WO2023023467A1 - Nucleic acid molecules and methods of using the same - Google Patents

Nucleic acid molecules and methods of using the same Download PDF

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Publication number
WO2023023467A1
WO2023023467A1 PCT/US2022/074905 US2022074905W WO2023023467A1 WO 2023023467 A1 WO2023023467 A1 WO 2023023467A1 US 2022074905 W US2022074905 W US 2022074905W WO 2023023467 A1 WO2023023467 A1 WO 2023023467A1
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nucleic acid
cell
acid molecule
receptor
protein
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PCT/US2022/074905
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French (fr)
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James Andorko
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Interius Biotherapeutics, Inc.
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Publication of WO2023023467A1 publication Critical patent/WO2023023467A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18211—Henipavirus, e.g. hendra virus
    • C12N2760/18222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • nucleic acid molecules are provided.
  • the nucleic acid molecule comprises a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal repeat (LTR) bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5 ’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein; a polynucleotide sequence encoding a central polypurine tract (cPPT); a polynucleotide sequence encoding a second constitutive promoter; a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.); and a polynucleotide encoding a posttranscriptional regulatory element.
  • the nucleotide sequences are operatively connected one another.
  • composition comprising the nucleic acid molecules provided herein are provided.
  • methods of delivering the molecule to a cell comprise contacting a cell with a nucleic acid sequence as provided herein into a cell of the subject or into the subject and said nucleic acid sequence is taken up by the cell in the subject.
  • methods of preparing a virus comprising a nucleic acid molecule encoding a target molecule are provided.
  • the methods comprise contacting a cell with the nucleic acid molecule as provided herein under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule.
  • methods of treating a disease in a subject comprise administering to the subject the virus prepared according to the methods provided herein.
  • the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
  • FIG. 1 illustrates a non-limiting schematic of a nucleic acid molecule, such as a plasmid, as described herein.
  • FIG. 2 illustrates a non-limiting schematic of a nucleic acid molecule, such as a plasmid, as described herein.
  • FIG. 3 illustrates non-limiting embodiments of a nucleic acid molecule, such as a plasmid, as described herein.
  • FIG. 4 illustrates non-limiting embodiments of a nucleic acid molecule, such as a plasmid, as described herein.
  • FIG. 5 illustrates non-limiting embodiments of a nucleic acid molecule, such as a plasmid, as described herein.
  • FIG. 6 illustrates viral particles produced from the nucleic acid molecules as provided for herein transducing human peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • FIGS. 6A-6B illustrate viral particles derived from the nucleic acid molecules of the present disclosure and pseudotyped with VSV-G delivering and expressing GFP and CAR-2A- NGFR transgenes, respectively.
  • FIGS. 6C-6D illustrate the ability of viral particles derived from the nucleic acid molecules of the as provided for herein and pseudotyped with Nipah virus structural proteins and a scFv that binds to a cell surface protein on a PBMC to deliver and express GFP and CAR-2A- NGFR transgenes, respectively.
  • FIGS. 6E-6F illustrate the ability of viral particles derived from the nucleic acid molecules of the present disclosure and pseudotyped with Nipah virus structural proteins and a VHH that binds to a cell surface protein on a PBMC to deliver and express GFP and CAR-2A- NGFR transgenes, respectively.
  • a nucleic acid molecule comprising: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal repeat (LTR) bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5 ’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein and/or a binder molecule; a polynucleotide sequence encoding a central polypurine tract (cPPT); a polynucleotide sequence encoding a second promoter; a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.); and a polynucleotide encoding a posttranscriptional regulatory element.
  • LTR long terminal repeat
  • nucleic acid molecule of embodiment 2, wherein the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
  • nucleic acid molecule of any one of embodiments 1-4, wherein the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
  • polycistronic e.g., bicistronic, tricistronic, quadci str onic
  • the constitutive promoter is selected from the group consisting of EF-1 alpha, Rous Sarcoma virus (RSV), SV-40, and Mason-Pfizer monkey virus-CTE.
  • tissue-specific promoter is selected from the group consisting of B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
  • tissue-specific promoter is selected from the group consisting of ZAP70 and CD4.
  • nucleic acid molecule of any one of embodiments 1-9 wherein the sequence encoding the second promoter encodes a constitutive promoter, an inducible promoter, or a tissue specific promoter.
  • nucleic acid molecule of embodiment 10, wherein the constitutive promoter is selected from the group consisting of EF-1 alpha, Rous Sarcoma virus (RSV), SV-40, and Mason-Pfizer monkey virus-CTE.
  • tissue-specific promoter is selected from the group consisting of B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
  • tissue-specific promoter is selected from the group consisting of ZAP70 and CD4.
  • CAR chimeric antigen receptor
  • nucleic acid molecule of embodiment 14, wherein the reporter gene is a fluorescent protein or an enzyme.
  • nucleic acid molecule of embodiment 16 wherein the cleavable linker is a P2A, T2A, E2A, or F2A linker.
  • nucleic acid molecule of embodiment 16 wherein the glycine linker comprises one more of the repeat of GGGGS, GSG, or any combination thereof.
  • nucleic acid molecule of embodiment 4 wherein the sequence encoding the intermediate elements encodes an Internal Ribosome Entry Site (IRES).
  • IRS Internal Ribosome Entry Site
  • nucleic acid molecule of embodiment 22 wherein the sequence encoding the partial gag encodes for the HIV-1 psi element, rev response element (RRE), and a Gp41 peptide.
  • Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R;
  • GPRC5D Guanylyl cyclase C (GCC); Heat shock protein 70-2 mutated (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexasaccharide portion of globoH glycoceramide (GloboH); High molecular weight-melanoma associated antigen (HMWMAA); HIV1 envelope glycoprotein; HLA; HLA-DOA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DM; HLA-DOB; HLA-DP; HLA-DQ; HLA-DR; HLA-G; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human Telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; IL1 IRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza
  • HIV-1 psi element, rev response element (RRE), and a Gp41 peptide d. a polynucleotide encoding a central polypurine tract (cPPT); e. a polynucleotide encoding an EF-1 alpha promoter; f. a polynucleotide encoding a first target molecule; g. optionally a polynucleotide encoding a first linker or first intermediate element; h. optionally a polynucleotide encoding a second target molecule; i.
  • cPPT central polypurine tract
  • WBV Woodchuck Hepatitis Virus
  • n is any integer (e.g., 0, 1, 2, 3, 4).
  • nucleic acid molecule of embodiment 28 wherein the nucleic acid molecule comprises one or more polynucleotides encoding one or more target molecules (e.g., n is 1, 2, 3, 4).
  • nucleic acid molecule of embodiment 29 wherein the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
  • nucleic acid molecule of any one of embodiments 29-32, wherein the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
  • CAR chimeric antigen receptor
  • nucleic acid molecule of embodiment 34 wherein the reporter gene is a fluorescent protein or an enzyme.
  • nucleic acid molecule of embodiment 34 wherein at least one of the first, second, or more target molecules encodes a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • nucleic acid molecule of embodiment 34 wherein the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte; Her2/Neu+ cancer cell; NKG2D+ natural killer cell; SLC1A3+ astrocyte; SLC7A10+ adip
  • Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R;
  • Lewis(Y) antigen Lewis(Y) antigen; Lews Ag; Livl; Locus K 9 (LY6K); Low conductance chloride channel; Lymphocyte antigen 6 complex; Lymphocyte antigen 75 (LY75); Lymphocyte-specific protein tyrosine kinase (LCK); Mammary gland differentiation antigen (NY-BR-1); Melanoma antigen recognized by T cells 1 (MelanA or MARTI); Melanoma- associated antigen 1 (MAGE-A1); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT- 2); Melanoma inhibitor of apoptosis (ML-IAP); Mesothelin; MPL; Mucin 1 cell surface associated (MUC1); N-Acetyl glucosaminyl-transf erase V (NA 17); Nectin-4; Neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-ace
  • Prostase Prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Prostatic acid phosphatase (PAP); Prostein; Protease Serine 21 (Testisin or PRSS21); Proteasome (Prosome Macropain) Subunit Beta Type 9 (LMP2); PTK7; Ras G12V; Ras Homolog Family Member C (RhoC); Rat sarcoma (Ras) mutant; Receptor for Advanced Gly cation Endproducts (RAGE-1); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Receptor tyrosine-protein kinase ERBB2 or Her-22/neu; Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); Sarcoma translocation breakpoints; Serine 2 (TMPRSS2) ETS fusion gene; Sialyl Lewis adhesion molecule (sLe
  • nucleic acid molecule of any one of embodiments 28-40, wherein the glycine linker comprises one more of the repeat of GGGGS, GSG, or any combination thereof.
  • IRS Internal Ribosome Entry Site
  • nucleic acid molecule of embodiment 28, wherein a)-k) are operatively connected to each other in a 5’ to 3’ direction.
  • nucleic acid molecule of embodiment 44 wherein a 3’ end of a) is operatively connected to 5’ end of b); a 3’ end of b) is operatively connected to a 5’ end of c); a 3’ end of c) is operatively connected to a 5’ end of d); a 3’ end of d) is operatively connected to a 5’ end of e); a 3’ end of e) is operatively connected to a 5’ end of f); optionally a 3’ end of f) is operatively connected to a 5’ end of g); optionally a 3’ end of g) is operatively connected to a 5’ end of h); optionally a 3’ end of h) is operatively connected to a 5’ end of i); optionally a 3’ end of i) is operatively connected to a 5’ end of j); a 3’ end of f) or optionally j) is operatively operative
  • nucleic acid molecule of embodiment 1-46 wherein the nucleic acid molecule is a non-integrating plasmid.
  • a host cell comprising the nucleic acid molecules of any one of embodiments 1-47.
  • a pharmaceutical composition comprising the host cell of embodiment 48.
  • a pharmaceutical composition comprising the nucleic acid molecules of any one of embodiments 1-47.
  • a method of delivering a molecule to a cell comprising contacting a cell with a nucleic acid sequence of any one of embodiments 1-47 into a cell of the subject or into the subject and said nucleic acid sequence is taken up by the cell in the subject.
  • nucleic acid sequence is introduced into the cell or subject by electroporation, injection, sonication, transfection, transduction, gene guns, encompassed by nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell.
  • a method of preparing a virus comprising a nucleic acid molecule encoding a target molecule comprising contacting a cell with the nucleic acid molecule of any one of embodiments 1-47 under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule.
  • the cell is a producer cell (e.g., HEK293, HEK293T, HEK293FT).
  • pseudotyped lentivirus is pseudotyped with a human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, immunodeficiency virus in simian (SIV), maedi / visna virus, Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, or SARS-CoV virus.
  • HAV-1 human immunodeficiency virus
  • HIV-2 HIV-2
  • equine infectious anemia virus immunodeficiency virus in simian
  • SIV immunodeficiency virus in simian
  • maedi / visna virus Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus
  • a method of treating a disease in a subject comprising administering to the subject the virus prepared according to any one of embodiments 53-58 to treat the disease.
  • the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
  • Embodiments provided for herein provide a platform that can, for example, allow for the integration of foreign nucleotides that can be expressed as immunogens or target molecules.
  • the expression of immunogens or molecules can be used, for example, in methods to prevent or treat cancer and/or infectious diseases.
  • nucleic acid molecules provided herein can be used to assemble a number of DNA fragments into one nucleic acid molecule. This can be done, for example, in spite of restriction sites redundancy found at the ends and within the DNA fragments.
  • the nucleic acid molecules provided herein can also comprise a nucleus uptake component, such as a nuclear localization signal (domain) that can facilitate the rapid plasmid integration or uptake within the nucleus.
  • the nucleic acid molecules provided for herein can cross the cell membrane and transfects the nucleus. Once that process occurs the foreign nucleotide is then expressed by the host’s cell transcription and translation process.
  • a “foreign nucleotide” is one that is introduced into the cell and is not native to the cell’s genome. Examples include any of the nucleic acid molecules provided for herein and examples also include plasmids or other types of vectors provided herein. After translation, the antigen when it has reached the cell surface can be presented in conjunction with a major histocompatibility complex protein class I or class II.
  • the antigen presenting cell such as a macrophage or dendritic cell, would take that foreign antigen and travel to a lymph node where the APC will present the antigenic target protein and/or fragment which then lead to an immune response to the antigen encoded for by the nucleic acid molecules provided herein.
  • APC antigen presenting cell
  • the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
  • the present disclosure modifies certain terms or values with the term “about,” however, the disclosure should also be understood to disclose the exact value as well and is simply not written out for convenience. For example, the phrase “about 9 to about 25” also discloses “9 to 25.” Additionally, a range, such the phrase “from X to Y” where X and Y are any integer includes the endpoints. For example, the phrase “from 1 to 5” means 1, 2, 3, 4, or 5.
  • administering when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient.
  • methods of administration that can be used to administer nucleic acid molecules, include, but are not limited to, transfection, electroporation, injection, sonication, or by any method in combination with other known techniques. Such combination techniques include heating and radiation.
  • the nucleic acid molecule is delivered to a muscle cell. This can be done, for example, by electroporation or other suitable technique. Electroporation of the nucleic acid molecule to the muscle or other tissue type can be done, for example, using an electroporation device.
  • the term “animal” as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals.
  • cloning is used in reference to the ligating process of a nucleic acid molecule into another nucleic acid molecule, such as a plasmid.
  • the cloned molecule can then be transferred into a host cell or subject for duplication, amplification, or administration.
  • cloning vector and “cloning vector plasmid” are used to refer to a circular DNA plasmid which contains in minimum an “origin of replication” (“ori”).
  • the origin of replication is the site of replication initiation.
  • the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the term “genetic construct” refers to the DNA or RNA molecule that comprises a nucleotide sequence which encodes the target molecule and which includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells.
  • the nucleic acid molecules provided herein are in the form of a plasmid or viral vector.
  • the genetic construct is a plasmid or a viral vector. In some embodiments, the genetic construct does not contain integration elements.
  • the plasmid does not contain, or is free of, any long terminal repeats, or inverted terminal repeats, or other sequences that would facilitate the integration of the plasmid into the subject’s, or cell’s, genome.
  • the plasmid is a non-integrating plasmid.
  • a non-integrating plasmid is a plasmid that is not designed to integrate into a genome of the subject or a cell that comes into contact the plasmid.
  • the non-integrating plasmid is contacted with a cell without any other components that would facilitate the integration of the plasmid into a genome.
  • FIG.l illustrates a non-limiting configuration of a nucleic acid molecule as described herein. Referring to FIG.
  • first promoter region that comprises, for example and without limiting, a RSV promoter that is operatively connected to a first element (El) that comprises a 5’ LTR, as provided for herein, that is operatively connected to a second element (E2) that comprises a polynucleotide encoding a viral structural protein, such as a gag polynucleotide, for example and without limiting, a partial gag polynucleotide, as provided for herein, that is operatively connected to a third element (E3) that comprises a central polypurine tract (cPPT), as provided for herein, that is operatively connected to a second promoter (P2) that comprises, for example and without limiting, an EF-1 alpha promoter that is operatively linked to a first target molecule (TM1), as provided for herein, optionally that is operatively connected to a first linker or intermediate element (L1/IE1), as provided for herein, optionally that
  • FIG.2 illustrates a non-limiting configuration of a nucleic acid molecule as described herein.
  • a first promoter region Pl
  • Pl a RSV promoter that is operatively connected to a first element (El) that comprises a 5’ LTR, as provided for herein, that is operatively connected to a second element (E2) that comprises a polynucleotide encoding a viral structural protein, such as a gag polynucleotide, for example and without limiting, a partial gag polynucleotide, as provided for herein, that is operatively connected to a third element (E3) that comprises a central polypurine tract (cPPT), as provided for herein, that is operatively connected to a second promoter (P2) that comprises, for example and without limiting, an EF-1 alpha promoter that is operatively linked to a first target molecule (TM1), as provided for herein,
  • TM1 first target molecule
  • DNA construct refers to a DNA molecule that is synthesized by the cloning steps that are consecutive with a cloning vector plasmid. This is the process that is commonly used as a means to direct gene expression to an appropriate mammalian host. This mammalian host could be cells that have been cultured in vitro or transgenic mice in vivo.
  • DNA fragment refers to any DNA molecule isolation that includes but is not limited to the different parts of the plasmid such as the intron, exon, reporter gene, poly(A) tail, and the different cloning sites. These DNA fragment could also include signal nucleotides, such as, the mRNA stabilization signal and the nuclear localization signal. Plasmid vector can comprise of natural and synthetic DNA fragments.
  • pellet region refers to the sequence of nucleotides that is not required for targeted gene expression, but is designed to increase the gene expression levels.
  • the term “expressible form” refers to gene constructs which contain the necessary regulatory elements operably linked to a coding sequence that encodes a target molecule, such that when present in the cell of the individual, the coding sequence will be expressed.
  • gene promoter or “promoter” as used herein refer to and is in reference to a sequence of nucleotides that is required for gene expression.
  • the term “genetic vaccine” refers to a pharmaceutical preparation that comprises a genetic construct that comprises a nucleotide sequence that encodes a target molecule and can include pharmaceutical preparations useful to invoke a therapeutic immune response.
  • the nucleotide sequence encodes a shRNA, siRNA, antisense, antibodies, hormones, and the like.
  • Other target molecules can also be encoded for as described herein, such as, but not limited to, adjuvants.
  • the term “genetic therapeutic” refers to a pharmaceutical preparation that comprises a genetic construct that comprises a nucleotide sequence that encodes a therapeutic or compensating protein.
  • inhibiting includes the administration of a plasmid or nucleic acid molecule to prevent the onset of the symptoms, alleviate the symptoms, reduce the symptoms, delay or decrease the progression of the disease and/or its symptoms, or eliminate the disease, condition or disorder.
  • ORI refers to sequences of nucleotides that can direct or lead to host cell duplication of a plasmid.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • poly(A) tail is in reference to the nucleotide sequence of adenine (A) nucleotides. These nucleotides are usually found at the terminal end of the messenger RNA molecule (mRNA). The poly(A) tail is incorporated at the 3’ of the end of the DNA construct that allows for enhancement of the gene expression of interest.
  • the nucleic acid molecules provided herein comprise a poly(A) tail.
  • tags refer to sequences of nucleotides that encode a protein or peptide region that is unique, which allow for it to be detected and distinguished from any endogenous counterpart.
  • Non-limiting examples of tags include His tag, GST tag, Calmodulin Binding Protein (CBP), Maltose-binding protein (MBP), myc tag, HA tag, FLAG tag, and the like.
  • target molecule can refer to a molecule or a protein against which an immune response can be elicited and is desired to be elicited against.
  • the target molecule e.g., a target protein
  • the immune response directed against the target molecule can be used to induce the immune response that will protect the individual against and treat the individual for the specific infection or disease with which the target molecule is associated.
  • the target molecule is a cell surface protein or a protein (antigen) that is secreted from the cell.
  • a “target molecule” can also refer to a protein that is expressed in, or secreted from, a cell.
  • the target molecule e.g., a target protein
  • nucleic acid molecules described herein can also be used to express different types of target molecules, such as nucleic acid molecules provided for herein, which includes, but are not limited to shRNA, siRNA, antisense, microRNAs, and the like.
  • the term “binder molecule” refers to any polypeptide or polynucleotide that may be used to bind a desired target.
  • the target is a cell, such as but not limited to an immune cell, and the binder molecule binds to the cell.
  • the target is a specific protein and the binder molecule binds to said protein.
  • Non-limiting examples of binder molecules and targets are provided for herein.
  • cytokine generally refers to proteins that are important in cell signaling and act through receptors.
  • the nucleic acid molecule comprises a sequence encoding a cytokine.
  • cytokines include IL-1, IL- 1 -like, IL- 1 alpha, IL-1 beta, IL-IRA, IL-18, CD132, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, CD131, IL-3, IL-5, GM-CSF, IL-6-like, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL- 14, IL- 16, IL- 17, IFN-alpha, IFN-beta, IFN-gamma, CD 154, LT-beta, TNF-alpha, TNF-beta, 4- 1BBL, ARIL, CD
  • the nucleic acid molecule comprises a sequence encoding a cytokine selected from the group comprising IL-1, IL- 1 -like, IL-1 alpha, IL-1 beta, IL-IRA, IL-18, CD132, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, CD131, IL-3, IL-5, GM- CSF, IL-6-like, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-alpha, IFN-beta, IFN-gamma, CD154, LT-beta, TNF-alpha, TNF-beta, 4-1BBL, ARIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-beta
  • terapéutica means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient.
  • embodiments are directed to the treatment of cancer or the decrease in proliferation of cells.
  • embodiments are directed to the treatment of infections or infectious agents.
  • a “therapeutically effective amount” or “effective amount” of a therapeutic is a predetermined amount calculated to achieve the desired effect, /. ⁇ ., stimulate an immune response.
  • the activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate.
  • treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results.
