoGenerator™ Flex-S system. The stop was pulled back on the right, the chip was slid left side in first, then the stop was released.

[0188] The NanoGenerator™ was turned on, “new recipe” was selected on the device, and a recipe was created based on the volume and aqueous / solvent ratio was desired. The creation of the recipe allowed for the adjustment of the channel, volume, and total flow rate. LNPs were produced using a total flow rate of 3 mL / min and with an aqueous solvent ratio of 3: 1. The volume that was input in the Flex-S recipe did not account for PBS dilution. For example, if the recipe was set to generate 200 pL of volume, the actual theoretical yield was greater, depending on how much PBS the formulation was diluted with.

[0189] Next, if the NanoGenerator™ had not yet been run, the machine was warmed up by clicking on the “play” button at the top right to start a blank run. This triggered the system to run, generating no product. The machine was then “zeroed” after a three minute wait.

[0190] 5 pL of pure ethanol were added to the mixing chip, in the top well, according to the desired channel that was designated to synthesize the LNPs; this prevented pre-mixing. Next, the collection tank was added directly onto the mixing chip, oriented with the channel that was being used.

[0191] To load the aqueous and solvent phases, the aqueous components (e.g., the versikine mRNA) were loaded in the left tank, the solvent components (e.g., the lipids) were loaded in the right tank, and the PBS buffer was loaded in the big collection tank. For example, for a 400 pL synthesis with an aqueous: solvent ratio 3: 1, at least 172.5 pL aqueous and 57.5 pL solvent were added to the left and right tank, respectively. And at least 25 pL of PBS were added to the collection tank for “in-line dilution.” Volumes greater than 25 pL of PBS can be added, as long as the combined volume generated by the desired recipe and the volume of PBS added does not exceed 1 ml.

[0192] Next, tubes were checked again to ensure that they were properly connected, then the recipe was run by clicking the green “play” button at the top right of the NanoGenerator™ device.

[0193] The product was deposited in the collection tank where PBS was added to and was generally a little less (e.g., about 5% to about 10% less) than the yield that was intended to be synthesized. The final product was collected as soon as possible and immediately transferred to a refrigerator for storage. Alternatively, the product was stored on ice if it was to be dialyzed thereafter. ii) NanoGeneratorl xtFlex-M platform

[0194] The versikine mRNA-LNPs were also generated using the NanoGenerator™ Flex-M system when a higher throughput of product was desired.

[0195] First, the cables for the Flex-M platform were set up. The tube attached to “Flow sensor water” on the Flex-M device itself (on the right side) was attached to flow sensor #1 on the NanoGenerator™, and the tube attached to “flow sensor solvent” was attached to flow sensor #2 on the NanoGenerator™. Next, the tubes labeled “water” and “solvent” were attached to the corresponding channels on the NanoGenerator™ such that the tube labeled “water” was attached to Ch #1 and the tube labeled “solvent” was attached to Ch #3. The software was switched to Flex-M and verified that it worked by clicking on the gear near the bottom right of the screen, then clicking on "‘about.” On the left side of the popup screen, a letter “S” or “M” was displayed depending on which mode the software as currently on (e.g., the letter “S” corresponds to the Flex-S software and the letter “M” corresponds to the Flex-M software). The modes were switched by clicking on the letter. The mode was switched to “M” in this instance, and it was selected by clicking “OK.”

[0196] The “about” tab was reopened and the option “check” was selected. This checked that the system was properly connected. After the check was completed, the tab was closed and re-opened again. It was verified that “Meter 1” on the tab contained “2000D” and “OK,” and “Meter 2” included “1000D” and “OK.”

[0197] Each mixing chip has four staggered herringbone connections - these herringbones connect one collection / outlet well to two inlet wells, which correspond to the aqueous and solvent phases. The mixing chip was aligned on the Flex-M such that the four outlet wells were on the left. The three tubes (with pegs at the end) were placed and secured such that they were on outlet / inlet wells connected by a staggered herringbone. The channel in which the product is mixed can be adjusted by moving the three tube pegs attached to the chip. If the channel is changed, it is important to keep the orientation consistent and to ensure that they are in the proper mixing alignment. There was no collection tank for the Flex-M device.

[0198] A 15 mL Falcon tube was added to each of the sections labeled “water,” “solvent,” and “collection” of the Flex-M device. A 50 mL Falcon tube was added to the section labeled “waste.”

[0199] A recipe was created for the Flex-M device. When using the Flex-M system, the same total flow rate, the same flow rate ratio, and the same aqueous: sol vent ratio were used as those used with the Flex-S device. The NanoGenerator™ Flex-M system allows for any volume between about 1.5 and about 15 mL to be synthesized. In contrast to the Flex-S device, the Flex-M device does not require an “in-line” PBS dilution.