  • untranslated region refers to the sequences of nucleotides that cover the nucleotide region that does not code for a protein found within a mRNA molecule. These regions that are not translated can be found at the 5’ and 3’ regions of the mRNA molecule.
  • the nucleic acid molecule provided herein that encodes a target molecule or a fragment of the target molecule comprises an untranslated region.
  • the nucleic acid molecule and methods disclosed herein can be utilized with or on a subject in need of such treatment, which can also be referred to as “in need thereof.”
  • the phrase “in need thereof’ means that the subject has been identified as having a need for the particular method or treatment and that the treatment has been given to the subject for that particular purpose with a specific intent.
  • the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a target molecule. In some embodiments, the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a viral structural protein that is operatively connected to a polynucleotide encoding a promoter that is operatively linked to a polynucleotide encoding a target molecule.
  • the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a binder molecule that is operatively connected to a polynucleotide encoding a promoter that is operatively linked to a polynucleotide encoding a target molecule.
  • the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a viral structural protein and a binder molecule that is operatively connected to a polynucleotide encoding a promoter that is operatively linked to a polynucleotide encoding a target molecule.
  • the nucleic acid sequence comprises: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein, a polynucleotide sequence encoding a central polypurine tract, a polynucleotide sequence encoding a second constitutive promoter, a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.), and a polynucleotide encoding a posttranscriptional regulatory element.
  • target molecules e.g. protein of interest, polypeptide, miRNA, shRNA, etc.
  • the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
  • the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
  • the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another.
  • the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
  • the nucleic acid sequence comprises: a sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, and optionally a sequence encoding an amino acid sequence of a second target molecule, wherein the sequences are operatively connected to one another.
  • the nucleic acid sequence comprises: a sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, and a sequence encoding an amino acid sequence of a third target molecule, wherein the sequences are operatively connected to one another.
  • the nucleic acid sequence comprises: a sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, a sequence encoding an amino acid sequence of a third target molecule, a sequence encoding an amino acid sequence of a third linker or intermediate element, and a sequence encoding an amino acid sequence of a fourth target molecule, wherein the sequences are operatively connected to one another.
  • the nucleic acid sequence comprises a polycistronic expression system.
  • the polycistronic expression system is a bicistronic expression system.
  • the polycistronic expression system is a tricistronic expression system.
  • the polycistronic expression system is a quadcistronic expression system.
  • the bicistronic expression system comprises a sequence encoding an amino acid sequence of a first target molecule operatively connected to a sequence encoding an amino acid sequence of a first linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a second target molecule.
  • the tricistronic expression system comprises a sequence encoding an amino acid sequence of a first target molecule operatively connected to a sequence encoding an amino acid sequence of a first linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a second target molecule operatively connected to a sequence encoding an amino acid sequence of a second linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a third target molecule.
  • the quadcistronic expression system comprises a sequence encoding an amino acid sequence of a first target molecule operatively connected to a sequence encoding an amino acid sequence of a first linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a second target molecule operatively connected to a sequence encoding an amino acid sequence of a second linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a third target molecule operatively connected to a sequence encoding an amino acid sequence of a third linker or intermediate element operatively linked to a sequence encoding an amino acid sequence of a fourth target molecule.
  • the polycistronic expression system comprises one or more sequence encoding an amino acid sequence of one or more target molecule operatively linked to one or more sequence encoding an amino acid sequence of a one or more linker or intermediate element.
  • the one or more sequence encoding one or more target molecule repeats “n” times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats “n-1” times.
  • “n” is an integer (e.g., 0, 1, 2, 3, 4).
  • the one or more sequence encoding one or more target molecules repeats 1 time, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 0 times.
  • the one or more sequence encoding one or more target molecules repeats 2 times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 1 time. In some embodiments, the one or more sequence encoding one or more target molecules repeats 3 times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 2 times. In some embodiments, the one or more sequence encoding one or more target molecules repeats 4 times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 3 times.
  • the first, second, third, fourth, or more polypeptides encoding the target molecule are different. In some embodiments, the first, second, third, fourth, or more polypeptides encoding the target molecule are the same. In some embodiments, the first, second, third, fourth, or more polypeptides encoding the first, second, third, fourth, or more target molecule are different. In some embodiments, the first, second, third, fourth, or more polypeptides encoding the first, second, third, fourth, or more target molecule are the same. In some embodiments, the first, second, third, fourth, or more target molecules are different. In some embodiments, the first, second, third, fourth, or more target molecules are the same.
  • the first, second, third, or more polypeptides encoding the linker or intermediate element are different. In some embodiments, the first, second, third, or more polypeptides encoding the linker or intermediate element are the same. In some embodiments, the first, second, third, or more polypeptides encoding the first, second, third, or more linker or intermediate element are different. In some embodiments, the first, second, third, or more polypeptides encoding the first, second, third, or more linker or intermediate element are the same. In some embodiments, the first, second, third, or more linker or intermediate element are different. In some embodiments, the first, second, third, or more linker or intermediate element are the same.
  • the nucleic acid sequentially comprises in a 5’ to 3’ direction: a. a polynucleotide encoding a RSV promoter; b. a polynucleotide encoding a 5’ LTR; c. a polynucleotide encoding a partial gag, wherein the partial gag further comprises HIV-1 psi element, rev response element (RRE), and a Gp41 peptide; d. a polynucleotide encoding a central polypurine tract (cPPT); e. a polynucleotide encoding an EF-1 alpha promoter; f.
  • a polynucleotide encoding a RSV promoter b. a polynucleotide encoding a 5’ LTR
  • a polynucleotide encoding a first target molecule g. optionally a polynucleotide encoding a first linker or first intermediate element
  • the nucleic acid molecule comprises one or more polynucleotides encoding one or more target molecules (e.g., n is 1, 2, 3, 4).
  • the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
  • the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
  • the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another.
  • the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci stronic) expression system.
  • the sequence encoding the first, second, or more target molecule encodes a reporter gene, a protein of interest, a chimeric antigen receptor (CAR), an armored CAR, a DAP-12, a cytokine, a siRNA, a shRNA, an antisense molecule, a microRNA, or a gene editing construct (e.g., CRISPR/CAS system).
  • a)-k) are operatively connected to each other in a 5’ to 3’ direction.
  • a 3’ end of a) is operatively connected to 5’ end of b); a 3’ end of b) is operatively connected to a 5’ end of c); a 3’ end of c) is operatively connected to a 5’ end of d); a 3’ end of d) is operatively connected to a 5’ end of e); a 3’ end of e) is operatively connected to a 5’ end of f); optionally a 3’ end of f) is operatively connected to a 5’ end of g); optionally a 3’ end of g) is operatively connected to a 5’ end of h); optionally a 3’ end of h) is operatively connected to a 5’ end of i); optionally a 3’ end of i) is operatively connected to a 5’ end of j); a 3’ end of f) or optionally j) is operatively connected to a 5’ end of a
  • the nucleic acid molecules provided for herein do not contain or do not comprise (e.g. free of) a sequence encoding a fragment of a target molecule, or alternatively, the nucleic acid molecule only encodes for one fragment of a target molecule if the full length protein is not encoded for by the nucleic acid molecule.
  • the nucleic acid sequence comprises a promoter.
  • the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
  • An “inducible promoter” is a promoter which activity can be regulated.
  • inducible promoters include, but are not limited to, chemically inducible promoters (e.g., tetracycline system, pLac promoter, pBad promoter, AlcA promoter, or LexA promoter), temperature inducible promoters (e.g., Hsp70-derived promoter, or Hsp90-derived promoter), and light inducible promoters (e.g., YFI system).
  • tissue-specific promoter is a promoter that limits the expression or largely limits the expression to a specific tissue type.
  • tissue-specific promoters include, but are not limited to, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (haematopoietic cells), CD68 (macrophages), desmin (muscle), elastase- 1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin (differentiating cells and healing tissue), Flt-1 (endothelial cells), GFAP (astrocytes), GPIIb (megakaryocytes), ICAM-2 (endothelial cells), mouse INF-beta (hematopoietic cells), Mb (muscle), Nphsl (podocytes), OG-2 (osteoblasts and odonblasts), SP-B (lung), SYN1 (neurons), WASP (hem
  • the promoter can also be a cell specific promoter so that the nucleic acid molecule’s expression is limited to a specific cell or subset of cells.
  • the promoter is a constitutive promoter.
  • constitutive promoters include, but are not limited to, CMV, EF-1 alpha, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GALI, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, and U6.
  • promoters that can be used include, but are not limited EF-1 alpha, SV40, Rous Sarcoma virus (RSV), Mason-Pfizer monkey virus-CTE, and CTE+rev.
  • the RSV promoter is encoded by a sequence of tgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaag caccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatg gattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaac (SEQ ID NO: 1)
  • the EF-1 alpha promoter is encoded by a sequence of cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttt tccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttcttttttcgcaacgggtttgccgccag aacacaggtaagtgccgtgtgtggttccgccag aacacaggtaagtgccgtgtgtggttccgcgccag aaca
  • promoters are known and can be incorporated into the nucleic acid molecule. However, this list is merely for example purposes only as there are numerous promoters that can be used to drive the expression of a target molecule (e.g., a protein, RNAi components) from the nucleic acid molecule. As provided for herein, the nucleic acid molecule encoding the promoter is operably connected to the nucleic acid molecule encoding the target molecule to control, regulate, or drive the expression of the target protein in a cell.
  • a target molecule e.g., a protein, RNAi components
  • the nucleic acid molecule comprises a nucleic acid molecule encoding a target molecule.
  • the target molecule is a protein of interest, antibodies, hormones, chimeric antigen receptors (CARs), armored CARs, receptors, binder molecules, DAP- 12, fusion proteins, reporter proteins, GFP, RFP, SEAP YFP, luciferase, betagalactosidase, and the like.
  • these target molecules can be expressed with or without the other elements of the plasmid, such as the linker, and the like.
  • the antibody is a single chain antibody.
  • the antibody is a single domain antibody (sdAb).
  • the antibody is a single chain fragment variable (scFv).
  • reporter gene refers to a polynucleotide sequence encoding a protein product that can generate, under appropriate conditions, a detectable signal that allows detection for indicating the presence and/or quantity of the reporter gene protein product.
  • reporter gene products include, but are not limited to, enzymes, bioluminescent molecules, or fluorescent molecules.
  • the reporter gene product is a GFP, RFP, SEAP YFP, luciferase, beta-galactosidase, and the like.
  • the target molecule can be an antibody that is expressed from the nucleic acid molecule (e.g. plasmid).
  • antibody as used herein is meant in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies and antibody fragments, such as scFv or hexabodies (PLOS Biology
  • humanized antibody is intended to include antibodies having variable region frameworks derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region can be derived from such human sequences, e.g., human germline sequences, or naturally occurring (e.g., allotypes) or mutated versions of human germline sequences.
  • the humanized antibodies may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the binder molecule binds to a cell.
  • the cell is any desired cell.
  • the cell is an immune cell.
  • the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD 19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte
  • the binder molecule is any polypeptide or polynucleotide that may be used to bind a desired target.
  • the binder molecule is any polypeptide, polynucleotide, or fragment thereof that binds to CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3,
  • the TBD binds to CD7. In some embodiments, the TBD binds to CD8. In some embodiments, the TBD is an antibody. It is to be understood that in the context of the present disclosure “antibody” not only refers to a “complete” antibody comprising two identical heavy chains, two identical light chains, and two antigen binding fragments, but also refers to antibodies of any isotype, fragments of antibodies including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, single domain light chain antibodies, bi-specific antibodies, multi-specific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
  • antibody not only refers to a “complete” antibody comprising two identical heavy chains, two identical light chains, and two antigen binding fragments, but also refers to antibodies of any isotype, fragments of antibodies including, but not limited to,
  • the antibody is selected from the group comprising a scFv, Fab, VHH, single domain antibody, and the like. In some embodiments, the antibody is a scFv. In some embodiments the antibody is a Fab. In some embodiments, the antibody is a VHH. In some embodiments, the antibody is a single domain antibody.
  • antibodies are proteins or polypeptides that exhibit binding specificity to a specific antigen.
  • Intact antibodies are heterotetrameric proteins, composed of two light chains and two heavy chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.
  • Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgAi, IgA2, IgGi, IgG2, IgGs and IgG4.
  • antibody fragment means a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, single chain antibody molecules and multispecific antibodies formed from at least two intact antibodies.
  • the antibody can be a single-chain variable fragment (scFv) antibody.
  • a peptide having a specific amino acid sequence can be encoded by different nucleic acid molecules because of the fact that the genetic code is degenerate.
  • the nucleic acid molecule ’s sequence is optimized.
  • the sequence can be optimized based upon codon usage and frequency depending upon the cell type that is being used or the subject that is being administered the nucleic acid molecule. Codon optimization can be useful to maximize protein expression. This can be done by optimizing the codon usage of mRNA sequences for mammalian cells. For example, changing the immunogen gene sequences encoding infectious target proteins used within nucleic acid molecule can be used to increase expression and the expressed protein immunogenicity. Methods of optimizing codon usage are known.
  • nucleic acid molecules provided for herein may have stop codons in the sequence.
  • stop codons could be replaced by degenerate stop codons. Stop codons are known to be U(T)AA, U(T)AG, and U(T)GA.
  • nucleic acid sequences shown above are simply for illustration purposes only and not intended to be limiting to those that encode for the relevant amino acid sequence of the target protein. Additionally, in some embodiments, less than the full length of the target protein is used. In some embodiments, at least 5, 10, 15, or 20 amino acid residues, independently, from the N- and/or C-terminus are not encoded for by the nucleic acid molecule. In some embodiments, the target protein is larger than the fragment of the target protein that is encoded for by the nucleic acid molecule.
  • the proteins encoded by the nucleic acid molecules provided herein comprise conservative substitutions. Conservative substitutions are known to the skilled artisan.
  • the nucleic acid molecule comprises a different nucleic acid molecule comprising a nucleic sequence that encodes for a fragment of the target protein.
  • the nucleic acid sequence comprises a sequence encoding a linker such as, but not limited to a cleavable linker, a glycine-serine or a glycine-alanine linker.
  • the cleavable linker is selected from P2A, T2A, E2A, and F2A.
  • the nucleic acid sequence comprises a sequence encoding a P2A linker.
  • the nucleic acid sequence comprises a sequence encoding a T2A linker.
  • the nucleic acid sequence comprises a sequence encoding a E2A linker.
  • the nucleic acid sequence comprises a sequence encoding a F2A linker.
  • P2A has the sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 3).
  • T2A has the sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 4).
  • E2A has the sequence of GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 5).
  • F2A has the sequence of GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6).
  • the glycineserine linker comprises the sequence of GGGGS (SEQ ID NO: 21), or GSG.
  • the linker can also comprise repeats of this sequence.
  • the linker comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS (SEQ ID NO: 21), or GSG repeats.
  • the linkers can also be mixed with one another.
  • the nucleic acid molecules provided for herein do not contain or do not comprise (e.g. free of) a sequence encoding a linker.
  • the nucleic acid sequence comprises a sequence encoding an intermediate element, such as, but not limited to an Internal Ribosome Entry Site (IRES).
  • IRES is encoded by a sequence of: gagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaag gtctgtgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcag gcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaggc ggcacacaaccccagtgccacgtttttt
  • the nucleic acid sequence comprises a sequence encoding a 5’ LTR and 3’ LTR.
  • 5’ LTR and 3’ LTR elements comprise an R region and a U5 region.
  • 5’ LTR is encoded by a sequence of: gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataa agcttgccttgagtgcttcaagtagtgtgtgccccgtctgtgtgtgtgactctggtaactagagatccctcagacccttttagt cagtgtggaaatctctagca (SEQ ID NO: 8)
  • 3’ LTR is encoded by a sequence of: tggaag
  • the nucleic acid sequence comprises a sequence encoding a viral structural protein.
  • the viral structural protein is encoded by a gag element.
  • the gag element is a partial gag element.
  • the partial gag element comprises a HIV-1 psi element, a rev response element (RRE), and a Gp41 peptide.
  • the partial gag element is encoded by a sequence of: cgaacagggacttgaaagcgaaagggaaaccagaggagctctcgacgcaggactcggcttgctgaagcgcgc acggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagag atgggtgcgtcagtattaagcgggggagaattagatcgcgatgggaaaaattcggttaaggccagggg gaaagaaaaaatataaattaaaacatatagtatgggcaagggagctagaacgattcgcagttaatcctggctgt tagaaacatcagaaggctgtagacaaatactgggacagctggctgt tagaaacatcagaagg
  • the nucleic acid sequence comprises a sequence encoding a central polypurine tract (cPPT).
  • the cPPT is encoded by a sequence of: tagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtgga tatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatg gccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgctgttggtgggcgg ggatcaagtactacagttaaggccgctgtggtgggcgg ggatcaagcaggagcaggaatttggcatgg ggatcaagcaggaattttgg
  • the nucleic acid sequence comprises a sequence encoding a posttranscriptional regulatory element.
  • the posttranscriptional regulatory element is a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).
  • the WPRE is encoded by a sequence of: atcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct gcttatgtggatacgct gcttatcatgctattgctttcccgtatggcttttcattttctcctcttgtataaatcctggttgctgtctctttat gaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgtgcactgtgttttgctgacgcaacccccactggttgggggg cattgccaccaccacctgtcagctccctttccgggactttttttcccctct
  • the nucleic acid sequence comprises a sequence encoding a polyadenylation signal.
  • the polyadenylation signal is an SV40 poly(A) signal.
  • the SV40 poly(A) signal is encoded by a sequence of: aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcat tctagttgtggtttgtccaaactcatcaatgtatctta (SEQ ID NO: 13)
  • the nucleic acid sequence comprises a sequence encoding an antibiotic resistance gene.
  • the antibiotic resistance gene confers resistance to an antibiotic selected from the group comprising kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.
  • the nucleic acid comprises a sequence encoding an ampicillin resistance gene.
  • the ampicillin resistance gene is encoded by a sequence of: atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctg gtgaaagtaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagat ccttgagagttttcgccccgaagaacgttttccaatgatgagcactttttaaagttctgctatgtggcgcggtattatcccgt attgacgccgggcaagagcaactcggtcgcgcgcatacactattctcagaatgacttggtcgcg
  • the ampicillin resistance gene is encoded by a sequence of: cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttca ataatattgaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttttgccttttttgcggcatttttgcctttttgttttgctcacccagaaacgctggtgaaagtaaagatgctgaagatcagtttgggtgcacgagtgggttacatcgaact ggatctcaacagcggtaagatccttgagagtttttcgccccg
  • the nucleic acid molecule comprises an origin of replication.
  • the origin of replication is a Fl origin of replication (ori).
  • the Fl ori is encoded by a sequence of: acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcg ccctagcgcccccgctcctttcgcttttctccctttctcgccacgttcgccggctttccccgtcaagctctaaatcggg ggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggtgatggtttcacgtagt gggccatcgccccttttacggcacctcg
  • the nucleic acid molecule comprises an SV40 origin of replication (ori).
  • the SV40 ori is encoded by a sequence of: atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc (SEQ ID NO: 17)
  • the nucleic acid molecule comprises a safety switch.
  • the safety switch is an inducible suicide gene, such as and without limiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD), Fas ligand, or cytochrome P450.
  • safety switch can include accessory tags. Non-limiting examples of accessory tags include a c-myc tag, CD20, CD52 (Campath), truncated EGFR gene (EGFRt) or a part or a combination thereof.
  • sequence encoding a target molecule may be operably linked to other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences, e.g., to mimic the native expression.
  • the Kozak consensus sequence is gccaccatgg (SEQ ID NO: 18).
  • the nucleic acid molecules provided for herein do not contain or do not comprise (e.g. free of) a sequence encoding a linker.
  • nucleic acid molecule can be operably linked to the sequence encoding the protein.
  • a pharmaceutical composition comprising the nucleic acid molecule comprising: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein, a polynucleotide sequence encoding a central polypurine tract, a polynucleotide sequence encoding a second constitutive promoter, a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.), and a polynucleotide encoding a posttranscriptional regulatory element.
  • target molecules e.g. protein of interest, polypeptide, miRNA, shRNA, etc.
  • a pharmaceutical composition comprises the sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, and optionally a sequence encoding an amino acid sequence of a second target molecule, wherein the sequences are operatively connected to one another.
  • a pharmaceutical composition comprises the sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, and a sequence encoding an amino acid sequence of a third target molecule, wherein the sequences are operatively connected to one another.
  • a pharmaceutical composition comprises the sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, a sequence encoding an amino acid sequence of a third target molecule, a sequence encoding an amino acid sequence of a third linker or intermediate element, and a sequence encoding an amino acid sequence of a fourth target molecule, wherein the sequences are operatively connected to one another.
  • proteins or peptides encoded by the nucleic acid molecules described herein are also provided herein.
  • nucleic acid molecules are provided, wherein the nucleic acid molecule comprises a polynucleotide encoding a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein.
  • the promoter is a RSV promoter. In some embodiments, the promoter is an EF-1 alpha promoter.
  • the nucleic acid molecule comprises a 5’LTR sequence. Examples of 5’LTR sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a partial gag sequence. Examples of partial gag sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a cPPT sequence. Examples of cPPT sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a second promoter. In some embodiments, the second promoter is a constitutive promoter. In some embodiments, the second promoter is an inducible promoter. In some embodiments, the second promoter is a tissuespecific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein.
  • the second promoter is a RSV promoter. In some embodiments, the second promoter is an EF-1 alpha promoter.
  • the second promoter is a tissue specific promoter selected from B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
  • the second promoter is a ZAP70 promoter.
  • the second promoter is a CD4 promoter.
  • the nucleic acid molecule comprises a polynucleotide encoding a target molecule.
  • the target molecule or protein can be any molecule or protein encoded for by a nucleic acid molecule that one chooses to be encoded for by the nucleic acid molecule.
  • the nucleic acid molecule comprises a WPRE element. Examples of WPRE element sequences are provided for herein.
  • the nucleic acid molecule comprises a 3’LTR. Examples of 3’LTR sequences are provided for herein.
  • the elements provided for herein can be operatively connected to one another. If all of the elements are present, the nucleic acid molecule can encode a protein comprising the amino acid sequence of a target molecule or protein.
  • nucleic acid molecules are provided, wherein the nucleic acid molecule comprises a polynucleotide encoding a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein.
  • the promoter is a RSV promoter. In some embodiments, the promoter is an EF-1 alpha promoter.
  • the nucleic acid molecule comprises a 5’LTR sequence. Examples of 5’LTR sequences are provided for herein.
  • the nucleic acid molecule comprises a gag sequence, for example and without limiting, a partial gag sequence. Examples of partial gag sequences are provided for herein.
  • the nucleic acid molecule comprises a cPPT sequence. Examples of cPPT sequences are provided for herein.
  • the nucleic acid molecule comprises a second promoter.
  • the second promoter is a constitutive promoter.
  • the second promoter is an inducible promoter.
  • the second promoter is a tissue-specific promoter.
  • the second promoter is a RSV promoter. In some embodiments, the second promoter is an EF-1 alpha promoter.
  • the second promoter is a tissue specific promoter selected from B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
  • the second promoter is a ZAP70 promoter.
  • the second promoter is a CD4 promoter.
  • the nucleic acid molecule comprises a polynucleotide encoding a target molecule.
  • target molecules and proteins are provided for herein. These examples are for illustrative purposes only and are intended to be non-limiting.
  • the target molecule or protein can be any molecule or protein encoded for by a nucleic acid molecule that one chooses to be encoded for by the nucleic acid molecule.
  • the nucleic acid molecule comprises a polynucleotide encoding a linker.
  • linkers are provided for herein.
  • the linker can be any peptide linker that can be encoded for by a nucleic acid molecule.
  • the nucleic acid molecule comprises a polynucleotide encoding an intermediate element.
  • intermediate elements are provided for herein.
  • the intermediate element can be an IRES element.
  • the nucleic acid molecule comprises a second polynucleotide encoding a second target molecule.
  • target molecules and proteins are provided for herein. These examples are for illustrative purposes only and are intended to be non-limiting.
  • the target molecule or protein can be any molecule or protein encoded for by a nucleic acid molecule that one chooses to be encoded for by the nucleic acid molecule.
  • the nucleic acid molecule comprises a posttranscriptional regulatory element, for example and without limiting, a WPRE element. Examples of WPRE element sequences are provided for herein.
  • the nucleic acid molecule comprises a 3’LTR. Examples of 3’LTR sequences are provided for herein.
  • the elements provided for herein can be operatively connected to one another. If all of the elements are present the nucleic acid molecule can encode a protein comprising the amino acid sequence of the first molecule or protein; the amino acid sequence of the linker, and the amino acid sequence of the second molecule or protein.
  • the nucleic acid molecule comprises a polycistronic expression system as provided for herein.
  • nucleic acid molecules are provided, wherein the nucleic acid molecule comprises a polynucleotide encoding a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein.
  • the promoter is a RSV promoter. In some embodiments, the promoter is an EF-1 alpha promoter.
  • the nucleic acid molecule comprises a 5’LTR sequence. Examples of 5’LTR sequences are provided for herein.
  • the nucleic acid molecule comprises a gag sequence, for example and without limiting, a partial gag sequence. Examples of partial gag sequences are provided for herein.
  • the nucleic acid molecule comprises a cPPT sequence. Examples of cPPT sequences are provided for herein.
  • the nucleic acid molecule comprises a second promoter.
  • the second promoter is a constitutive promoter.
  • the second promoter is an inducible promoter.
  • the second promoter is a tissue-specific promoter.
  • the second promoter is a RSV promoter. In some embodiments, the second promoter is an EF-1 alpha promoter.
  • the second promoter is a tissue specific promoter selected from B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
  • the second promoter is a ZAP70 promoter.
  • the second promoter is a CD4 promoter.
  • the nucleic acid molecule comprises a polycistronic expression system as provided for herein.
  • the polycistronic expression system allows for expression of one or more target molecule and one or more linker or intermediate element. Examples of polycistronic expression systems are provided for herein.
  • the nucleic acid molecule comprises a posttranscriptional regulatory element, for example and without limiting, a WPRE element. Examples of WPRE element sequences are provided for herein.
  • the nucleic acid molecule comprises a 3’LTR. Examples of 3’LTR sequences are provided for herein.
  • the elements provided for herein can be operatively connected to one another. If all of the elements are present the nucleic acid molecule can encode a protein comprising the amino acid sequence of the first molecule or protein; the amino acid sequence of the first or more linker, and the amino acid sequence of the second or more molecule or protein.
  • the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, and a sequence encoding a first target molecule, wherein the sequences are operatively connected to one another.
  • cPPT central polypurine tract
  • the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, and optionally a sequence encoding a second target molecule, wherein the sequences are operatively connected to one another.
  • cPPT central polypurine tract
  • the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, and optionally a sequence encoding a third target molecule, wherein the sequences are operatively connected to one another.
  • cPPT central polypurine tract
  • the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, optionally a sequence encoding a third target molecule, optionally a sequence encoding a third linker or intermediate element, and optionally a sequence encoding a fourth target molecule, wherein the sequences are operatively connected to one another.
  • the linker or intermediate element is as provided for herein.
  • the target molecule is as provided for herein.
  • the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, and a sequence encoding a first target molecule, wherein the sequences are operatively connected to one another.
  • the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, and optionally a sequence encoding a second target molecule, wherein the sequences are operatively connected to one another.
  • cPPT central polypurine tract
  • the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, and optionally a sequence encoding a third target molecule, wherein the sequences are operatively connected to one another.
  • cPPT central polypurine tract
  • the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, optionally a sequence encoding a third target molecule, optionally a sequence encoding a third linker or intermediate element, and optionally a sequence encoding a fourth target molecule, wherein the sequences are operatively connected to one another.
  • linkers and intermediate elements are as provided for herein.
  • the linker or intermediate element is as provided for herein.
  • the target molecule is as provided for herein.
  • the nucleic acid molecule comprises a sequence of gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtg cttcaagtagtgtgtgccccgtctgttgtgtgtgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgc ccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcacggcaagaggc gaggggcggcgactggtgagtacgccaaaa
  • the nucleic acid molecule comprises a sequence that is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to that of SEQ ID NO: 19.
  • the nucleic acid molecule comprises a sequence of: tgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaagcaccgtgcatgccg attggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcatt gcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggttagaccagatctgagcctgggagctctctggctggttagaccagatctgagcctgggagctc
  • the nucleic acid molecules provided for herein can be provided as a single molecule, for example as a plasmid.
  • each element is encoded by a single polynucleotide sequence (or double stranded molecule, such as, but not limited to a plasmid) as opposed to distinct nucleotide molecules encoding for different elements.
  • the use of a single molecule, such as a plasmid permits the user to efficiently introduce the target molecule into the relevant cell or cellular environment.
  • the nucleic acid molecules provided herein comprise one or more of a polynucleotide encoding a constitutive promoter; a polynucleotide encoding a target molecule, which can also be referred to as a molecule of interest; and a polynucleotide encoding a nuclear localization signal; wherein the sequences can be, or are, operatively connected one another.
  • the nucleic acid molecules provided herein comprise a polynucleotide encoding a constitutive promoter; a polynucleotide encoding a target molecule, which can also be referred to as a molecule of interest; and a polynucleotide encoding a nuclear localization signal; wherein the sequences are operatively connected one another.
  • the molecule of interest can be any molecule that can be encoded by the nucleic acid molecule.
  • the target molecule can be a chimeric antigen receptor (CAR) that can be expressed in a T-cell or other type of cell and function in the T-cell.
  • CAR chimeric antigen receptor
  • the nucleic acid molecule which can be in the form of a plasmid, can be used to deliver a target molecule to a cell of interest.
  • the cell is an immune cell, such as a T-cell, dendritic cell, NK cell, a TIL, a MIL, and the like.
  • the nucleic acid molecule is used to deliver a target molecule that can be expressed in a cell.
  • the nucleic acid molecule which can be a plasmid, can be used to express a protein or nucleic acid molecule in a cell.
  • the target molecule can be a nucleic acid molecule that encodes for a protein, an antisense nucleic acid molecule, a siRNA molecule, a microRNA, an antibody, a receptor, or any other type of molecule that can be encoded for by a nucleic acid molecule, such as those described herein.
  • Other examples of products that can be encoded for are hormones, gene products, and the like.
  • the target molecule is a chimeric antigen receptor (CAR).
  • CARs that can be used include those that are comprise an extracellular region that bind to PD-1, PD-L1, BSMA, PSMA, and the like.
  • the CAR comprises a CD19 extracellular binding domain.
  • the CAR comprises a 4-1BB intracellular region.
  • the CAR comprises a CD3( ⁇ intracellular signaling domain.
  • the CAR comprises a CD28 intracellular domain.
  • the transmembrane domain of the CAR is a CD3( ⁇ transmembrane domain or a CD28 transmembrane domain.
  • the CAR is an armored CAR.
  • CAR- modified T-cell potency may be further enhanced through the introduction of additional genes, including those encoding proliferative cytokines (i.e., IL-12) or costimulatory ligands (i.e., 4- 1BBL), thus producing "armored" CAR-modified T-cells.
  • the nucleic acid molecule e.g. plasmid, is a non-integrating nucleic acid molecule.
  • the nucleic acid molecules provided for herein are administered to a subject and taken up by the cells.
  • the cells are treated with the nucleic acid molecule (e.g. plasmid) ex-vivo and then administered back to a subject to express the molecules in vivo.
  • the nucleic acid molecules are complexed with nanoparticles to deliver the nucleic acid molecule to a specific cell type.
  • the nucleic acid molecule can be encapsulated or complexed with a lipid nanoparticle, a polymer nanoparticle, liposome, a neutral liposome, a biodegradable polymer matrix e.g. hydrogel), and the like.
  • Example of nanoparticles are described in Xiao et al., Molecular Therapy: Methods & Clinical Development Vol. 12 March 2019, pp. 1-18, which is hereby incorporated by reference in its entirety.
  • polymers that can be used include, but are not limited to, polyetherimide (PEI), lactosylated polylysine (PLL), polyacrylic acid (PAA), poly(aliphatic ester) (PAE), and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA). These polymers can be modified by chemical modification or can be free of modifications.
  • Other polymers include, but are not limited to, chitosan (e.g.
  • cationic chitosan poly(ethyleneglycol)-modified chitosan (PEG-CS), carboxymethyl dextran (CMD)-chitosan, gelatin (e.g. cationic gelatin), dextran (e.g. cationic dextran), cellulose (e.g. cationic cellulose), cyclodextrin (e.g. cationic cyclodextrin).
  • PEG-CS poly(ethyleneglycol)-modified chitosan
  • CMD carboxymethyl dextran
  • gelatin e.g. cationic gelatin
  • dextran e.g. cationic dextran
  • cellulose e.g. cationic cellulose
  • cyclodextrin e.g. cationic cyclodextrin
  • the nucleic acid molecule is not encapsulated with a carrier or nanoparticle.
  • the pharmaceutical composition is free of a nanoparticle that encapsulates the nucleic acid molecule.
  • the nucleic acid molecule can comprise a promoter, such as a constitutive promoter.
  • constitutive promoters include, but are not limited to EF-1 alpha, SV40, Rous Sarcoma virus, and Mason-Pfizer monkey virus-CTE.
  • the promoter is EF-1 alpha.
  • the promoter is SV40.
  • the promoter is RSV.
  • the promoter is the Mason-Pfizer monkey virus-CTE.
  • the promoter is a tissue specific promoter.
  • Tissue specific promoters are known in the art, and any such tissue specific promoter may be utilized in any of the embodiments of the present disclosure.
  • the tissue specific promoter is as provided for herein.
  • tissue specific promoter examples include, but are not limited to, B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, and NSE/RU5’.
  • the tissue specific promoter is B29.
  • the tissue specific promoter is CD14.
  • the tissue specific promoter is CD43.
  • the tissue specific promoter is CD45.
  • the tissue specific promoter is CD68. In some embodiments, the tissue specific promoter is desmin. In some embodiments, the tissue specific promoter is elastase-1. In some embodiments, the tissue specific promoter is endoglin. In some embodiments, the tissue specific promoter is fibronectin. In some embodiments, the tissue specific promoter is Flt-1. In some embodiments, the tissue specific promoter is GFAP. In some embodiments, the tissue specific promoter is GPIIb. In some embodiments, the tissue specific promoter is ICAM-2. In some embodiments, the tissue specific promoter is mouse INF-beta. In some embodiments, the tissue specific promoter is Mb. In some embodiments, the tissue specific promoter is Nphsl.
  • the tissue specific promoter is OG-2. In some embodiments, the tissue specific promoter is SP-B. In some embodiments, the tissue specific promoter is SYN1. In some embodiments, the tissue specific promoter is WASP. In some embodiments, the tissue specific promoter is SV40/bAlb. In some embodiments, the tissue specific promoter is SV40/hAlb. In some embodiments, the tissue specific promoter is SV40/CD43. In some embodiments, the tissue specific promoter is SV40/CD45. In some embodiments, the tissue specific promoter is NSE/RU5’. In some embodiments, the tissue specific promoter is an immune cell specific promoter. Examples of immune cell specific promoters include, but are not limited to, ZAP70 and CD4. In some embodiments, the tissue specific promoter is the ZAP70 promoter. In some embodiments, the tissue specific promoter is the CD4 promoter.
  • the nucleic acid molecules can also further comprise a nucleic acid sequence that encodes for one or more adjuvants.
  • the adjuvant is IL-12.
  • the nucleic acid molecule encodes one or more of the group consisting of anti- CD40 antibody, GM-CSF, bevacizumab, interferon-alpha, interferon-beta, poly-(I:C) and derivatives, RNA interleukin (IL)-l, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23, and the like.
  • any of the nucleic acid molecules provided for herein can be a plasmid or other type of circular DNA sequence, such that it can be used to express its products in a cell.
  • compositions comprising the nucleic acid molecules described herein are provided.
  • examples of pharmaceutical compositions are provided for herein.
  • methods of delivering a molecule to a cell comprise contacting a cell with a virus comprising a nucleic acid molecule, as provided herein, or a nucleic acid molecule as provided for herein into a cell of the subject or into the subject and said virus comprising a nucleic acid molecule, as provided herein, or nucleic acid sequence is taken up by the cell in the subject.
  • the nucleic acid molecule is then expressed and the target molecules or other expression cassettes are expressed in the cell.
  • the nucleic acid sequence is introduced into the cell or subject by electroporation, injection, sonication, transfection, transduction, gene guns, encompassed by nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell.
  • the cell or tissue that the nucleic acid molecule is delivered to is skin, muscle, breast, lung, pancreas, brain, ovarian, uterine, endometrial, colon, prostate, esophageal, gum, tongue, throat, liver, eye, thymus, or kidney tissue or cell.
  • methods of preparing a virus comprising a nucleic acid molecule encoding a target molecule are provided.
  • the method comprises contacting a cell with the nucleic acid molecule, as provided for herein, under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule.
  • the cell is a producer cell.
  • producer cells include HEK293 and derivatives, Sf9, and CAP (CEVEC).
  • the virus is a lentivirus.
  • the lentivirus is a pseudotyped lentivirus.
  • a pseudotyped lentivirus is a lentiviral particle having one or more envelope glycoproteins that are encoded by a virus that is distinct from the lentiviral genome.
  • the envelope protein can be, for example and without limitation, from a different virus or from a non-viral origin.
  • the envelope protein can be a native envelope protein or an envelope protein that is modified, mutated or engineered as described herein.
  • the pseudotyped lentivirus can comprise, for example, fusion (F) or attachment (G) protein of paramyxovirus in addition to membrane proteins having hemagglutinin activity such as H protein of paramyxovirus.
  • the paramyxovirus is a Nipah virus or a Measles virus.
  • the pseudotyped lentivirus is pseudotyped with Nipah virus fusion protein (NiV-F), Nipah virus attachment protein (NiV-G), Measles virus hemagglutinin (MV-H), or Measles virus fusion protein (MV-F).
  • the pseudotyped lentivirus is pseudotyped with Nipah virus fusion protein (NiV-F). In some embodiments, the pseudotyped lentivirus is pseudotyped with Nipah virus attachment protein (NiV-G). In some embodiments, the pseudotyped lentivirus is pseudotyped with Measles virus hemagglutinin (MV-H). In some embodiments, the pseudotyped lentivirus is pseudotyped with Measles virus fusion protein (MV-F).
  • Nipah and Measles viruses and elements thereof can be found in PCT Publication No. WO2017182585, U.S. Publication No. 20190144885, U.S. Patent No.
  • the pseudotyped lentivirus is pseudotyped with a human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, immunodeficiency virus in simian (SIV), maedi / visna virus, Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, and SARS-CoV virus.
  • HAV-1 human immunodeficiency virus
  • HIV-2 feline immunodeficiency virus
  • FIV feline immunodeficiency virus
  • SIV immunodeficiency virus in simian
  • maedi / visna virus Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus
  • Dengue virus Hepatitis B virus
  • Rabies virus Semliki Forest virus
  • the pseudotyped lentivirus can comprise, additionally or alternatively, envelope proteins derived from other viruses.
  • preferred envelope proteins include those derived from viruses infectious to human cells.
  • proteins include, but not limited to, amphotropic envelope proteins of retrovirus, G protein of vesicular stomatitis virus (VSV-G), etc.
  • proteins of viruses belonging to the herpes viridae include, for example, gB, gD, gH and gp85 proteins of herpes simplex virus, gp350 and gp220 proteins of EB virus, etc.
  • proteins of viruses belonging to the hepadna viridae include S protein of hepatitis B virus, etc.
  • compositions and molecules described herein can be used to treat a disease.
  • diseases that can be treated include, but are not limited to, immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
  • the compositions and molecules described herein can be used to treat a cancer.
  • diseases that can be treated include, but are not limited to, brain, breast, lung, ovarian, endometrial, colon, lung, skin (e.g. melanoma), blood, lymphoma, leukemia, and the like.
  • compositions described herein can further comprise a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical compositions comprise about 1 ng to about 10,000 pg of the nucleic acid molecule.
  • the pharmaceutical compositions can be formulated according to the mode of administration to be used.
  • One having ordinary skill in the art can readily formulate a pharmaceutical composition that comprises a genetic construct or nucleic acid molecule as described herein.
  • an isotonic formulation can be used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like.
  • the pharmaceutical composition comprises a stabilizer.
  • stabilizers include, but are not limited to, gelatin and albumin.
  • the pharmaceutical preparations can be provided as sterile and pyrogen free.
  • methods of inducing an immune response against a target molecule in a subject comprise introducing the virus comprising the nucleic acid, as provided herein, described herein into the subject.
  • the virus is introduced in a cell of the subject.
  • the nucleic acid molecule can also be taken up by the cell. Once inside the cell the cell’s machinery can be used to express the target molecule (e.g., a target protein) and the construct that is encoded for by the nucleic acid molecule.
  • the nucleic acid molecule is as described herein.
  • the method of introduction or administration can be any method, including the methods described herein.
  • the nucleic acid molecule is introduced by electroporation or injection. In some embodiments, the nucleic acid molecule is introduced into, or administered to, the subject by sonication, transfection, transduction, gene guns, nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell and the like.
  • bupivacaine or other similar adjuvant is used to help facilitate the induction of an immune response.
  • the nucleic acid molecule is administered to a tissue of the subject.
  • the tissue is skin, muscle, liver, fat, nerve, kidney, and the like.
  • methods of treating a disease are provided.
  • the methods of treating disease in a subject introducing, or administering, a virus comprising the nucleic acid, as provided herein, described herein to the subject.
  • the virus is introduced directly into a cell.