[0200] Once the desired recipe was set up, a test run with w ater and ethanol in the “w ater” and “solvent” tubes, respectively, was performed. Afterwards, the aqueous phase (e.g., the versikine mRNA) was loaded into the “water” tube, the lipids (or solvent phase) were loaded into the “solvent” tube, and the device was run. Product (e g., the LNPs) was collected in the “collection” tube. The product was transferred to ice as soon as possible thereafter.

[0201] Next, the system was w ashed by running the same recipe with water and ethanol. iii) Dialysis and Concentration of Versikine mRNA-LNPs

[0202] After synthesis with either the Flex-S or Flex-M systems, the LNPs encapsulating versikine mRNA (i.e., versikine mRNA-LNPs) were dialyzed against lx PBS at a pH 7.4. This brought the LNPs close to physiological pH and was a crucial step for future in vivo administration.

[0203] The large, 2 ml Slide-a-Lyzer™ dialysis cassette was used for mRNA-LNP volumes of about 0.2 mL to about 2 mL, and the small. 0.5 ml Slide-a-Lyzer™ dialysis cassette was used for mRNA-LNP volumes or about 50 to about 500 pL. A small amount of PBS was first added to the Slide-a-Lyzer™ dialysis cassettes to ensure that they didn’t leak. The Slide-a- Lyzer™ dialysis cassettes were then emptied.

[0204] 14 ml or 44 ml of PBS were added to a conical vial when using the small Slide-a- Lyzer™ dialysis cassette or the Slide-a-Lyzer™ large dialysis cassette, respectively. The mRNA-LNP product was added to the Slide-a-Lyzer™ cassette. Then, the Slide-a-Lyzer™ cassette containing the mRNA-LNP product was placed in the conical vial containing PBS, submerged in PBS, and put in a cold room (e.g.. at a temperature of about 4 °C) on a shaker to dialyze for 2 hours.

[0205] After 2 hours, the PBS was replaced with fresh PBS, and the cassette was allowed to dialyze overnight in the cold room. Next, the mRNA-LNP product was removed from the Slide-a-Lyzer™ cassette and stored in the refrigerator (e.g., at a temperature of about 4 °C) for long-term storage or stored on ice for immediate use.

[0206] Prior to concentrating the mRNA-LNP product, the dialyzed mRNA-LNPs were filtered with a 0.2 pm polyethersulfone (PES) filter. The mRNA-LNP product w as then concentrated with an Amicon® ultra centrifugal filter, 100K molecular weight cutoff (MWCO). The dialyzed mRNA-LNP product was added to the filter device of the centrifugal concentrator, the components of the concentrator were then assembled, and the filter device was inserted into one of the provided microcentrifuge collection tubes facing up. The capped filter device then was inserted into a centrifuge rotor, ensuring that the white membrane was facing the center of the rotor, the concentrator is poking out the well of the centrifuge as little as possible, and that collection tube containing the filter device was properly counterbalanced.

[0207] The filter device was spun (e.g., in a swinging bucket rotor) for about 30 mins, at 4000 g and at 4 degrees Celsius. The assembled device was removed from the centrifuge, and the Amicon® Ultra filter device was separated from the microcentrifuge tube. To recover the concentrated solute, the Amicon® Ultra filter device was placed upside down in a clean microcentrifuge tube. The assembled device was then placed in the centrifuge, aligning open cap towards the center of the rotor and properly counterbalanced with a similar device. The assembled device was spun for 2 minutes at 1,000 x g to transfer the concentrated sample from the filter device to the collection tube. The concentrated mRNA-LNP product was collected from the collection tube and immediately stored in the refrigerator (e.g., at a temperature of about 4 °C) for long-term storage or stored on ice for immediate use.

[0208] C) Characterization and Quality) Control of Versikine mRNA-LNPs

[0209] The Malvern® Zetasizer® and its software were used to perform dynamic light scattering (DLS) and zeta potential measurements for the LNPs and / or mRNA-LNPs. Dynamic light scattering yielded readouts for particle size and poly dispersity index (PDI), whereas zeta potential described the surface charge of LNP in their buffer solution.

[0210] For DLS, a standard operating procedure (SOP) was created on the Malvern® software with the following properties: ZEN0118 cuvette, 90 degree scattering angle, temperature: 25 degrees Celsius, equilibration time: 120 seconds, 3 measurements of automatic duration, material absorbance: 0.01, material refractive index: 1.45, dispersant refractive index: 1.33, dispersant viscosity: 0.894.

[0211] 1 pL of concentrated LNP and 49 pL of PBS were added to a Disposable low volume polystyrene cuvette (e.g., Malvern® part number #:ZEN0118). The cuvette was placed in the Zetasizer® machine, and the SOP was run.