  • the nucleic acid molecule is introduced directly into a cell.
  • the virus comprising the nucleic acid molecule is taken up by the cell and expressed in the cell.
  • the target protein and the fragment of the target protein is a protein that is overexpressed, or specifically expressed, in a cancer cell.
  • the nucleic acid molecule is administered by electroporation, injection, sonication, transfection, transduction, and the like. In some embodiments, the nucleic acid molecule is administered to the skin, muscle or other tissue of the subject. In some embodiments, the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
  • methods of treating an infectious agent or an infection in a subject comprise introducing/administering the virus produced as described herein to the subject.
  • the virus is administered directly into a cell of the subject.
  • the virus can be taken up by the cell and viral nucleic acid molecule, also referred to as the nucleic acid molecule and as provided herein, can be expressed in the cell.
  • viral nucleic acid molecule also referred to as the nucleic acid molecule and as provided herein
  • the subject when the nucleic acid molecule is expressed the subject’s immune response will recognize the target protein and the fragment of the target protein as foreign and an immune response will be generated. The generated immune response can treat or prevent the infection.
  • the generated immune response can treat or prevent the infectious agent from causing a disease or will inhibit the growth of the infectious agent to ameliorate symptoms of the infection.
  • the methods of administration can be any of the methods described herein.
  • the virus can be administered to the skin, muscle, fat, kidney, or other tissue of the subject. In some embodiments, the virus is administered to the mucosa of the subject.
  • the nucleic acid molecule is introduced into the subject or the cell by electroporation, injection, sonication, transfection, and transduction.
  • the target protein is a HIV or influenza protein, such as Gpl20, Gag, Nef, Tat, hemagglutinin (HA), neuraminidase (NA), and the like.
  • HIV or influenza protein such as Gpl20, Gag, Nef, Tat, hemagglutinin (HA), neuraminidase (NA), and the like.
  • a cell comprising the nucleic acid molecules provided for herein are provided.
  • the cell is an isolated cell.
  • the cell is ex-vivo and not in a subject.
  • the cell can be any cell type, such as a T-cell, a muscle cell, a skin cell, a brain cell, and the like.
  • Example 1 Plasmid production.
  • the plasmid comprised of a polynucleotide bounded by long terminal repeats is mixed with both a plasmid encoding envelope fusion proteins (pseudotype) and one or more plasmids encoding polynucleotides required for packaging the proviral plasmid into a viral particle.
  • the plasmid mixture is complexed with a reagent, such as calcium phosphate or a lipid transfection mixture, which facilitates cellular uptake into the packaging cells.
  • the packaging cells consisting of the plasmid mixture then produce viral vector particles which can be purified from the packaging cells by mechanisms known to one skilled in the art (centrifugation; binding, washing and elution to purification columns; gradient purification; etc.). Purified viral vector particles can be supplemented with other components to become the pharmaceutical composition described herein.
  • the pharmaceutical composition can be applied to cells ex vivo or introduced into a subject in vivo to allow transduction with the vector delivering the polynucleotide encoding the target molecule(s) into the cells described herein. After transduction, the vector will express the target molecules.
  • Example 2 Viral vectors generated from plasmid transduce human PBMCs.
  • LTR-containing plasmids were designed encoding a self-inactivating lentiviral vector genome with a transgene of either green fluorescent protein (GFP) as a cytoplasmic reporter or two transmembrane proteins separated by a self-cleaving peptide sequence, CAR-2A- NGFR.
  • GFP green fluorescent protein
  • the lentivector genomes and pseudotyping plasmids were transfected along with a packaging plasmid set consisting of a human-simian chimeric gag-pol and HIV-1 rev in HEK293T cells.
  • Viral supernatant was purified by centrifugation over a 20% sucrose cushion and resuspended in X-Vivo media. Resuspended virus was analyzed by a p24 assay to determine viral protein content in solution and calculate a physical titer. 10-fold dilutions by p24 mass of virus were administered to SupTl cells to determine a functional titer by flow cytometry.
  • Vectors were then administered to activated human PBMCs to assess transduction and transgene expression.
  • Flow cytometry was utilized to assess the fraction of CD4+ cells with either GFP expression in the cytoplasm or NGFR protein expression on the surface at seven days after transduction (FIG. 6 A-F).
  • the examples provided for herein demonstrate the flexibility of the nucleic molecules described herein to deliver molecules and have them expressed. This can be used to deliver a molecule to a cell or tissue or can be used to induce an immune response against a variety of target molecules.
  • the nucleic acid molecules can also be used to generate viral vectors that can be used to, for example, transduce, PBMCs.
  • the examples described herein are exemplary in manner and are not intended, nor should they be used, to limit the scope of the embodiments. Each and every reference, publication, accession number, patent, document, etc., is hereby incorporated by reference in its entirety for its intended purpose.

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Abstract

Nucleic acid molecules are provided herein that can be used, for example, to produce viruses that can be used, for example to treat cancer or infections, or to induce an immune response in a subject, or to deliver or express a target molecule in or from a cell.

Description

Nucleic Acid Molecules and Methods of Using the Same
Background
[0001] Immunotherapy and gene therapy has revolutionized the treatments of various diseases. However, the therapies are still imperfect and thus, there is a need to provide new therapies that overcome the disadvantages and shortcoming still present today. The present disclosure fulfills these needs as well as others.
Summary
[0002] In some embodiments, nucleic acid molecules are provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal repeat (LTR) bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5 ’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein; a polynucleotide sequence encoding a central polypurine tract (cPPT); a polynucleotide sequence encoding a second constitutive promoter; a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.); and a polynucleotide encoding a posttranscriptional regulatory element. In some embodiments, the nucleotide sequences are operatively connected one another.
[0003] In some embodiments, pharmaceutical composition comprising the nucleic acid molecules provided herein are provided.
[0004] In some embodiments, methods of delivering the molecule to a cell are provided. In some embodiments, the methods comprise contacting a cell with a nucleic acid sequence as provided herein into a cell of the subject or into the subject and said nucleic acid sequence is taken up by the cell in the subject. In some embodiments, methods of preparing a virus comprising a nucleic acid molecule encoding a target molecule are provided. In some embodiments, the methods comprise contacting a cell with the nucleic acid molecule as provided herein under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule.
[0005] In some embodiments, methods of treating a disease in a subject are provided. In some embodiments, the methods comprise administering to the subject the virus prepared according to the methods provided herein. In some embodiments, the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
Brief Description of Drawings
[0006] FIG. 1 illustrates a non-limiting schematic of a nucleic acid molecule, such as a plasmid, as described herein.
[0007] FIG. 2 illustrates a non-limiting schematic of a nucleic acid molecule, such as a plasmid, as described herein.
[0008] FIG. 3 illustrates non-limiting embodiments of a nucleic acid molecule, such as a plasmid, as described herein.
[0009] FIG. 4 illustrates non-limiting embodiments of a nucleic acid molecule, such as a plasmid, as described herein.
[0010] FIG. 5 illustrates non-limiting embodiments of a nucleic acid molecule, such as a plasmid, as described herein.
[0011] FIG. 6 illustrates viral particles produced from the nucleic acid molecules as provided for herein transducing human peripheral blood mononuclear cells (PBMCs).
[0012] FIGS. 6A-6B illustrate viral particles derived from the nucleic acid molecules of the present disclosure and pseudotyped with VSV-G delivering and expressing GFP and CAR-2A- NGFR transgenes, respectively.
[0013] FIGS. 6C-6D illustrate the ability of viral particles derived from the nucleic acid molecules of the as provided for herein and pseudotyped with Nipah virus structural proteins and a scFv that binds to a cell surface protein on a PBMC to deliver and express GFP and CAR-2A- NGFR transgenes, respectively.
[0014] FIGS. 6E-6F illustrate the ability of viral particles derived from the nucleic acid molecules of the present disclosure and pseudotyped with Nipah virus structural proteins and a VHH that binds to a cell surface protein on a PBMC to deliver and express GFP and CAR-2A- NGFR transgenes, respectively.
Enumerated List of Embodiments
1. A nucleic acid molecule comprising: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal repeat (LTR) bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5 ’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein and/or a binder molecule; a polynucleotide sequence encoding a central polypurine tract (cPPT); a polynucleotide sequence encoding a second promoter; a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.); and a polynucleotide encoding a posttranscriptional regulatory element.
2. The nucleic acid molecule of embodiment 1, wherein the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
3. The nucleic acid molecule of embodiment 2, wherein the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
4. The nucleic acid molecule of embodiments 2 or 3, wherein the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another.
5. The nucleic acid molecule of any one of embodiments 1-4, wherein the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
6. The nucleic acid molecule of any one of embodiments 1-5, wherein the sequence encoding the first promoter encodes a constitutive promoter, an inducible promoter, or a tissuespecific promoter.
7. The nucleic acid molecule of embodiment 6, wherein the constitutive promoter is selected from the group consisting of EF-1 alpha, Rous Sarcoma virus (RSV), SV-40, and Mason-Pfizer monkey virus-CTE.
8. The nucleic acid molecule of embodiment 6, wherein the tissue-specific promoter is selected from the group consisting of B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
9. The nucleic acid molecule of embodiment 6, wherein the tissue-specific promoter is selected from the group consisting of ZAP70 and CD4.
10. The nucleic acid molecule of any one of embodiments 1-9, wherein the sequence encoding the second promoter encodes a constitutive promoter, an inducible promoter, or a tissue specific promoter.
11. The nucleic acid molecule of embodiment 10, wherein the constitutive promoter is selected from the group consisting of EF-1 alpha, Rous Sarcoma virus (RSV), SV-40, and Mason-Pfizer monkey virus-CTE.
12. The nucleic acid molecule of embodiment 10, wherein the tissue-specific promoter is selected from the group consisting of B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
13. The nucleic acid molecule of embodiment 10, wherein the tissue-specific promoter is selected from the group consisting of ZAP70 and CD4.
14. The nucleic acid molecule of embodiment 1, wherein the sequence encoding the target molecule encodes a reporter gene, a protein of interest, a chimeric antigen receptor (CAR), an armored CAR, a DAP-12, a cytokine, a siRNA, a shRNA, an antisense molecule, a microRNA, or a gene editing construct (e.g., CRISPR/CAS system).
15. The nucleic acid molecule of embodiment 14, wherein the reporter gene is a fluorescent protein or an enzyme.
16. The nucleic acid molecule of embodiment 4, wherein the sequence encoding the linker encodes a glycine-serine linker or a cleavable linker.
17. The nucleic acid molecule of embodiment 16, wherein the cleavable linker is a P2A, T2A, E2A, or F2A linker.
18. The nucleic acid molecule of embodiment 16, wherein the glycine linker comprises one more of the repeat of GGGGS, GSG, or any combination thereof.
19. The nucleic acid molecule of embodiment 4, wherein the sequence encoding the intermediate elements encodes an Internal Ribosome Entry Site (IRES). 20. The nucleic acid molecule of any of embodiments 1-19, wherein the nucleic acid molecule optionally comprises a safety switch.
21. The nucleic acid molecule of embodiment 1, wherein the sequence encoding the viral structural protein encodes a gag protein.
22. The nucleic acid molecule of embodiment 1, wherein the sequence encoding the viral structural protein encodes a partial gag protein.
23. The nucleic acid molecule of embodiment 22, wherein the sequence encoding the partial gag encodes for the HIV-1 psi element, rev response element (RRE), and a Gp41 peptide.
24. The nucleic acid molecule of embodiment 1, wherein the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte; Her2/Neu+ cancer cell; NKG2D+ natural killer cell; SLC1A3+ astrocyte; SLC7A10+ adipocyte.
25. The nucleic acid molecule of embodiment 1, wherein the binder molecule binds to CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, A glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors; A glycosylated CD43 epitope expressed on non-hematopoietic cancers; A kinase anchor protein 4 (AKAP-4); Adrenoceptor beta 3 (ADRB3); AFP; Anaplastic lymphoma kinase (ALK); Androgen receptor; Angiopoietin-binding cell surface receptor 2 (Tie 2); Auto antibody to desmoglein 1 (Dsgl); Auto antibody to desmoglein 3 (Dsg3); B7H3 (CD276); Biotin; Bone marrow stromal cell antigen 2 (BST2); BST1/CD157; Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la);
Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R;
CD276/B7H3; CD300 molecule-like family member f (CD300LF); CDH1-CD324; CDH6; CDH17; CDH19; Chromosome X open reading frame 61 (CXORF61); Claudin 6 (CLDN6); Claudinl8.2 (CLD18A2 or CLDN18A.2); CMV pp65; C-MYC epitope Tag; Cripto; CS1 (also referred to as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24); CSF2RA (GM-CSFR- alpha); C-type lectin domain family 12 member A (CLEC12A); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); Cyclin Bl; Cytochrome P450 IB 1 (CYP1B 1); DLL3; EBV-EBNA3c; EGF-bke module- containing mucin-like hormone receptor-like 2 (EMR2); Elongation factor 2 mutated (ELF2M); Ephrin B2; Ephrin type-A receptor 2 (EphA2); Epidermal growth factor receptor (EGFR); Epidermal growth factor receptor variant III (EGFRviii); Epithelial cell adhesion molecule (EPCAM); ERG; ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Fc fragment of IgA receptor (FCAR or CD89); Fc receptor-like 5 (FCRL5); Fibroblast activation protein alpha (FAP); FITC; Fms Like Tyrosine Kinase 3 (FLT3); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Follicle stimulating hormone receptor (FSHR); Fos-related antigen 1; Fucosyl-GMl; G protein coupled receptor class C group 5 member D (GPRC5D); G protein-coupled receptor 20 (GPR20); GAD; Ganglioside G2 (GD2) ; Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer); Ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l- 4)bDGlcp(l-l)Cer); GD3; GFRalpha4; Glycoprotein 100 (gplOO); Glypican-3 (GPC3); Gonadotropin Hormone receptor (CGHR or GR); GpA33; GpNMB;
GPRC5D; Guanylyl cyclase C (GCC); Heat shock protein 70-2 mutated (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexasaccharide portion of globoH glycoceramide (GloboH); High molecular weight-melanoma associated antigen (HMWMAA); HIV1 envelope glycoprotein; HLA; HLA-DOA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DM; HLA-DOB; HLA-DP; HLA-DQ; HLA-DR; HLA-G; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human Telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; IL1 IRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza A hemagglutinin (HA); Insulin-like growth factor 1 receptor (IGF -I receptor); Interleukin 11 receptor alpha (IL-llRa); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Intestinal carboxyl esterase; KIT (CD117); KSHV K8.1; KSHV-gH; LAMP1 ; Legumain; Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1); Leutenizing hormone receptor (LHR); Lewis(Y) antigen; Lews Ag; Livl; Locus K 9 (LY6K); Low conductance chloride channel; Lymphocyte antigen 6 complex; Lymphocyte antigen 75 (LY75); Lymphocyte-specific protein tyrosine kinase (LCK); Mammary gland differentiation antigen (NY-BR-1); Melanoma antigen recognized by T cells 1 (MelanA or MARTI); Melanoma- associated antigen 1 (MAGE-A1); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT- 2); Melanoma inhibitor of apoptosis (ML-IAP); Mesothelin; MPL; Mucin 1 cell surface associated (MUC1); N-Acetyl glucosaminyl-transf erase V (NA 17); Nectin-4; Neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-acetyl-GD2 ganglioside (OAcGD2); Olfactory receptor 51E2 (OR51E2); Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); P53 mutant; Paired box protein Pax-3 (PAX3); Paired box protein Pax-5 (PAX5); Pannexin 3 (PANX3); PDL1; P-glycoprotein; Placenta-specific 1 (PLAC1); Platelet-derived growth factor receptor beta (PDGFR-beta); Polysialic acid; Proacrosin binding protein sp32 (OY-TES1); Prostase; Prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Prostatic acid phosphatase (PAP);
Prostein; Protease Serine 21 (Testisin or PRSS21); Proteasome (Prosome Macropain) Subunit Beta Type 9 (LMP2); PTK7; Ras G12V; Ras Homolog Family Member C (RhoC); Rat sarcoma (Ras) mutant; Receptor for Advanced Gly cation Endproducts (RAGE-1); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Receptor tyrosine-protein kinase ERBB2 or Her-22/neu; Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); Sarcoma translocation breakpoints; Serine 2 (TMPRSS2) ETS fusion gene; Sialyl Lewis adhesion molecule (sLe); SLAMF4; SLAMF6; Slea (CAI 9.9 or Sialyl Lewis Antigen); Sperm protein 17 (SPA17); Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Stage-specific embryonic antigen-4 (SSEA-4); STEAP1; Survivin; Synovial sarcoma X breakpoint 2 (SSX2); TCR Gamma Alternate Reading Frame Protein (TARP); TCR-betal chain; TCR-beta2 chain; TCR-delta chain; TCR-gamma chain; TCRgamma-delta; Telomerase; TGFbetaR2; The antigen recognized by TNT antibody; Thyroid stimulating hormone receptor (TSHR); Timl-/HVCR1; Tissue Factor 1 (TF1); Tn ag; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); TNF receptor family member B cell maturation (BCMA); Transglutaminase 5 (TGS5); Transmembrane protease; TROP2; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7-related (TEM7R); Tumor protein p53 (p53); Tumor-associated glycoprotein 72 (TAG72); Tyrosinase; Tyrosinase-related protein 2 (TRP-2); Uroplakin 2 (UPK2); Vascular endothelial growth factor receptor 2 (VEGFR2); V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Wilms tumor protein (WT1); or X Antigen Family Member 1 A (XAGE1).
26. The nucleic acid of any one of embodiments 1-25, wherein the nucleic acid molecule comprises an antibiotic resistance gene.
27. The nucleic acid of embodiment 26, wherein the antibiotic resistance gene confers resistance to an antibiotic selected from the group comprising kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.
28. A nucleic acid molecule sequentially comprising in a 5’ to 3’ direction: a. a polynucleotide encoding a RSV promoter; b. a polynucleotide encoding a 5’ LTR; c. a polynucleotide encoding a partial gag, wherein the partial gag further comprises
HIV-1 psi element, rev response element (RRE), and a Gp41 peptide; d. a polynucleotide encoding a central polypurine tract (cPPT); e. a polynucleotide encoding an EF-1 alpha promoter; f. a polynucleotide encoding a first target molecule; g. optionally a polynucleotide encoding a first linker or first intermediate element; h. optionally a polynucleotide encoding a second target molecule; i. optionally a polynucleotide encoding a second or more (e.g., third, fourth, fifth) linker or second or more (e.g., third, fourth, fifth) intermediate element; j. optionally a polynucleotide encoding a third or more (e.g., fourth, fifth, sixth) target molecule; k. a polynucleotide encoding a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE); and l. a polynucleotide encoding a 3’ LTR; wherein the sequences are operatively connected to one another, and optionally wherein g)-j) repeat n times, wherein n is any integer (e.g., 0, 1, 2, 3, 4).
29. The nucleic acid molecule of embodiment 28, wherein the nucleic acid molecule comprises one or more polynucleotides encoding one or more target molecules (e.g., n is 1, 2, 3, 4). 30. The nucleic acid molecule of embodiment 29, wherein the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
31. The nucleic acid molecule of any one of embodiments 29-30, wherein the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
32. The nucleic acid molecule of any one of embodiments 29-31, wherein the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another.
33. The nucleic acid molecule of any one of embodiments 29-32, wherein the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
34. The nucleic acid molecule of any one of embodiments 28-33, wherein the sequence encoding the first, second, or more target molecule encodes a reporter gene, a protein of interest, a chimeric antigen receptor (CAR), an armored CAR, a DAP- 12, a cytokine, a binder molecule, a siRNA, a shRNA, an antisense molecule, a microRNA, or a gene editing construct (e.g., CRISPR/CAS system).
35. The nucleic acid molecule of embodiment 34, wherein the reporter gene is a fluorescent protein or an enzyme.
36. The nucleic acid molecule of embodiment 34, wherein at least one of the first, second, or more target molecules encodes a chimeric antigen receptor (CAR).
37. The nucleic acid molecule of embodiment 34, wherein the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte; Her2/Neu+ cancer cell; NKG2D+ natural killer cell; SLC1A3+ astrocyte; SLC7A10+ adipocyte. 38. The nucleic acid molecule of embodiment 34, wherein the binder molecule binds to CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, A glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors; A glycosylated CD43 epitope expressed on non-hematopoietic cancers; A kinase anchor protein 4 (AKAP-4); Adrenoceptor beta 3 (ADRB3); AFP; Anaplastic lymphoma kinase (ALK); Androgen receptor; Angiopoietin-binding cell surface receptor 2 (Tie 2); Auto antibody to desmoglein 1 (Dsgl); Auto antibody to desmoglein 3 (Dsg3); B7H3 (CD276); Biotin; Bone marrow stromal cell antigen 2 (BST2); BST1/CD157; Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la);
Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R;
CD276/B7H3; CD300 molecule-like family member f (CD300LF); CDH1-CD324; CDH6; CDH17; CDH19; Chromosome X open reading frame 61 (CXORF61); Claudin 6 (CLDN6); Claudinl8.2 (CLD18A2 or CLDN18A.2); CMV pp65; C-MYC epitope Tag; Cripto; CS1 (also referred to as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24); CSF2RA (GM-CSFR- alpha); C-type lectin domain family 12 member A (CLEC12A); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); Cyclin Bl; Cytochrome P450 IB 1 (CYP1B 1); DLL3; EBV-EBNA3c; EGF-bke module- containing mucin-like hormone receptor-like 2 (EMR2); Elongation factor 2 mutated (ELF2M); Ephrin B2; Ephrin type-A receptor 2 (EphA2); Epidermal growth factor receptor (EGFR); Epidermal growth factor receptor variant III (EGFRviii); Epithelial cell adhesion molecule (EPCAM); ERG; ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Fc fragment of IgA receptor (FCAR or CD89); Fc receptor-like 5 (FCRL5); Fibroblast activation protein alpha (FAP); FITC; Fms Like Tyrosine Kinase 3 (FLT3); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Follicle stimulating hormone receptor (FSHR); Fos-related antigen 1; Fucosyl-GMl; G protein coupled receptor class C group 5 member D (GPRC5D); G protein-coupled receptor 20 (GPR20); GAD; Ganglioside G2 (GD2) ; Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer); Ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l- 4)bDGlcp(l-l)Cer); GD3; GFRalpha4; Glycoprotein 100 (gplOO); Glypican-3 (GPC3); Gonadotropin Hormone receptor (CGHR or GR); GpA33; GpNMB; GPRC5D; Guanylyl cyclase C (GCC); Heat shock protein 70-2 mutated (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexasaccharide portion of globoH glycoceramide (GloboH); High molecular weight-melanoma associated antigen (HMWMAA); HIV1 envelope glycoprotein; HLA; HLA-DOA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DM; HLA-DOB; HLA-DP; HLA-DQ; HLA-DR; HLA-G; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human Telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; IL1 IRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza A hemagglutinin (HA); Insulin-like growth factor 1 receptor (IGF -I receptor); Interleukin 11 receptor alpha (IL-llRa); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Intestinal carboxyl esterase; KIT (CD117); KSHV K8.1; KSHV-gH; LAMP1 ; Legumain; Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1); Leutenizing hormone receptor (LHR);
Lewis(Y) antigen; Lews Ag; Livl; Locus K 9 (LY6K); Low conductance chloride channel; Lymphocyte antigen 6 complex; Lymphocyte antigen 75 (LY75); Lymphocyte-specific protein tyrosine kinase (LCK); Mammary gland differentiation antigen (NY-BR-1); Melanoma antigen recognized by T cells 1 (MelanA or MARTI); Melanoma- associated antigen 1 (MAGE-A1); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT- 2); Melanoma inhibitor of apoptosis (ML-IAP); Mesothelin; MPL; Mucin 1 cell surface associated (MUC1); N-Acetyl glucosaminyl-transf erase V (NA 17); Nectin-4; Neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-acetyl-GD2 ganglioside (OAcGD2); Olfactory receptor 51E2 (OR51E2); Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); P53 mutant; Paired box protein Pax-3 (PAX3); Paired box protein Pax-5 (PAX5); Pannexin 3 (PANX3); PDL1; P-glycoprotein; Placenta-specific 1 (PLAC1); Platelet-derived growth factor receptor beta (PDGFR-beta); Polysialic acid; Proacrosin binding protein sp32 (OY-TES1);
Prostase; Prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Prostatic acid phosphatase (PAP); Prostein; Protease Serine 21 (Testisin or PRSS21); Proteasome (Prosome Macropain) Subunit Beta Type 9 (LMP2); PTK7; Ras G12V; Ras Homolog Family Member C (RhoC); Rat sarcoma (Ras) mutant; Receptor for Advanced Gly cation Endproducts (RAGE-1); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Receptor tyrosine-protein kinase ERBB2 or Her-22/neu; Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); Sarcoma translocation breakpoints; Serine 2 (TMPRSS2) ETS fusion gene; Sialyl Lewis adhesion molecule (sLe); SLAMF4; SLAMF6; Slea (CAI 9.9 or Sialyl Lewis Antigen); Sperm protein 17 (SPA17); Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Stage-specific embryonic antigen-4 (SSEA-4); STEAP1; Survivin; Synovial sarcoma X breakpoint 2 (SSX2); TCR Gamma Alternate Reading Frame Protein (TARP); TCR-betal chain; TCR-beta2 chain; TCR-delta chain; TCR-gamma chain; TCRgamma-delta; Telomerase; TGFbetaR2; The antigen recognized by TNT antibody; Thyroid stimulating hormone receptor (TSHR); Timl-/HVCR1; Tissue Factor 1 (TF1); Tn ag; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); TNF receptor family member B cell maturation (BCMA); Transglutaminase 5 (TGS5); Transmembrane protease; TROP2; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7-related (TEM7R); Tumor protein p53 (p53); Tumor-associated glycoprotein 72 (TAG72); Tyrosinase; Tyrosinase-related protein 2 (TRP-2); Uroplakin 2 (UPK2); Vascular endothelial growth factor receptor 2 (VEGFR2); V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Wilms tumor protein (WT1); or X Antigen Family Member 1 A (XAGE1).
39. The nucleic acid molecule of any one of embodiments 28-38, wherein the sequence encoding the first, second or more linker encodes a glycine-serine linker or a cleavable linker.
40. The nucleic acid molecule of any one of embodiments 28-39, wherein the cleavable linker is a P2A, T2A, E2A, or F2A linker.
41. The nucleic acid molecule of any one of embodiments 28-40, wherein the glycine linker comprises one more of the repeat of GGGGS, GSG, or any combination thereof.
42. The nucleic acid molecule of any one of embodiments 28-41, wherein the sequence encoding the first, second or more intermediate element encodes an Internal Ribosome Entry Site (IRES).
43. The nucleic acid molecule of any one of embodiments 28-42, wherein the nucleic acid molecule optionally comprises a safety switch.
44. The nucleic acid molecule of embodiment 28, wherein a)-k) are operatively connected to each other in a 5’ to 3’ direction.
45. The nucleic acid molecule of embodiment 44, wherein a 3’ end of a) is operatively connected to 5’ end of b); a 3’ end of b) is operatively connected to a 5’ end of c); a 3’ end of c) is operatively connected to a 5’ end of d); a 3’ end of d) is operatively connected to a 5’ end of e); a 3’ end of e) is operatively connected to a 5’ end of f); optionally a 3’ end of f) is operatively connected to a 5’ end of g); optionally a 3’ end of g) is operatively connected to a 5’ end of h); optionally a 3’ end of h) is operatively connected to a 5’ end of i); optionally a 3’ end of i) is operatively connected to a 5’ end of j); a 3’ end of f) or optionally j) is operatively connected to a 5’ end of k); and a 3’ end of k) is operatively connected to a 5’ end of 1).
46. The nucleic acid molecule of any one of embodiment 1-45, wherein the nucleic acid molecule is a plasmid.
47. The nucleic acid molecule of embodiment 1-46, wherein the nucleic acid molecule is a non-integrating plasmid.
48. A host cell comprising the nucleic acid molecules of any one of embodiments 1-47.
49. A pharmaceutical composition comprising the host cell of embodiment 48.
50. A pharmaceutical composition comprising the nucleic acid molecules of any one of embodiments 1-47.
51. A method of delivering a molecule to a cell, the method comprising contacting a cell with a nucleic acid sequence of any one of embodiments 1-47 into a cell of the subject or into the subject and said nucleic acid sequence is taken up by the cell in the subject.
52. The method of embodiment 51, wherein the nucleic acid sequence is introduced into the cell or subject by electroporation, injection, sonication, transfection, transduction, gene guns, encompassed by nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell.
53. A method of preparing a virus comprising a nucleic acid molecule encoding a target molecule, the method comprising contacting a cell with the nucleic acid molecule of any one of embodiments 1-47 under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule. 54. The method of embodiment 53, wherein the cell is a producer cell (e.g., HEK293, HEK293T, HEK293FT).
55. The method of any one of embodiments 53 or 54, wherein the virus is a lentivirus.
56. The method of any one of embodiments 53-55, wherein the virus is a pseudotyped lentivirus.
57. The method of embodiment 56, wherein the pseudotyped lentivirus is pseudotyped with VSV-G, Nipah virus fusion protein (NiV-F), Nipah virus attachment protein (NiV-G), Measles virus hemagglutinin (MV-H), or Measles virus fusion protein (MV-F).
58. The method of embodiment 56, wherein the pseudotyped lentivirus is pseudotyped with a human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, immunodeficiency virus in simian (SIV), maedi / visna virus, Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, or SARS-CoV virus.
59. A method of treating a disease in a subject, the method comprising administering to the subject the virus prepared according to any one of embodiments 53-58 to treat the disease.
60. The method of embodiment 59, wherein the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
61. The method of embodiment 59, wherein the subject is in need of treatment.
Detailed Description
[0015] Embodiments provided for herein provide a platform that can, for example, allow for the integration of foreign nucleotides that can be expressed as immunogens or target molecules. The expression of immunogens or molecules can be used, for example, in methods to prevent or treat cancer and/or infectious diseases. In some embodiments, nucleic acid molecules provided herein can be used to assemble a number of DNA fragments into one nucleic acid molecule. This can be done, for example, in spite of restriction sites redundancy found at the ends and within the DNA fragments. The nucleic acid molecules provided herein can also comprise a nucleus uptake component, such as a nuclear localization signal (domain) that can facilitate the rapid plasmid integration or uptake within the nucleus. This uptake can lead to an immunological response. [0016] For example, the nucleic acid molecules provided for herein can cross the cell membrane and transfects the nucleus. Once that process occurs the foreign nucleotide is then expressed by the host’s cell transcription and translation process. A “foreign nucleotide” is one that is introduced into the cell and is not native to the cell’s genome. Examples include any of the nucleic acid molecules provided for herein and examples also include plasmids or other types of vectors provided herein. After translation, the antigen when it has reached the cell surface can be presented in conjunction with a major histocompatibility complex protein class I or class II. Without being bound by any particular theory, the antigen presenting cell (APC), such as a macrophage or dendritic cell, would take that foreign antigen and travel to a lymph node where the APC will present the antigenic target protein and/or fragment which then lead to an immune response to the antigen encoded for by the nucleic acid molecules provided herein.
[0017] It must also be noted that as used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
[0018] As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. The present disclosure modifies certain terms or values with the term “about,” however, the disclosure should also be understood to disclose the exact value as well and is simply not written out for convenience. For example, the phrase “about 9 to about 25” also discloses “9 to 25.” Additionally, a range, such the phrase “from X to Y” where X and Y are any integer includes the endpoints. For example, the phrase “from 1 to 5” means 1, 2, 3, 4, or 5.
[0019] “Administering” when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient. Nonlimiting examples of methods of administration that can be used to administer nucleic acid molecules, include, but are not limited to, transfection, electroporation, injection, sonication, or by any method in combination with other known techniques. Such combination techniques include heating and radiation. In some embodiments, the nucleic acid molecule is delivered to a muscle cell. This can be done, for example, by electroporation or other suitable technique. Electroporation of the nucleic acid molecule to the muscle or other tissue type can be done, for example, using an electroporation device. [0020] The term “animal” as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals.
[0021] The term “cloning” is used in reference to the ligating process of a nucleic acid molecule into another nucleic acid molecule, such as a plasmid. The cloned molecule can then be transferred into a host cell or subject for duplication, amplification, or administration.
[0022] The terms “cloning vector” and “cloning vector plasmid” are used to refer to a circular DNA plasmid which contains in minimum an “origin of replication” (“ori”). The origin of replication is the site of replication initiation.
[0023] As used herein, the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[0024] As used herein, the term “genetic construct” refers to the DNA or RNA molecule that comprises a nucleotide sequence which encodes the target molecule and which includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells. In some embodiments, the nucleic acid molecules provided herein are in the form of a plasmid or viral vector. In some embodiments, the genetic construct is a plasmid or a viral vector. In some embodiments, the genetic construct does not contain integration elements. In some embodiments, the plasmid does not contain, or is free of, any long terminal repeats, or inverted terminal repeats, or other sequences that would facilitate the integration of the plasmid into the subject’s, or cell’s, genome. In some embodiments, the plasmid is a non-integrating plasmid. A non-integrating plasmid is a plasmid that is not designed to integrate into a genome of the subject or a cell that comes into contact the plasmid. In some embodiments, the non-integrating plasmid is contacted with a cell without any other components that would facilitate the integration of the plasmid into a genome. For example, plasmids can be used in conjunction with gene editing platforms, such as CRISPR, that can be used to integrate portions of the plasmid into the genome. Therefore, in some embodiments, the plasmid can be used without CRISPR or CRISPR like enzyme, such as Cas9 and the like. [0025] For example, FIG.l illustrates a non-limiting configuration of a nucleic acid molecule as described herein. Referring to FIG. l, it illustrates a first promoter region (Pl) that comprises, for example and without limiting, a RSV promoter that is operatively connected to a first element (El) that comprises a 5’ LTR, as provided for herein, that is operatively connected to a second element (E2) that comprises a polynucleotide encoding a viral structural protein, such as a gag polynucleotide, for example and without limiting, a partial gag polynucleotide, as provided for herein, that is operatively connected to a third element (E3) that comprises a central polypurine tract (cPPT), as provided for herein, that is operatively connected to a second promoter (P2) that comprises, for example and without limiting, an EF-1 alpha promoter that is operatively linked to a first target molecule (TM1), as provided for herein, optionally that is operatively connected to a first linker or intermediate element (L1/IE1), as provided for herein, optionally that is operatively connected to a second target molecule (TM2), as provided for herein, wherein the first linker or intermediate element and the second target molecule can repeat “n” times, wherein “n” is any integer (e.g., 0, 1, 2, 3, 4), that is operatively linked to a fourth element (E4) that comprises a posttranscriptional regulatory element, for example and without limiting, a WPRE element, as provided for herein, that is operatively linked to a fifth element (E5) that comprises a 3’ LTR, as provided for herein.
[0026] FIG.2 illustrates a non-limiting configuration of a nucleic acid molecule as described herein. Referring to FIG.2, it illustrates a first promoter region (Pl) that comprises, for example and without limiting, a RSV promoter that is operatively connected to a first element (El) that comprises a 5’ LTR, as provided for herein, that is operatively connected to a second element (E2) that comprises a polynucleotide encoding a viral structural protein, such as a gag polynucleotide, for example and without limiting, a partial gag polynucleotide, as provided for herein, that is operatively connected to a third element (E3) that comprises a central polypurine tract (cPPT), as provided for herein, that is operatively connected to a second promoter (P2) that comprises, for example and without limiting, an EF-1 alpha promoter that is operatively linked to a first target molecule (TM1), as provided for herein, optionally that is operatively connected to a first linker or intermediate element (L1/IE1), as provided for herein, optionally that is operatively connected to a second target molecule (TM2), as provided for herein, wherein the first linker or intermediate element and the second target molecule can repeat “n” times, wherein “n” is any integer (e.g., 0, 1, 2, 3, 4), that is operatively linked to a fourth element (E4) that comprises a posttranscriptional regulatory element, for example and without limiting, a WPRE element, as provided for herein, that is operatively linked to a fifth element (E5) that comprises a 3’ LTR, as provided for herein, that is operatively connected to a sixth element (E6) that comprises a SV40 poly(A) signal, as provided for herein, that is operatively connected to a seventh element (E7) that comprises a SV40 origin of replication, as provided for herein, that is operatively connected to an eighth element (E8) that comprises an Fl origin of replication, as provided for herein, that is operatively connected to a ninth element (E9) that comprises an antibiotic resistance gene, as provided for herein. In some embodiments, (L1/IE1) and (TM2) can repeat 1, 2, 3, or 4 times encoding 1, 2, 3, or 4 target molecules that are the same or different from each other.
[0027] As used herein, “DNA construct” refers to a DNA molecule that is synthesized by the cloning steps that are consecutive with a cloning vector plasmid. This is the process that is commonly used as a means to direct gene expression to an appropriate mammalian host. This mammalian host could be cells that have been cultured in vitro or transgenic mice in vivo.
[0028] The term “DNA fragment” refers to any DNA molecule isolation that includes but is not limited to the different parts of the plasmid such as the intron, exon, reporter gene, poly(A) tail, and the different cloning sites. These DNA fragment could also include signal nucleotides, such as, the mRNA stabilization signal and the nuclear localization signal. Plasmid vector can comprise of natural and synthetic DNA fragments.
[0029] The term “enhancer region” refers to the sequence of nucleotides that is not required for targeted gene expression, but is designed to increase the gene expression levels.
[0030] As used herein, the term “expressible form” refers to gene constructs which contain the necessary regulatory elements operably linked to a coding sequence that encodes a target molecule, such that when present in the cell of the individual, the coding sequence will be expressed.
[0031] The terms “gene promoter” or “promoter” as used herein refer to and is in reference to a sequence of nucleotides that is required for gene expression.
[0032] As used herein, the term “genetic vaccine” refers to a pharmaceutical preparation that comprises a genetic construct that comprises a nucleotide sequence that encodes a target molecule and can include pharmaceutical preparations useful to invoke a therapeutic immune response. In some embodiments, the nucleotide sequence encodes a shRNA, siRNA, antisense, antibodies, hormones, and the like. Other target molecules can also be encoded for as described herein, such as, but not limited to, adjuvants.
[0033] As used herein, the term “genetic therapeutic” refers to a pharmaceutical preparation that comprises a genetic construct that comprises a nucleotide sequence that encodes a therapeutic or compensating protein.
[0034] The term “inhibiting” includes the administration of a plasmid or nucleic acid molecule to prevent the onset of the symptoms, alleviate the symptoms, reduce the symptoms, delay or decrease the progression of the disease and/or its symptoms, or eliminate the disease, condition or disorder.
[0035] As used herein, the terms “origin of replication” or “ORI” refer to sequences of nucleotides that can direct or lead to host cell duplication of a plasmid.
[0036] By “pharmaceutically acceptable,” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[0037] As used herein, the term “poly(A) tail” is in reference to the nucleotide sequence of adenine (A) nucleotides. These nucleotides are usually found at the terminal end of the messenger RNA molecule (mRNA). The poly(A) tail is incorporated at the 3’ of the end of the DNA construct that allows for enhancement of the gene expression of interest. In some embodiments, the nucleic acid molecules provided herein comprise a poly(A) tail.
[0038] As used herein, the terms “tag sequence” or “Tag” refer to sequences of nucleotides that encode a protein or peptide region that is unique, which allow for it to be detected and distinguished from any endogenous counterpart. Non-limiting examples of tags include His tag, GST tag, Calmodulin Binding Protein (CBP), Maltose-binding protein (MBP), myc tag, HA tag, FLAG tag, and the like.
[0039] As used herein, the term “target molecule” can refer to a molecule or a protein against which an immune response can be elicited and is desired to be elicited against. The target molecule (e.g., a target protein) can be, for example, an immunogenic protein or fragment thereof, which shares at least an epitope with a protein from the pathogen or undesirable celltype such as a cancer cell. The immune response directed against the target molecule can be used to induce the immune response that will protect the individual against and treat the individual for the specific infection or disease with which the target molecule is associated. In some embodiments, the target molecule is a cell surface protein or a protein (antigen) that is secreted from the cell.
[0040] A “target molecule” can also refer to a protein that is expressed in, or secreted from, a cell. The target molecule (e.g., a target protein) can be a protein of interest, a receptor, an antibody, a chimeric antigen receptor (CAR), an armored CAR, a DAP- 12, a cytokine, a binder molecule, a hormone, a siRNA, a shRNA, an antisense molecule, a microRNA, a gene editing construct (e.g., CRISPR/CAS system), and the like.
[0041] The nucleic acid molecules described herein can also be used to express different types of target molecules, such as nucleic acid molecules provided for herein, which includes, but are not limited to shRNA, siRNA, antisense, microRNAs, and the like.
[0042] As used herein, the term “binder molecule” refers to any polypeptide or polynucleotide that may be used to bind a desired target. In some embodiments, the target is a cell, such as but not limited to an immune cell, and the binder molecule binds to the cell. In some embodiments, the target is a specific protein and the binder molecule binds to said protein. Non-limiting examples of binder molecules and targets are provided for herein.