[0212] Size readouts were given in four different readouts (all in nanometers (nm)): Z- average, intensity mean, number mean, and volume mean. The number mean readout was used to determine the size of the LNPs and / or mRNA-LNPs.

[0213] For zeta potential measurements, the folded capillary zeta cell cuvettes (e.g., Malvern® part number #: DTS1070) were used, and the SOP was set to run three, 100-read measurements.

[0214] The software’s readouts were switched to display “zeta,” and the SOP was run for zeta potential. Zeta potential was given in millivolt (mV) units. A a near neutral zeta potential (about -10 mV to about 10 mV) is ideal for LNPs.

[0215] LNP-versikine rnRNA particles were then dialyzed to neutral pH (pH7.4) against 1,000 volumes of PBS overnight. LNP-versikine mRNA particles were concentrated using ultrafiltration, filter-sterilized and stored at 4 °C for 1 week or lyophilized and stored at -80 deg C for long-term use. Finally, LNP were routinely characterized for size, zeta potential (surface charge), encapsulation efficiency, concentration and endotoxin contamination. Typical targets: size 80-100nm in diameter, greater than 90% mRNA encapsulation and <0.25 EU / mL endotoxin.

[0216] OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A versikine nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 1.

2. A versikine nucleic acid molecule comprising SEQ ID NO: 1.

3. The versikine nucleic acid molecule of claim 1 or 2, wherein one or more nucleotides of the versikine nucleic acid molecule comprises one or more chemically modified nucleotides.

4. The versikine nucleic acid molecule of claim 3, wherein the one or more chemically modified nucleotides comprises N 1 -methyladenosine (ml A), N6-methyladenosine (m6A). or adenosine to inosine (A-to-I) modification, pseudouracil. Ml -pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphoramidates, 2 -O-Me nucleoside analog, 2’F-RNA modification, 2’-deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F-ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-O-MOE (2’-O- (2-methoxy ethyl)) modification.

5. The versikine nucleic acid molecule of claim 4, comprising 5-methoxyuridine (5moU) or pseudouridine.

6. A pharmaceutical composition comprising: the versikine nucleic acid molecule of any one of claims 1-5, and a lipid nanoparticle.

7. The pharmaceutical composition of claim 6, wherein the versikine nucleic acid molecule is encapsulated in the lipid nanoparticle.

8. The pharmaceutical composition of claim 6 or 7, wherein one or more nucleotides of the versikine nucleic acid molecule comprises one or more chemically-modified nucleotides.

9. The pharmaceutical composition of claim 8. wherein the one or more chemically- modified nucleotides comprises N1 -methyladenosine (mlA), N6-methyladenosine (m6A), or adenosine to inosine (A-to-I) modification, pseudouracil, Ml-pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine. phosphorothioate (PS) linkage modification. N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphoramidates, 2’-0-Me nucleoside analog, 2’F-RNA modification, 2’-deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F-ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2'-0-M0E (2’-O- (2-methoxyethyl)) modification.

10. The pharmaceutical composition of any one of claims 6-9, wherein the lipid nanoparticle comprises ALC-0315, 1,2 DSPC, cholesterol, ALC-0159, SM-102, DMG-PEG(2000), D-Lin-MC3-DMA. or any combinations thereof.

11. The pharmaceutical composition of any one of claims 6-10, wherein the lipid nanoparticle comprises SM-102, 1,2 DSPC, cholesterol, and DMG-PEG(2000) at a molar ratio of 50: 10:38.5: 1.5, respectively.

12. An isolated cell comprising the versikine nucleic acid molecule of any one of claims 1-5.

13. A method for encapsulating the versikine nucleic acid molecule of any one of claims 1-5 in a lipid nanoparticle, the method comprising:(a) generating the nucleic acid molecule by using a transcription vector,(b) mixing the nucleic acid molecule with a plurality’ of lipid nanoparticles, thereby- forming lipid nanoparticles encapsulating the nucleic acid molecule.

14. The method of claim 13, wherein the generating step (a) comprises using a transcription buffer comprising ATP, CTP, GTP, UTP, or a chemically modified nucleotide.

15. The method of claim 14, wherein the chemically modified nucleotide comprises Nl- methyladenosine (mlA). N6-methyladenosine (m6A). or adenosine to inosine (A-to-I) modification, pseudouracil, Ml -pseudouracil, 5-methoxyuridine (5moU), orN4- acetylcytidine, phosphorothioate (PS) linkage modification, N3’ phosphoramidate (NP) linkage modification, Boranophosphate intemucleotide linkage modification, Phosphonoacetate (PACE) linkage modification, Morpholino phosphorami dates, 2'- O-Me nucleoside analog. 2’F-RNA modification, 2’-deoxy-2’-fluoro-P-D-arabino nucleic acid (2’F-ANA) modification, Locked nucleic acid (LNA), unlocked nucleic acid (UNA) modification, or 2’-0-M0E (2’-O-(2-methoxy ethyl)) modification.