[0043] As used herein, the term “cytokine” generally refers to proteins that are important in cell signaling and act through receptors. In some embodiments, the nucleic acid molecule comprises a sequence encoding a cytokine. Non-limiting examples of cytokines include IL-1, IL- 1 -like, IL- 1 alpha, IL-1 beta, IL-IRA, IL-18, CD132, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, CD131, IL-3, IL-5, GM-CSF, IL-6-like, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL- 14, IL- 16, IL- 17, IFN-alpha, IFN-beta, IFN-gamma, CD 154, LT-beta, TNF-alpha, TNF-beta, 4- 1BBL, ARIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-beta, TGF-betal, TGF-beta2, TGF-beta3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, and CXCL8 (formerly IL- 18). In some embodiments, the nucleic acid molecule comprises a sequence encoding a cytokine selected from the group comprising IL-1, IL- 1 -like, IL-1 alpha, IL-1 beta, IL-IRA, IL-18, CD132, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, CD131, IL-3, IL-5, GM- CSF, IL-6-like, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-alpha, IFN-beta, IFN-gamma, CD154, LT-beta, TNF-alpha, TNF-beta, 4-1BBL, ARIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-beta, TGF-betal, TGF-beta2, TGF-beta3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, and CXCL8 (formerly IL- 18). [0044] As used herein, the term “therapeutic” means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. In part, embodiments are directed to the treatment of cancer or the decrease in proliferation of cells. In part, embodiments are directed to the treatment of infections or infectious agents.
[0045] A “therapeutically effective amount” or “effective amount” of a therapeutic is a predetermined amount calculated to achieve the desired effect, /.< ., stimulate an immune response. The activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate.
[0046] The terms “treat,” “treated,” or “treating” as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results.
[0047] The term “untranslated region” refers to the sequences of nucleotides that cover the nucleotide region that does not code for a protein found within a mRNA molecule. These regions that are not translated can be found at the 5’ and 3’ regions of the mRNA molecule. In some embodiments, the nucleic acid molecule provided herein that encodes a target molecule or a fragment of the target molecule comprises an untranslated region.
[0048] In some embodiments, the nucleic acid molecule and methods disclosed herein can be utilized with or on a subject in need of such treatment, which can also be referred to as “in need thereof.” As used herein, the phrase “in need thereof’ means that the subject has been identified as having a need for the particular method or treatment and that the treatment has been given to the subject for that particular purpose with a specific intent.
[0049] In some embodiments, the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a target molecule. In some embodiments, the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a viral structural protein that is operatively connected to a polynucleotide encoding a promoter that is operatively linked to a polynucleotide encoding a target molecule. In some embodiments, the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a binder molecule that is operatively connected to a polynucleotide encoding a promoter that is operatively linked to a polynucleotide encoding a target molecule. In some embodiments, the nucleic acid sequence comprises a polynucleotide encoding a promoter that is operatively connected to a polynucleotide encoding a viral structural protein and a binder molecule that is operatively connected to a polynucleotide encoding a promoter that is operatively linked to a polynucleotide encoding a target molecule. In some embodiments, the nucleic acid sequence comprises: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein, a polynucleotide sequence encoding a central polypurine tract, a polynucleotide sequence encoding a second constitutive promoter, a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.), and a polynucleotide encoding a posttranscriptional regulatory element.
[0050] In some embodiments, the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule. In some embodiments, the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules. In some embodiments, the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
[0051] In some embodiments, the nucleic acid sequence comprises: a sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, and optionally a sequence encoding an amino acid sequence of a second target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid sequence comprises: a sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, and a sequence encoding an amino acid sequence of a third target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid sequence comprises: a sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, a sequence encoding an amino acid sequence of a third target molecule, a sequence encoding an amino acid sequence of a third linker or intermediate element, and a sequence encoding an amino acid sequence of a fourth target molecule, wherein the sequences are operatively connected to one another.
[0052] In some embodiments, the nucleic acid sequence comprises a polycistronic expression system. In some embodiments, the polycistronic expression system is a bicistronic expression system. In some embodiments, the polycistronic expression system is a tricistronic expression system. In some embodiments, the polycistronic expression system is a quadcistronic expression system. In some embodiments, the bicistronic expression system comprises a sequence encoding an amino acid sequence of a first target molecule operatively connected to a sequence encoding an amino acid sequence of a first linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a second target molecule. In some embodiments, the tricistronic expression system comprises a sequence encoding an amino acid sequence of a first target molecule operatively connected to a sequence encoding an amino acid sequence of a first linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a second target molecule operatively connected to a sequence encoding an amino acid sequence of a second linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a third target molecule. In some embodiments, the quadcistronic expression system comprises a sequence encoding an amino acid sequence of a first target molecule operatively connected to a sequence encoding an amino acid sequence of a first linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a second target molecule operatively connected to a sequence encoding an amino acid sequence of a second linker or intermediate element operatively connected to a sequence encoding an amino acid sequence of a third target molecule operatively connected to a sequence encoding an amino acid sequence of a third linker or intermediate element operatively linked to a sequence encoding an amino acid sequence of a fourth target molecule. In some embodiments, the polycistronic expression system comprises one or more sequence encoding an amino acid sequence of one or more target molecule operatively linked to one or more sequence encoding an amino acid sequence of a one or more linker or intermediate element. In some embodiments, the one or more sequence encoding one or more target molecule repeats “n” times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats “n-1” times. In some embodiments, “n” is an integer (e.g., 0, 1, 2, 3, 4). In some embodiments, the one or more sequence encoding one or more target molecules repeats 1 time, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 0 times. In some embodiments, the one or more sequence encoding one or more target molecules repeats 2 times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 1 time. In some embodiments, the one or more sequence encoding one or more target molecules repeats 3 times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 2 times. In some embodiments, the one or more sequence encoding one or more target molecules repeats 4 times, and the one or more sequence encoding an amino acid sequence of one or more linker or intermediate element repeats 3 times.
[0053] In some embodiments, the first, second, third, fourth, or more polypeptides encoding the target molecule are different. In some embodiments, the first, second, third, fourth, or more polypeptides encoding the target molecule are the same. In some embodiments, the first, second, third, fourth, or more polypeptides encoding the first, second, third, fourth, or more target molecule are different. In some embodiments, the first, second, third, fourth, or more polypeptides encoding the first, second, third, fourth, or more target molecule are the same. In some embodiments, the first, second, third, fourth, or more target molecules are different. In some embodiments, the first, second, third, fourth, or more target molecules are the same.
[0054] In some embodiments, the first, second, third, or more polypeptides encoding the linker or intermediate element are different. In some embodiments, the first, second, third, or more polypeptides encoding the linker or intermediate element are the same. In some embodiments, the first, second, third, or more polypeptides encoding the first, second, third, or more linker or intermediate element are different. In some embodiments, the first, second, third, or more polypeptides encoding the first, second, third, or more linker or intermediate element are the same. In some embodiments, the first, second, third, or more linker or intermediate element are different. In some embodiments, the first, second, third, or more linker or intermediate element are the same.
[0055] In some embodiments, the nucleic acid sequentially comprises in a 5’ to 3’ direction: a. a polynucleotide encoding a RSV promoter; b. a polynucleotide encoding a 5’ LTR; c. a polynucleotide encoding a partial gag, wherein the partial gag further comprises HIV-1 psi element, rev response element (RRE), and a Gp41 peptide; d. a polynucleotide encoding a central polypurine tract (cPPT); e. a polynucleotide encoding an EF-1 alpha promoter; f. a polynucleotide encoding a first target molecule; g. optionally a polynucleotide encoding a first linker or first intermediate element; h. optionally a polynucleotide encoding a second target molecule; i. optionally a polynucleotide encoding a second or more (e.g., third, fourth, fifth) linker or second or more (e.g., third, fourth, fifth) intermediate element; j. optionally a polynucleotide encoding a third or more (e.g., fourth, fifth, sixth) target molecule; k. a polynucleotide encoding a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE); and l. a polynucleotide encoding a 3’ LTR; wherein the sequences are operatively connected to one another, and optionally wherein g)-j) repeat n times, wherein n is any integer (e.g., 0, 1, 2, 3, 4). In some embodiments, the nucleic acid molecule comprises one or more polynucleotides encoding one or more target molecules (e.g., n is 1, 2, 3, 4). In some embodiments, the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule. In some embodiments, the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules. In some embodiments, wherein the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci stronic) expression system. In some embodiments, the sequence encoding the first, second, or more target molecule encodes a reporter gene, a protein of interest, a chimeric antigen receptor (CAR), an armored CAR, a DAP-12, a cytokine, a siRNA, a shRNA, an antisense molecule, a microRNA, or a gene editing construct (e.g., CRISPR/CAS system). In some embodiments, a)-k) are operatively connected to each other in a 5’ to 3’ direction. In some embodiments, a 3’ end of a) is operatively connected to 5’ end of b); a 3’ end of b) is operatively connected to a 5’ end of c); a 3’ end of c) is operatively connected to a 5’ end of d); a 3’ end of d) is operatively connected to a 5’ end of e); a 3’ end of e) is operatively connected to a 5’ end of f); optionally a 3’ end of f) is operatively connected to a 5’ end of g); optionally a 3’ end of g) is operatively connected to a 5’ end of h); optionally a 3’ end of h) is operatively connected to a 5’ end of i); optionally a 3’ end of i) is operatively connected to a 5’ end of j); a 3’ end of f) or optionally j) is operatively connected to a 5’ end of k); and a 3’ end of k) is operatively connected to a 5’ end of 1).
[0056] In some embodiments, the nucleic acid molecules provided for herein do not contain or do not comprise (e.g. free of) a sequence encoding a fragment of a target molecule, or alternatively, the nucleic acid molecule only encodes for one fragment of a target molecule if the full length protein is not encoded for by the nucleic acid molecule.
[0057] In some embodiments, the nucleic acid sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter. An “inducible promoter” is a promoter which activity can be regulated. Non-limiting examples of inducible promoters include, but are not limited to, chemically inducible promoters (e.g., tetracycline system, pLac promoter, pBad promoter, AlcA promoter, or LexA promoter), temperature inducible promoters (e.g., Hsp70-derived promoter, or Hsp90-derived promoter), and light inducible promoters (e.g., YFI system). A “tissue-specific promoter” is a promoter that limits the expression or largely limits the expression to a specific tissue type. Non-limiting examples of tissue-specific promoters include, but are not limited to, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (haematopoietic cells), CD68 (macrophages), desmin (muscle), elastase- 1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin (differentiating cells and healing tissue), Flt-1 (endothelial cells), GFAP (astrocytes), GPIIb (megakaryocytes), ICAM-2 (endothelial cells), mouse INF-beta (hematopoietic cells), Mb (muscle), Nphsl (podocytes), OG-2 (osteoblasts and odonblasts), SP-B (lung), SYN1 (neurons), WASP (hematopoietic cells), SV40/bAlb (liver), SV40/hAlb (liver), SV40/CD43 (leukocytes and platelets), SV40/CD45 (hematopoietic cells), and NSE/RU5’ (mature neurons). The promoter can also be a cell specific promoter so that the nucleic acid molecule’s expression is limited to a specific cell or subset of cells. In some embodiments, the promoter is a constitutive promoter. Non-limiting examples of constitutive promoters include, but are not limited to, CMV, EF-1 alpha, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, GALI, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, and U6. Examples of promoters that can be used include, but are not limited EF-1 alpha, SV40, Rous Sarcoma virus (RSV), Mason-Pfizer monkey virus-CTE, and CTE+rev.
[0058] In some embodiments, the RSV promoter is encoded by a sequence of tgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaag caccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatg gattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaac (SEQ ID NO: 1)
[0059] In some embodiments, the EF-1 alpha promoter is encoded by a sequence of cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttt tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccag aacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactt ccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgctt aaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggca ccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggca agatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggccc gtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtct caagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcc cggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggac gcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttc atgtgactccactgagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttggagtacgtcgtctttaggtt ggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttg atgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttct tccatttcaggtgtcgtga (SEQ ID NO: 2) These non-limiting examples of promoters can also be referred to as constitutive promoters. These non-limiting examples of promoters are known and can be incorporated into the nucleic acid molecule. However, this list is merely for example purposes only as there are numerous promoters that can be used to drive the expression of a target molecule (e.g., a protein, RNAi components) from the nucleic acid molecule. As provided for herein, the nucleic acid molecule encoding the promoter is operably connected to the nucleic acid molecule encoding the target molecule to control, regulate, or drive the expression of the target protein in a cell.
[0060] In some embodiments, the nucleic acid molecule comprises a nucleic acid molecule encoding a target molecule. In some embodiments, the target molecule is a protein of interest, antibodies, hormones, chimeric antigen receptors (CARs), armored CARs, receptors, binder molecules, DAP- 12, fusion proteins, reporter proteins, GFP, RFP, SEAP YFP, luciferase, betagalactosidase, and the like. In some embodiments, these target molecules can be expressed with or without the other elements of the plasmid, such as the linker, and the like. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody is a single domain antibody (sdAb). In some embodiments, the antibody is a single chain fragment variable (scFv).
[0061] As used herein, a “reporter gene” refers to a polynucleotide sequence encoding a protein product that can generate, under appropriate conditions, a detectable signal that allows detection for indicating the presence and/or quantity of the reporter gene protein product. Non-limiting examples of reporter gene products include, but are not limited to, enzymes, bioluminescent molecules, or fluorescent molecules. In some embodiments, the reporter gene product is a GFP, RFP, SEAP YFP, luciferase, beta-galactosidase, and the like.
[0062] As provided for herein, the target molecule can be an antibody that is expressed from the nucleic acid molecule (e.g. plasmid). The term “antibody” as used herein is meant in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies and antibody fragments, such as scFv or hexabodies (PLOS Biology | DOI: 10.1371/journal.pbio.1002344 January 6, 2016, which is hereby incorporated by reference in its entirety).
[0063] The term “humanized antibody”, “engineered antibody”, “human framework adapted”, and “HF A” as used herein, is intended to include antibodies having variable region frameworks derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region can be derived from such human sequences, e.g., human germline sequences, or naturally occurring (e.g., allotypes) or mutated versions of human germline sequences. The humanized antibodies may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
[0064] In some embodiments, the binder molecule binds to a cell. In some embodiments, the cell is any desired cell. In some embodiments, the cell is an immune cell. In some embodiments, the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD 19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte; Her2/Neu+ cancer cell; NKG2D+ natural killer cell; SLC1A3+ astrocyte; SLC7A10+ adipocyte.
[0065] In some embodiments, the binder molecule is any polypeptide or polynucleotide that may be used to bind a desired target. In some embodiments, the binder molecule is any polypeptide, polynucleotide, or fragment thereof that binds to CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, A glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors; A glycosylated CD43 epitope expressed on non-hematopoietic cancers; A kinase anchor protein 4 (AKAP-4); Adrenoceptor beta 3 (ADRB3); AFP; Anaplastic lymphoma kinase (ALK); Androgen receptor; Angiopoietin-binding cell surface receptor 2 (Tie 2); Auto antibody to desmoglein 1 (Dsgl); Auto antibody to desmoglein 3 (Dsg3); B7H3 (CD276); Biotin; Bone marrow stromal cell antigen 2 (BST2); BST1/CD157; Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la); Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R; CD276/B7H3; CD300 molecule-like family member f (CD300LF); CDH1-CD324; CDH6; CDH17; CDH19; Chromosome X open reading frame 61 (CXORF61); Claudin 6 (CLDN6); Claudinl8.2 (CLD18A2 or CLDN18A.2); CMV pp65; C-MYC epitope Tag; Cripto; CS1 (also referred to as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24); CSF2RA (GM-CSFR-alpha); C-type lectin domain family 12 member A (CLEC12A); C-type lectin-like molecule-1 (CLL-1 or CLECL1); Cyclin Bl; Cytochrome P450 IB 1 (CYP1B 1); DLL3; EBV-EBNA3c; EGF-bke module- containing mucin-like hormone receptor-like 2 (EMR2); Elongation factor 2 mutated (ELF2M); Ephrin B2; Ephrin type-A receptor 2 (EphA2); Epidermal growth factor receptor (EGFR); Epidermal growth factor receptor variant III (EGFRviii); Epithelial cell adhesion molecule (EPCAM); ERG; ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Fc fragment of IgA receptor (FCAR or CD89); Fc receptor-like 5 (FCRL5); Fibroblast activation protein alpha (FAP); FITC; Fms Like Tyrosine Kinase 3 (FLT3); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Follicle stimulating hormone receptor (FSHR); Fos-related antigen 1; Fucosyl-GMl; G protein coupled receptor class C group 5 member D (GPRC5D); G protein-coupled receptor 20 (GPR20); GAD; Ganglioside G2 (GD2) ; Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l- l)Cer); Ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l- 4)bDGlcp(l-l)Cer); GD3; GFRalpha4; Glycoprotein 100 (gplOO); Glypican-3 (GPC3); Gonadotropin Hormone receptor (CGHR or GR); GpA33; GpNMB; GPRC5D; Guanylyl cyclase C (GCC); Heat shock protein 70-2 mutated (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexasaccharide portion of globoH glycoceramide (GloboH); High molecular weight-melanoma associated antigen (HMWMAA); HIV1 envelope glycoprotein; HLA; HLA-DOA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DM; HLA-DOB; HLA-DP; HLA-DQ; HLA-DR; HLA-G; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human Telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; IL1 IRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza A hemagglutinin (HA); Insulin-like growth factor 1 receptor (IGF -I receptor); Interleukin 11 receptor alpha (IL-llRa); Interleukin- 13 receptor subunit alpha-2 (IL- 13Ra2 or CD213A2); Intestinal carboxyl esterase; KIT (CD117); KSHV K8.1; KSHV-gH; LAMP1 ; Legumain; Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Leutenizing hormone receptor (LHR); Lewis(Y) antigen; Lews Ag; Livl; Locus K 9 (LY6K); Low conductance chloride channel; Lymphocyte antigen 6 complex; Lymphocyte antigen 75 (LY75); Lymphocyte-specific protein tyrosine kinase (LCK); Mammary gland differentiation antigen (NY-BR-1); Melanoma antigen recognized by T cells 1 (MelanA or MARTI); Melanoma- associated antigen 1 (MAGE-A1); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Melanoma inhibitor of apoptosis (ML-IAP); Mesothelin; MPL; Mucin 1 cell surface associated (MUC1); N-Acetyl glucosaminyl-transferase V (NA17); Nectin-4; Neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-acetyl-GD2 ganglioside (OAcGD2); Olfactory receptor 51E2 (OR51E2); Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); P53 mutant; Paired box protein Pax-3 (PAX3); Paired box protein Pax-5 (PAX5); Pannexin 3 (PANX3); PDL1; P-glycoprotein; Placenta-specific 1 (PLAC1); Platelet-derived growth factor receptor beta (PDGFR-beta); Polysialic acid; Proacrosin binding protein sp32 (OY-TES1); Prostase; Prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Prostatic acid phosphatase (PAP); Prostein; Protease Serine 21 (Testisin or PRSS21); Proteasome (Prosome Macropain) Subunit Beta Type 9 (LMP2); PTK7; Ras G12V; Ras Homolog Family Member C (RhoC); Rat sarcoma (Ras) mutant; Receptor for Advanced Gly cation Endproducts (RAGE-1); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Receptor tyrosine-protein kinase ERBB2 or Her-22/neu; Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); Sarcoma translocation breakpoints; Serine 2 (TMPRSS2) ETS fusion gene; Sialyl Lewis adhesion molecule (sLe); SLAMF4; SLAMF6; Slea (CAI 9.9 or Sialyl Lewis Antigen); Sperm protein 17 (SPA17); Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Stage-specific embryonic antigen-4 (S SEA-4); STEAP1; Survivin; Synovial sarcoma X breakpoint 2 (SSX2); TCR Gamma Alternate Reading Frame Protein (TARP); TCR-betal chain; TCR-beta2 chain; TCR-delta chain; TCR-gamma chain; TCRgamma-delta; Telomerase; TGFbetaR2; The antigen recognized by TNT antibody; Thyroid stimulating hormone receptor (TSHR); Timl-/HVCR1; Tissue Factor 1 (TF1); Tn ag; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); TNF receptor family member B cell maturation (BCMA); Transglutaminase 5 (TGS5); Transmembrane protease; TROP2; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7-related (TEM7R); Tumor protein p53 (p53); Tumor-associated glycoprotein 72 (TAG72); Tyrosinase; Tyrosinase-related protein 2 (TRP-2); Uroplakin 2 (UPK2); Vascular endothelial growth factor receptor 2 (VEGFR2); V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Wilms tumor protein (WT1); or X Antigen Family Member 1A (XAGE1). In some embodiments, the TBD binds to CD7. In some embodiments, the TBD binds to CD8. In some embodiments, the TBD is an antibody. It is to be understood that in the context of the present disclosure “antibody” not only refers to a “complete” antibody comprising two identical heavy chains, two identical light chains, and two antigen binding fragments, but also refers to antibodies of any isotype, fragments of antibodies including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, single domain light chain antibodies, bi-specific antibodies, multi-specific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In some embodiments, the antibody is selected from the group comprising a scFv, Fab, VHH, single domain antibody, and the like. In some embodiments, the antibody is a scFv. In some embodiments the antibody is a Fab. In some embodiments, the antibody is a VHH. In some embodiments, the antibody is a single domain antibody.
[0066] In general, antibodies are proteins or polypeptides that exhibit binding specificity to a specific antigen. Intact antibodies are heterotetrameric proteins, composed of two light chains and two heavy chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAi, IgA2, IgGi, IgG2, IgGs and IgG4.
[0067] The term “antibody fragment” means a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, single chain antibody molecules and multispecific antibodies formed from at least two intact antibodies. In some embodiments, the antibody can be a single-chain variable fragment (scFv) antibody.
[0068] As is known to the skilled artisan, a peptide having a specific amino acid sequence can be encoded by different nucleic acid molecules because of the fact that the genetic code is degenerate. In some embodiments, the nucleic acid molecule’s sequence is optimized. The sequence can be optimized based upon codon usage and frequency depending upon the cell type that is being used or the subject that is being administered the nucleic acid molecule. Codon optimization can be useful to maximize protein expression. This can be done by optimizing the codon usage of mRNA sequences for mammalian cells. For example, changing the immunogen gene sequences encoding infectious target proteins used within nucleic acid molecule can be used to increase expression and the expressed protein immunogenicity. Methods of optimizing codon usage are known. Additionally, the nucleic acid molecules provided for herein may have stop codons in the sequence. One of skill in the art would understand that the stop codons could be replaced by degenerate stop codons. Stop codons are known to be U(T)AA, U(T)AG, and U(T)GA.
[0069] Thus, the nucleic acid sequences shown above are simply for illustration purposes only and not intended to be limiting to those that encode for the relevant amino acid sequence of the target protein. Additionally, in some embodiments, less than the full length of the target protein is used. In some embodiments, at least 5, 10, 15, or 20 amino acid residues, independently, from the N- and/or C-terminus are not encoded for by the nucleic acid molecule. In some embodiments, the target protein is larger than the fragment of the target protein that is encoded for by the nucleic acid molecule.
[0070] In some embodiments, the proteins encoded by the nucleic acid molecules provided herein comprise conservative substitutions. Conservative substitutions are known to the skilled artisan.
[0071] In some embodiments, the nucleic acid molecule comprises a different nucleic acid molecule comprising a nucleic sequence that encodes for a fragment of the target protein.
[0072] In some embodiments, the nucleic acid sequence comprises a sequence encoding a linker such as, but not limited to a cleavable linker, a glycine-serine or a glycine-alanine linker. In some embodiments, the cleavable linker is selected from P2A, T2A, E2A, and F2A. In some embodiments, the nucleic acid sequence comprises a sequence encoding a P2A linker. In some embodiments, the nucleic acid sequence comprises a sequence encoding a T2A linker. In some embodiments, the nucleic acid sequence comprises a sequence encoding a E2A linker. In some embodiments, the nucleic acid sequence comprises a sequence encoding a F2A linker. In some embodiments, P2A has the sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 3). In some embodiments, T2A has the sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 4). In some embodiments, E2A has the sequence of GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 5). In some embodiments, F2A has the sequence of GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6). In some embodiments, the glycineserine linker comprises the sequence of GGGGS (SEQ ID NO: 21), or GSG. The linker can also comprise repeats of this sequence. In some embodiments, the linker comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS (SEQ ID NO: 21), or GSG repeats. The linkers can also be mixed with one another. In some embodiments, the nucleic acid molecules provided for herein do not contain or do not comprise (e.g. free of) a sequence encoding a linker.
[0073] In some embodiments, the nucleic acid sequence comprises a sequence encoding an intermediate element, such as, but not limited to an Internal Ribosome Entry Site (IRES). In some embodiments, IRES is encoded by a sequence of: gagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaag gtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcag gcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggc ggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaa ggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgt ttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa (SEQ ID NO: 7)
[0074] In some embodiments, the nucleic acid sequence comprises a sequence encoding a 5’ LTR and 3’ LTR. In some embodiments, 5’ LTR and 3’ LTR elements comprise an R region and a U5 region. In some embodiments, 5’ LTR is encoded by a sequence of: gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataa agcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagt cagtgtggaaaatctctagca (SEQ ID NO: 8) [0075] In some embodiments, 3’ LTR is encoded by a sequence of: tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctga gcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgt gtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagca (SEQ ID NO: 9)
[0076] In some embodiments, the nucleic acid sequence comprises a sequence encoding a viral structural protein. In some embodiments, the viral structural protein is encoded by a gag element. In some embodiments, the gag element is a partial gag element. In some embodiments, the partial gag element comprises a HIV-1 psi element, a rev response element (RRE), and a Gp41 peptide. In some embodiments, the partial gag element is encoded by a sequence of: cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgc acggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagag atgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggg gaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgt tagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttaga tcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagaca agatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggagg agatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccac caaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttggga gcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgc agcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcag ctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaa actcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacct ggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaa gaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtg gtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagt taggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaata gaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtat (SEQ ID NO: 10)
[0077] In some embodiments, the nucleic acid sequence comprises a sequence encoding a central polypurine tract (cPPT). In some embodiments, the cPPT is encoded by a sequence of: tagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtgga tatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatg gccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcgg ggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaaga aaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaa aagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaa agaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggct (SEQ ID NO: 11)
[0078] In some embodiments, the nucleic acid sequence comprises a sequence encoding a posttranscriptional regulatory element. In some embodiments, the posttranscriptional regulatory element is a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE is encoded by a sequence of: atcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttat gaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttgggg cattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcct gccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcc tttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatcca gcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcgg atctccctttgggccgcctccccgcctg (SEQ ID NO: 12)
[0079] In some embodiments, the nucleic acid sequence comprises a sequence encoding a polyadenylation signal. In some embodiments, the polyadenylation signal is an SV40 poly(A) signal. In some embodiments, the SV40 poly(A) signal is encoded by a sequence of: aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcat tctagttgtggtttgtccaaactcatcaatgtatctta (SEQ ID NO: 13)
[0080] In some embodiments, the nucleic acid sequence comprises a sequence encoding an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene confers resistance to an antibiotic selected from the group comprising kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol. In some embodiments, the nucleic acid comprises a sequence encoding an ampicillin resistance gene. In some embodiments, the ampicillin resistance gene is encoded by a sequence of: atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctg gtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagat ccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgt attgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcaca gaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggc caacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcg ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatg gcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggag gcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtg agcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgg ggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaa (SEQ ID NO: 14)
[0081] In some embodiments, the ampicillin resistance gene is encoded by a sequence of: cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttca ataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgt ttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaact ggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgactt ggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataac catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaa catgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgac accacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaa caattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattg ctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgt atcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctc actgattaagcattggtaa (SEQ ID NO: 15)
[0082] In some embodiments, the nucleic acid molecule comprises an origin of replication. In some embodiments, the origin of replication is a Fl origin of replication (ori). In some embodiments, the Fl ori is encoded by a sequence of: acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcg ccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggg ggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagt gggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact ggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatga gctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaattt (SEQ ID NO: 16)
[0083] In some embodiments, the nucleic acid molecule comprises an SV40 origin of replication (ori). In some embodiments, the SV40 ori is encoded by a sequence of: atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc (SEQ ID NO: 17)
[0084] In some embodiments, the nucleic acid molecule comprises a safety switch. Without wishing to be bound to a particular theory, the basic concept of safety switches and suicide genes is described in (Jones et al., Front Pharmacol.; 5: 254. Doi: 10.3389). In some embodiments, the safety switch is an inducible suicide gene, such as and without limiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD), Fas ligand, or cytochrome P450. In some embodiments, safety switch can include accessory tags. Non-limiting examples of accessory tags include a c-myc tag, CD20, CD52 (Campath), truncated EGFR gene (EGFRt) or a part or a combination thereof.
[0085] In some embodiment, the sequence encoding a target molecule may be operably linked to other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences, e.g., to mimic the native expression. In some embodiments, the Kozak consensus sequence is gccaccatgg (SEQ ID NO: 18).
[0086] In some embodiments, the nucleic acid molecules provided for herein do not contain or do not comprise (e.g. free of) a sequence encoding a linker.
[0087] The other elements, which may or may not be present, of the nucleic acid molecule can be operably linked to the sequence encoding the protein.
[0088] In some embodiments, a pharmaceutical composition is provided that comprises the nucleic acid molecule comprising: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein, a polynucleotide sequence encoding a central polypurine tract, a polynucleotide sequence encoding a second constitutive promoter, a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.), and a polynucleotide encoding a posttranscriptional regulatory element.
[0089] In some embodiments, a pharmaceutical composition is provided that comprises the sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, and optionally a sequence encoding an amino acid sequence of a second target molecule, wherein the sequences are operatively connected to one another. In some embodiments, a pharmaceutical composition is provided that comprises the sequence encoding a promoter, a sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, and a sequence encoding an amino acid sequence of a third target molecule, wherein the sequences are operatively connected to one another. In some embodiments, a pharmaceutical composition is provided that comprises the sequence encoding an amino acid sequence of a first target molecule, optionally a sequence encoding an amino acid sequence of a first linker or intermediate element, optionally a sequence encoding an amino acid sequence of a second target molecule, optionally a sequence encoding an amino acid sequence of a second linker or intermediate element, a sequence encoding an amino acid sequence of a third target molecule, a sequence encoding an amino acid sequence of a third linker or intermediate element, and a sequence encoding an amino acid sequence of a fourth target molecule, wherein the sequences are operatively connected to one another.
[0090] Also provided herein are the proteins or peptides encoded by the nucleic acid molecules described herein.
[0091] In some embodiments, nucleic acid molecules are provided, wherein the nucleic acid molecule comprises a polynucleotide encoding a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein. In some embodiments, the promoter is a RSV promoter. In some embodiments, the promoter is an EF-1 alpha promoter. In some embodiments, the nucleic acid molecule comprises a 5’LTR sequence. Examples of 5’LTR sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a partial gag sequence. Examples of partial gag sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a cPPT sequence. Examples of cPPT sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a second promoter. In some embodiments, the second promoter is a constitutive promoter. In some embodiments, the second promoter is an inducible promoter. In some embodiments, the second promoter is a tissuespecific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein. In some embodiments, the second promoter is a RSV promoter. In some embodiments, the second promoter is an EF-1 alpha promoter. In some embodiments, the second promoter is a tissue specific promoter selected from B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4. In some embodiments, the second promoter is a ZAP70 promoter. In some embodiments, the second promoter is a CD4 promoter. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a target molecule. Examples of the target molecules and proteins are provided for herein. These examples are for illustrative purposes only and are intended to be non-limiting. The target molecule or protein can be any molecule or protein encoded for by a nucleic acid molecule that one chooses to be encoded for by the nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a WPRE element. Examples of WPRE element sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a 3’LTR. Examples of 3’LTR sequences are provided for herein. The elements provided for herein can be operatively connected to one another. If all of the elements are present, the nucleic acid molecule can encode a protein comprising the amino acid sequence of a target molecule or protein.
[0092] In some embodiments, nucleic acid molecules are provided, wherein the nucleic acid molecule comprises a polynucleotide encoding a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein. In some embodiments, the promoter is a RSV promoter. In some embodiments, the promoter is an EF-1 alpha promoter. In some embodiments, the nucleic acid molecule comprises a 5’LTR sequence. Examples of 5’LTR sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a gag sequence, for example and without limiting, a partial gag sequence. Examples of partial gag sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a cPPT sequence. Examples of cPPT sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a second promoter. In some embodiments, the second promoter is a constitutive promoter. In some embodiments, the second promoter is an inducible promoter. In some embodiments, the second promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissuespecific promoters that can be used are provided for herein. In some embodiments, the second promoter is a RSV promoter. In some embodiments, the second promoter is an EF-1 alpha promoter. In some embodiments, the second promoter is a tissue specific promoter selected from B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4. In some embodiments, the second promoter is a ZAP70 promoter. In some embodiments, the second promoter is a CD4 promoter. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a target molecule. Examples of the target molecules and proteins are provided for herein. These examples are for illustrative purposes only and are intended to be non-limiting. The target molecule or protein can be any molecule or protein encoded for by a nucleic acid molecule that one chooses to be encoded for by the nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a linker. Non-limiting examples of linkers are provided for herein. For example, the linker can be any peptide linker that can be encoded for by a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an intermediate element. Non-limiting examples of intermediate elements are provided for herein. For example, the intermediate element can be an IRES element. In some embodiments, the nucleic acid molecule comprises a second polynucleotide encoding a second target molecule. Examples of the target molecules and proteins are provided for herein. These examples are for illustrative purposes only and are intended to be non-limiting. The target molecule or protein can be any molecule or protein encoded for by a nucleic acid molecule that one chooses to be encoded for by the nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a posttranscriptional regulatory element, for example and without limiting, a WPRE element. Examples of WPRE element sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a 3’LTR. Examples of 3’LTR sequences are provided for herein. The elements provided for herein can be operatively connected to one another. If all of the elements are present the nucleic acid molecule can encode a protein comprising the amino acid sequence of the first molecule or protein; the amino acid sequence of the linker, and the amino acid sequence of the second molecule or protein.
[0093] In some embodiments, the nucleic acid molecule comprises a polycistronic expression system as provided for herein.
[0094] In some embodiments, nucleic acid molecules are provided, wherein the nucleic acid molecule comprises a polynucleotide encoding a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissue-specific promoters that can be used are provided for herein. In some embodiments, the promoter is a RSV promoter. In some embodiments, the promoter is an EF-1 alpha promoter. In some embodiments, the nucleic acid molecule comprises a 5’LTR sequence. Examples of 5’LTR sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a gag sequence, for example and without limiting, a partial gag sequence. Examples of partial gag sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a cPPT sequence. Examples of cPPT sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a second promoter. In some embodiments, the second promoter is a constitutive promoter. In some embodiments, the second promoter is an inducible promoter. In some embodiments, the second promoter is a tissue-specific promoter. Non-limiting examples of constitutive promoters that can be used are provided for herein. Non-limiting examples of inducible promoters that can be used are provided for herein. Non-limiting examples of tissuespecific promoters that can be used are provided for herein. In some embodiments, the second promoter is a RSV promoter. In some embodiments, the second promoter is an EF-1 alpha promoter. In some embodiments, the second promoter is a tissue specific promoter selected from B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4. In some embodiments, the second promoter is a ZAP70 promoter. In some embodiments, the second promoter is a CD4 promoter. In some embodiments, the nucleic acid molecule comprises a polycistronic expression system as provided for herein. In some embodiments, the polycistronic expression system allows for expression of one or more target molecule and one or more linker or intermediate element. Examples of polycistronic expression systems are provided for herein. In some embodiments, the nucleic acid molecule comprises a posttranscriptional regulatory element, for example and without limiting, a WPRE element. Examples of WPRE element sequences are provided for herein. In some embodiments, the nucleic acid molecule comprises a 3’LTR. Examples of 3’LTR sequences are provided for herein. The elements provided for herein can be operatively connected to one another. If all of the elements are present the nucleic acid molecule can encode a protein comprising the amino acid sequence of the first molecule or protein; the amino acid sequence of the first or more linker, and the amino acid sequence of the second or more molecule or protein.
[0095] In some embodiments, the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, and a sequence encoding a first target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, and optionally a sequence encoding a second target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, and optionally a sequence encoding a third target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises: a sequence encoding a first promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding a second promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, optionally a sequence encoding a third target molecule, optionally a sequence encoding a third linker or intermediate element, and optionally a sequence encoding a fourth target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the linker or intermediate element is as provided for herein. In some embodiments, the target molecule is as provided for herein.
[0096] In some embodiments, the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, and a sequence encoding a first target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, and optionally a sequence encoding a second target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, and optionally a sequence encoding a third target molecule, wherein the sequences are operatively connected to one another. In some embodiments, the nucleic acid molecule comprises: a sequence encoding a RSV promoter, a sequence encoding a 5’ LTR, a sequence encoding a partial gag, a sequence encoding a central polypurine tract (cPPT), a sequence encoding an EF-1 alpha promoter, a sequence encoding a first target molecule, optionally a sequence encoding a first linker or intermediate element, optionally a sequence encoding a second target molecule, optionally a sequence encoding a second linker or intermediate element, optionally a sequence encoding a third target molecule, optionally a sequence encoding a third linker or intermediate element, and optionally a sequence encoding a fourth target molecule, wherein the sequences are operatively connected to one another. In some embodiments, linkers and intermediate elements are as provided for herein. In some embodiments, the linker or intermediate element is as provided for herein. In some embodiments, the target molecule is as provided for herein.
[0097] In some embodiments, the nucleic acid molecule comprises a sequence of gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtg cttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgc ccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggc gaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagc gggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagca gggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcag acaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagct ttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatg agggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtgg tgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatga cgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttg caactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgg ggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctg gatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaa gaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggag gcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaac cccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatc tcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagcc agtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaa aaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcatt ccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatctt aagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagac ataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatcca gtttggctgcatacgcgtcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggg gtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtg ggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggtt cccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgg gttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggc cgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctt tttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcg tcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctc tggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatg gccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaa aagggcctttccgtcctcagccgtcgcttcatgtgactccactgagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttggagt acgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttg atgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtc gtgagctagctctagaggatccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgag ctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttca tctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgacc acatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactaca agacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacat cctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaa gatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctg cccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgac cgccgccgggatcactctcggcatggacgagctgtacaagtaaagcggccgcgtcgacaatcaacctctggattacaaaatttgtgaaaga ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttct cctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaa cccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccg cctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccttggctgc tcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgct gccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctc ggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactccca acgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactg cttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagt cagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaact tgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtcca aactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttt tttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagctagggacgtaccc aattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatc gccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta gcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccga tttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcccttt gacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatttt gccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcactt ttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttca ataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccaga aacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgag agttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagc aactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagag aattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttt tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacga tgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggagg cggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttca tttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccg tagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtt tgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagtt aggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg tcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgga gcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcg ccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcct ggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgt tggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactca ttaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc atgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtct tgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatc gtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctag ctcgatacataaac (SEQ ID NO: 19)
[0098] In some embodiments, the nucleic acid molecule comprises a sequence that is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to that of SEQ ID NO: 19.
[0099] In some embodiments, the nucleic acid molecule comprises a sequence of: tgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccg attggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcatt gcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagg gaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctca gacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacg caggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctag aaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaa agaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggc tgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtg tgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagca agcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaacca ttaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttg ggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaa caatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtg gaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttgg agtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactcctta attgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataaca aattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttag gcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggag agagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtaca catttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaac agcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggc cgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaa agaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggg gggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaa attcaaaattttcgggtttattacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcagtgggcagag cgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgg gaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgca acgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaatta cttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagcccctt cgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgat aagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggta tttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccacc gagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaa ggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggc gctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccactgagtacc gggcgccgtccaggcacctcgattagttctcgtgcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttcccca cactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctca agcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgagctagctctagaggatccaccggtcgccaccatggtgagcaagg gcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgaggg cgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgacca ccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgt ccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgc atcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatc atggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccact accagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccc caacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaagcg gccgcgtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgc tttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgt tgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccggga ctttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac aattccgtggtgttgtcggggaagctgacgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgt cccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacga gtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagcca ctttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagac cagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcc cgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag (SEQ ID NO: 20) [00100] In some embodiments, the nucleic acid molecule comprises a sequence that is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to that of SEQ ID NO: 20.
[00101] The nucleic acid molecules provided for herein can be provided as a single molecule, for example as a plasmid. In some embodiments, each element is encoded by a single polynucleotide sequence (or double stranded molecule, such as, but not limited to a plasmid) as opposed to distinct nucleotide molecules encoding for different elements. The use of a single molecule, such as a plasmid, permits the user to efficiently introduce the target molecule into the relevant cell or cellular environment.
[00102] In some embodiments, the nucleic acid molecules provided herein comprise one or more of a polynucleotide encoding a constitutive promoter; a polynucleotide encoding a target molecule, which can also be referred to as a molecule of interest; and a polynucleotide encoding a nuclear localization signal; wherein the sequences can be, or are, operatively connected one another. In some embodiments, the nucleic acid molecules provided herein comprise a polynucleotide encoding a constitutive promoter; a polynucleotide encoding a target molecule, which can also be referred to as a molecule of interest; and a polynucleotide encoding a nuclear localization signal; wherein the sequences are operatively connected one another. The molecule of interest (target molecule) can be any molecule that can be encoded by the nucleic acid molecule. For example, the target molecule can be a chimeric antigen receptor (CAR) that can be expressed in a T-cell or other type of cell and function in the T-cell. Thus, the nucleic acid molecule, which can be in the form of a plasmid, can be used to deliver a target molecule to a cell of interest. In some embodiments, the cell is an immune cell, such as a T-cell, dendritic cell, NK cell, a TIL, a MIL, and the like.