16. The method of claim 13, wherein a lipid nanoparticle of the plurality of lipid nanoparticles comprises ALC-0315, 1,2 DSPC, cholesterol, ALC-0159, SM-102, DMG-PEG(2000), D-Lin-MC3-DMA, or any combinations thereof.

17. The method of claim 16, wherein the lipid nanoparticle comprises SM-102, 1,2 DSPC, cholesterol, and DMG-PEG(2000) at a molar ratio of 50:10:38.5:1.5, respectively.

18. The method of claim 13. wherein a lipid nanoparticle of the plurality of lipid nanoparticles comprises phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), dimyristoyl phosphatidylglyerol (DMPG) and sphingomyelin (SM), l,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2- dioleoyloxy-3-dimethylammoniumpropane (DODAP) and analogues, l,2-di-(9Z- octadecenoyl)-sn-glycero-3 -phosphoethanol amine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3-phosphocholine (DOPC), phosphatidy lcholine (PC) and dimyristoyl phosphatidylcholine (DMPC), 1.2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA). l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), dioctadecyldi- methylammonium (DODA(Br)ZDDAB), dioctadecyldimethylammoniumchloride (DODAC), 1 ,2-dimyristoyloxypropyl-l ,3-dimethylhy droxy ethylammonium (DMRIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l- propanamium trifluoroacetate (DOSPA) analogues, cholesterol, sphingomyelin (SM), or any combinations thereof.

19. A method for sensitizing a response to an immunotherapy in a subject in need thereof, the method comprising: administering the versikine nucleic acid molecule of any one of claims 1-5, or the pharmaceutical composition of any one of claims 6-11 and an immunotherapeutic agent.

20. The method of claim 19. wherein the immunotherapy comprises an anti-IL4 immunotherapy.

21. The method of any one of claims 19-20, wherein the administering comprises delivery of the versikine nucleic acid molecule or pharmaceutical composition into a cell.

22. The method of claim 21, wherein the cell is a dendritic cell.

23. The method of claim 21, wherein the cell is an antigen-presenting cell.

24. The method of claim 21, wherein the delivery of the nucleic acid molecule comprises a virus-based delivery.

25. The method of claim 24. wherein the virus-based delivery is a lentivirus or an adenovirus.

26. The method of any one of claims 19-25, wherein the subject has a tumor.

27. The method of claim 26, w herein the tumor is a refractory tumor.

28. A method for treating cancer in a subject in need thereof, the method comprising: administering a versikine nucleic acid molecule of any one of claims 1-5 or the pharmaceutical composition of any one of claims 6-11, thereby treating the cancer.

29. The method of claim 28, wherein the subject is co-administered one or more agents that target an IL-4 pathway.

30. The method of claim 29, wherein the one or more agents that target the IL-4 pathway comprise dupilumab or pasolizumab.

31. The method of claim 30, wherein the one or more agents that target the IL-4 pathway is pitrakinra.

32. The method of claim 29. wherein the co-administration comprises administering the one or more agents that target the IL-4 pathway prior to administering the versikine nucleic acid molecule or the pharmaceutical composition.

33. The method of claim 29, wherein the co-administration comprises administering the one or more agents that target the IL-4 pathway after administering the versikine nucleic acid molecule or the pharmaceutical composition.

34. The method of claim 29, wherein the co-administration comprises administering the one or more agents that target the IL-4 pathway and the versikine nucleic acid molecule or the pharmaceutical composition at the same time.

35. The method of claim 28. wherein the subject is co-administered one or more agents that inhibit PD-1 and / or PD-L1.

36. The method of claim 35, wherein one or more agents that inhibit PD-1 and / or PD-L1 comprise an antibody inhibitor.

37. The method of claim 36, wherein the antibody inhibitor comprise cemiplimab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, or any combination thereof.

38. The method of claim 35, wherein the co-administration comprises administering the one or more agents that inhibit PD-1 and / or PD-L1 and the versikine nucleic acid molecule or the pharmaceutical composition prior to, after, and / or while simultaneously administering the versikine nucleic acid molecule or the pharmaceutical composition.

39. The method of any one of claims 28-38, wherein the cancer is a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer.

40. The method of claim 39, wherein the cancer is solid tumor.

41. The method of any one of claims 28-40, wherein the tumor is a refractory tumor.

42. The method of any one of claims 28-41, wherein the administering of the versikine nucleic acid molecule or the pharmaceutical composition comprises, buccal, dermal, enteral, intra-arterial, intradermal, intragastric. intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-tumoral, mucosal, nasal, oral, subcutaneous, or sublingual administration.

43. The method of claim 42, wherein administering comprises intra-tumoral or intravenous administration.

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