[00103] In some embodiments, the nucleic acid molecule is used to deliver a target molecule that can be expressed in a cell. Thus, the nucleic acid molecule, which can be a plasmid, can be used to express a protein or nucleic acid molecule in a cell. The target molecule can be a nucleic acid molecule that encodes for a protein, an antisense nucleic acid molecule, a siRNA molecule, a microRNA, an antibody, a receptor, or any other type of molecule that can be encoded for by a nucleic acid molecule, such as those described herein. Other examples of products that can be encoded for are hormones, gene products, and the like. The specific structure of the gene product is not necessarily critical, but instead shows that various embodiments that the nucleic acid molecules, such as a plasmid, can be used for. In some embodiments, the target molecule is a chimeric antigen receptor (CAR). Examples of CARs that can be used include those that are comprise an extracellular region that bind to PD-1, PD-L1, BSMA, PSMA, and the like. In some embodiments, the CAR comprises a CD19 extracellular binding domain. In some embodiments, the CAR comprises a 4-1BB intracellular region. In some embodiments, the CAR comprises a CD3(^ intracellular signaling domain. In some embodiments, the CAR comprises a CD28 intracellular domain. In some embodiments, the transmembrane domain of the CAR is a CD3(^ transmembrane domain or a CD28 transmembrane domain. In some embodiments, the CAR is an armored CAR. In some embodiments, CAR- modified T-cell potency may be further enhanced through the introduction of additional genes, including those encoding proliferative cytokines (i.e., IL-12) or costimulatory ligands (i.e., 4- 1BBL), thus producing "armored" CAR-modified T-cells. As described herein, in some embodiments, the nucleic acid molecule, e.g. plasmid, is a non-integrating nucleic acid molecule. [00104] In some embodiments, the nucleic acid molecules provided for herein are administered to a subject and taken up by the cells. In some embodiments, the cells are treated with the nucleic acid molecule (e.g. plasmid) ex-vivo and then administered back to a subject to express the molecules in vivo. In some embodiments, the nucleic acid molecules are complexed with nanoparticles to deliver the nucleic acid molecule to a specific cell type. For example, the nucleic acid molecule can be encapsulated or complexed with a lipid nanoparticle, a polymer nanoparticle, liposome, a neutral liposome, a biodegradable polymer matrix e.g. hydrogel), and the like. Example of nanoparticles are described in Xiao et al., Molecular Therapy: Methods & Clinical Development Vol. 12 March 2019, pp. 1-18, which is hereby incorporated by reference in its entirety. Examples of polymers that can be used include, but are not limited to, polyetherimide (PEI), lactosylated polylysine (PLL), polyacrylic acid (PAA), poly(aliphatic ester) (PAE), and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA). These polymers can be modified by chemical modification or can be free of modifications. Other polymers include, but are not limited to, chitosan (e.g. cationic chitosan), poly(ethyleneglycol)-modified chitosan (PEG-CS), carboxymethyl dextran (CMD)-chitosan, gelatin (e.g. cationic gelatin), dextran (e.g. cationic dextran), cellulose (e.g. cationic cellulose), cyclodextrin (e.g. cationic cyclodextrin).
[00105] In some embodiments, the nucleic acid molecule is not encapsulated with a carrier or nanoparticle. In some embodiments, the pharmaceutical composition is free of a nanoparticle that encapsulates the nucleic acid molecule.
[00106] As described herein, the nucleic acid molecule can comprise a promoter, such as a constitutive promoter. Examples of constitutive promoters include, but are not limited to EF-1 alpha, SV40, Rous Sarcoma virus, and Mason-Pfizer monkey virus-CTE. In some embodiments, the promoter is EF-1 alpha. In some embodiments, the promoter is SV40. In some embodiments, the promoter is RSV. In some embodiments, the promoter is the Mason-Pfizer monkey virus-CTE.
[00107] In some embodiments, the promoter is a tissue specific promoter. Tissue specific promoters are known in the art, and any such tissue specific promoter may be utilized in any of the embodiments of the present disclosure. In some embodiments, the tissue specific promoter is as provided for herein. Examples of tissue specific promoter include, but are not limited to, B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, and NSE/RU5’. In some embodiments, the tissue specific promoter is B29. In some embodiments, the tissue specific promoter is CD14. In some embodiments, the tissue specific promoter is CD43. In some embodiments, the tissue specific promoter is CD45. In some embodiments, the tissue specific promoter is CD68. In some embodiments, the tissue specific promoter is desmin. In some embodiments, the tissue specific promoter is elastase-1. In some embodiments, the tissue specific promoter is endoglin. In some embodiments, the tissue specific promoter is fibronectin. In some embodiments, the tissue specific promoter is Flt-1. In some embodiments, the tissue specific promoter is GFAP. In some embodiments, the tissue specific promoter is GPIIb. In some embodiments, the tissue specific promoter is ICAM-2. In some embodiments, the tissue specific promoter is mouse INF-beta. In some embodiments, the tissue specific promoter is Mb. In some embodiments, the tissue specific promoter is Nphsl. In some embodiments, the tissue specific promoter is OG-2. In some embodiments, the tissue specific promoter is SP-B. In some embodiments, the tissue specific promoter is SYN1. In some embodiments, the tissue specific promoter is WASP. In some embodiments, the tissue specific promoter is SV40/bAlb. In some embodiments, the tissue specific promoter is SV40/hAlb. In some embodiments, the tissue specific promoter is SV40/CD43. In some embodiments, the tissue specific promoter is SV40/CD45. In some embodiments, the tissue specific promoter is NSE/RU5’. In some embodiments, the tissue specific promoter is an immune cell specific promoter. Examples of immune cell specific promoters include, but are not limited to, ZAP70 and CD4. In some embodiments, the tissue specific promoter is the ZAP70 promoter. In some embodiments, the tissue specific promoter is the CD4 promoter.
[00108] The nucleic acid molecules can also further comprise a nucleic acid sequence that encodes for one or more adjuvants. In some embodiments, the adjuvant is IL-12. In some embodiments, the nucleic acid molecule encodes one or more of the group consisting of anti- CD40 antibody, GM-CSF, bevacizumab, interferon-alpha, interferon-beta, poly-(I:C) and derivatives, RNA interleukin (IL)-l, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23, and the like.
[00109] For the avoidance of doubt, any of the nucleic acid molecules provided for herein can be a plasmid or other type of circular DNA sequence, such that it can be used to express its products in a cell.
[00110] In some embodiments, pharmaceutical compositions comprising the nucleic acid molecules described herein are provided. Examples of pharmaceutical compositions are provided for herein.
[00111] In some embodiments, methods of delivering a molecule to a cell are provided. In some embodiments, the methods comprise contacting a cell with a virus comprising a nucleic acid molecule, as provided herein, or a nucleic acid molecule as provided for herein into a cell of the subject or into the subject and said virus comprising a nucleic acid molecule, as provided herein, or nucleic acid sequence is taken up by the cell in the subject. The nucleic acid molecule is then expressed and the target molecules or other expression cassettes are expressed in the cell. In some embodiments, the nucleic acid sequence is introduced into the cell or subject by electroporation, injection, sonication, transfection, transduction, gene guns, encompassed by nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell. In some embodiments, the cell or tissue that the nucleic acid molecule is delivered to is skin, muscle, breast, lung, pancreas, brain, ovarian, uterine, endometrial, colon, prostate, esophageal, gum, tongue, throat, liver, eye, thymus, or kidney tissue or cell.
[00112] In some embodiments, methods of preparing a virus comprising a nucleic acid molecule encoding a target molecule are provided. In some embodiments, the method comprises contacting a cell with the nucleic acid molecule, as provided for herein, under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule. In some embodiments, the cell is a producer cell. Non-limiting examples of producer cells include HEK293 and derivatives, Sf9, and CAP (CEVEC).
[00113] In some embodiment, the virus is a lentivirus. In some embodiments, the lentivirus is a pseudotyped lentivirus. A pseudotyped lentivirus is a lentiviral particle having one or more envelope glycoproteins that are encoded by a virus that is distinct from the lentiviral genome. The envelope protein can be, for example and without limitation, from a different virus or from a non-viral origin. The envelope protein can be a native envelope protein or an envelope protein that is modified, mutated or engineered as described herein. The pseudotyped lentivirus can comprise, for example, fusion (F) or attachment (G) protein of paramyxovirus in addition to membrane proteins having hemagglutinin activity such as H protein of paramyxovirus. In some embodiments, the paramyxovirus is a Nipah virus or a Measles virus. In some embodiments, the pseudotyped lentivirus is pseudotyped with Nipah virus fusion protein (NiV-F), Nipah virus attachment protein (NiV-G), Measles virus hemagglutinin (MV-H), or Measles virus fusion protein (MV-F). In some embodiments, the pseudotyped lentivirus is pseudotyped with Nipah virus fusion protein (NiV-F). In some embodiments, the pseudotyped lentivirus is pseudotyped with Nipah virus attachment protein (NiV-G). In some embodiments, the pseudotyped lentivirus is pseudotyped with Measles virus hemagglutinin (MV-H). In some embodiments, the pseudotyped lentivirus is pseudotyped with Measles virus fusion protein (MV-F). Non-limiting examples of Nipah and Measles viruses and elements thereof can be found in PCT Publication No. WO2017182585, U.S. Publication No. 20190144885, U.S. Patent No. 9,862,791, and U.S. Patent No. 10,415,057, all of which are incorporated by reference in their entirety. In some embodiments, the pseudotyped lentivirus is pseudotyped with a human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, immunodeficiency virus in simian (SIV), maedi / visna virus, Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, and SARS-CoV virus.
[00114] In some embodiments, the pseudotyped lentivirus can comprise, additionally or alternatively, envelope proteins derived from other viruses. For example, preferred envelope proteins include those derived from viruses infectious to human cells. Such proteins include, but not limited to, amphotropic envelope proteins of retrovirus, G protein of vesicular stomatitis virus (VSV-G), etc. Such proteins of viruses belonging to the herpes viridae include, for example, gB, gD, gH and gp85 proteins of herpes simplex virus, gp350 and gp220 proteins of EB virus, etc. Such proteins of viruses belonging to the hepadna viridae include S protein of hepatitis B virus, etc.
[00115] These examples are non-limiting and the molecules and compositions provided for herein can be used in any tissue or cell type desired by the user.
[00116] In some embodiments, the compositions and molecules described herein can be used to treat a disease. Examples of diseases that can be treated include, but are not limited to, immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof. In some embodiments, the compositions and molecules described herein can be used to treat a cancer. Examples of diseases that can be treated include, but are not limited to, brain, breast, lung, ovarian, endometrial, colon, lung, skin (e.g. melanoma), blood, lymphoma, leukemia, and the like.
[00117] Pharmaceutical compositions described herein can further comprise a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical compositions comprise about 1 ng to about 10,000 pg of the nucleic acid molecule. The pharmaceutical compositions can be formulated according to the mode of administration to be used. One having ordinary skill in the art can readily formulate a pharmaceutical composition that comprises a genetic construct or nucleic acid molecule as described herein. In cases where intramuscular injection is the chosen mode of administration, an isotonic formulation can be used. In some embodiments, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. In some cases, isotonic solutions such as phosphate buffered saline are used. In some embodiments, the pharmaceutical composition comprises a stabilizer. Examples of stabilizers include, but are not limited to, gelatin and albumin. The pharmaceutical preparations can be provided as sterile and pyrogen free.
[00118] In some embodiments, methods of inducing an immune response against a target molecule in a subject are provided. In some embodiments, the methods comprise introducing the virus comprising the nucleic acid, as provided herein, described herein into the subject. In some embodiments, the virus is introduced in a cell of the subject. The nucleic acid molecule can also be taken up by the cell. Once inside the cell the cell’s machinery can be used to express the target molecule (e.g., a target protein) and the construct that is encoded for by the nucleic acid molecule. In some embodiments, the nucleic acid molecule is as described herein. The method of introduction or administration can be any method, including the methods described herein. In some embodiments, the nucleic acid molecule is introduced by electroporation or injection. In some embodiments, the nucleic acid molecule is introduced into, or administered to, the subject by sonication, transfection, transduction, gene guns, nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell and the like.
[00119] In some embodiments, bupivacaine or other similar adjuvant is used to help facilitate the induction of an immune response.
[00120] In some embodiments, the nucleic acid molecule is administered to a tissue of the subject. In some embodiments, the tissue is skin, muscle, liver, fat, nerve, kidney, and the like.
[00121] In some embodiments, methods of treating a disease are provided. In some embodiments, the methods of treating disease in a subject introducing, or administering, a virus comprising the nucleic acid, as provided herein, described herein to the subject. In some embodiments, the virus is introduced directly into a cell. In some embodiments, the nucleic acid molecule is introduced directly into a cell. In some embodiments, the virus comprising the nucleic acid molecule is taken up by the cell and expressed in the cell. In some embodiments, the target protein and the fragment of the target protein is a protein that is overexpressed, or specifically expressed, in a cancer cell. In some embodiments, the nucleic acid molecule is administered by electroporation, injection, sonication, transfection, transduction, and the like. In some embodiments, the nucleic acid molecule is administered to the skin, muscle or other tissue of the subject. In some embodiments, the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
[00122] In some embodiments, methods of treating an infectious agent or an infection in a subject are provided. In some embodiments, the methods comprise introducing/administering the virus produced as described herein to the subject. In some embodiments, the virus is administered directly into a cell of the subject. As described throughout, the virus can be taken up by the cell and viral nucleic acid molecule, also referred to as the nucleic acid molecule and as provided herein, can be expressed in the cell. Without being bound by any particular theory, when the nucleic acid molecule is expressed the subject’s immune response will recognize the target protein and the fragment of the target protein as foreign and an immune response will be generated. The generated immune response can treat or prevent the infection. In some embodiments, the generated immune response can treat or prevent the infectious agent from causing a disease or will inhibit the growth of the infectious agent to ameliorate symptoms of the infection. The methods of administration can be any of the methods described herein. In some embodiments, the virus can be administered to the skin, muscle, fat, kidney, or other tissue of the subject. In some embodiments, the virus is administered to the mucosa of the subject. In some embodiments, the nucleic acid molecule is introduced into the subject or the cell by electroporation, injection, sonication, transfection, and transduction.
[00123] In some embodiments, the target protein is a HIV or influenza protein, such as Gpl20, Gag, Nef, Tat, hemagglutinin (HA), neuraminidase (NA), and the like.
[00124] In some embodiments, a cell comprising the nucleic acid molecules provided for herein are provided. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is ex-vivo and not in a subject. The cell can be any cell type, such as a T-cell, a muscle cell, a skin cell, a brain cell, and the like. [00125] The following examples are illustrative, but not limiting, of the compositions and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.
[00126] EXAMPLES
[00127] Example 1. Plasmid production.
[00128] The plasmid comprised of a polynucleotide bounded by long terminal repeats (proviral plasmid) is mixed with both a plasmid encoding envelope fusion proteins (pseudotype) and one or more plasmids encoding polynucleotides required for packaging the proviral plasmid into a viral particle. The plasmid mixture is complexed with a reagent, such as calcium phosphate or a lipid transfection mixture, which facilitates cellular uptake into the packaging cells. The packaging cells consisting of the plasmid mixture then produce viral vector particles which can be purified from the packaging cells by mechanisms known to one skilled in the art (centrifugation; binding, washing and elution to purification columns; gradient purification; etc.). Purified viral vector particles can be supplemented with other components to become the pharmaceutical composition described herein. The pharmaceutical composition can be applied to cells ex vivo or introduced into a subject in vivo to allow transduction with the vector delivering the polynucleotide encoding the target molecule(s) into the cells described herein. After transduction, the vector will express the target molecules.
[00129] Example 2. Viral vectors generated from plasmid transduce human PBMCs.
[00130] LTR-containing plasmids were designed encoding a self-inactivating lentiviral vector genome with a transgene of either green fluorescent protein (GFP) as a cytoplasmic reporter or two transmembrane proteins separated by a self-cleaving peptide sequence, CAR-2A- NGFR.
[00131] Pseudotyping of either genome was performed with one of three glycoproteins: (1) VSV-G, (2) Nipah Virus F protein and a truncated Nipah Virus G protein with a scFv that binds to a cell surface, or (3) Nipah Virus F protein and a truncated Nipah Virus G protein with a VHH that binds to a cell surface.
[00132] The lentivector genomes and pseudotyping plasmids were transfected along with a packaging plasmid set consisting of a human-simian chimeric gag-pol and HIV-1 rev in HEK293T cells. [00133] Viral supernatant was purified by centrifugation over a 20% sucrose cushion and resuspended in X-Vivo media. Resuspended virus was analyzed by a p24 assay to determine viral protein content in solution and calculate a physical titer. 10-fold dilutions by p24 mass of virus were administered to SupTl cells to determine a functional titer by flow cytometry.
[00134] Vectors were then administered to activated human PBMCs to assess transduction and transgene expression. Flow cytometry was utilized to assess the fraction of CD4+ cells with either GFP expression in the cytoplasm or NGFR protein expression on the surface at seven days after transduction (FIG. 6 A-F).
[00135] Physical titers were within an order of magnitude for either genome and all pseudotyping glycoproteins (see Table 1 below). Vectors pseudotyped with VSV-G had significantly higher functional titers in SupTl. VSV-G pseudotyped GFP genomes showed the highest transduction in PBMCs compared to the other constructs evaluated. Additionally, the 2nd transmembrane protein NGFR was detected in PBMCs (FIG. 6A). Combined, this data indicates cytoplasmic or polycistronic peptides can be expressed from the plasmid, and the plasmid can be utilized with multiple pseudotyping strategies.
Table 1: Titers in SupTl Cells
Figure imgf000060_0001
[00136] The examples provided for herein demonstrate the flexibility of the nucleic molecules described herein to deliver molecules and have them expressed. This can be used to deliver a molecule to a cell or tissue or can be used to induce an immune response against a variety of target molecules. The nucleic acid molecules can also be used to generate viral vectors that can be used to, for example, transduce, PBMCs. [00137] The examples described herein are exemplary in manner and are not intended, nor should they be used, to limit the scope of the embodiments. Each and every reference, publication, accession number, patent, document, etc., is hereby incorporated by reference in its entirety for its intended purpose.
[00138] This description is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and it is not intended to limit the scope of the embodiments described herein. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. However, in case of conflict, the patent specification, including definitions, will prevail.
[00139] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modification can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting.

Claims

What is claimed is:
1. A nucleic acid molecule comprising: a polynucleotide encoding a first promoter upstream of a 5’ and 3’ long terminal repeat (LTR) bounded polynucleotide sequence, wherein the polynucleotide sequence bounded by a 5 ’LTR and a 3’ LTR comprises: a polynucleotide sequence encoding a viral structural protein and/or a binder molecule; a polynucleotide sequence encoding a central polypurine tract (cPPT); a polynucleotide sequence encoding a second promoter; a polynucleotide encoding one or more target molecules (e.g. protein of interest, polypeptide, miRNA, shRNA, etc.); and a polynucleotide encoding a posttranscriptional regulatory element.
2. The nucleic acid molecule of claim 1, wherein the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
3. The nucleic acid molecule of claim 2, wherein the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
4. The nucleic acid molecule of claims 2 or 3, wherein the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another.
5. The nucleic acid molecule of any one of claims 1-4, wherein the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
6. The nucleic acid molecule of any one of claims 1-5, wherein the sequence encoding the first promoter encodes a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
7. The nucleic acid molecule of claim 6, wherein the constitutive promoter is selected from the group consisting of EF-1 alpha, Rous Sarcoma virus (RSV), SV-40, and Mason-Pfizer monkey virus-CTE.
8. The nucleic acid molecule of claim 6, wherein the tissue-specific promoter is selected from the group consisting of B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
9. The nucleic acid molecule of claim 6, wherein the tissue-specific promoter is selected from the group consisting of ZAP70 and CD4.
10. The nucleic acid molecule of any one of claims 1-9, wherein the sequence encoding the second promoter encodes a constitutive promoter, an inducible promoter, or a tissue specific promoter.
11. The nucleic acid molecule of claim 10, wherein the constitutive promoter is selected from the group consisting of EF-1 alpha, Rous Sarcoma virus (RSV), SV-40, and Mason-Pfizer monkey virus-CTE.
12. The nucleic acid molecule of claim 10, wherein the tissue-specific promoter is selected from the group consisting of B29, CD14, CD43, CD45, CD68, desmin, elastase-1, endoglin, fibronectin, Flt-1, GFAP, GPIIb, ICAM-2, mouse INF-beta, Mb, Nphsl, OG-2, SP-B, SYN1, WASP, SV40/bAlb, SV40/hAlb, SV40/CD43, SV40/CD45, NSE/RU5’, ZAP70 and CD4.
13. The nucleic acid molecule of claim 10, wherein the tissue-specific promoter is selected from the group consisting of ZAP70 and CD4.
14. The nucleic acid molecule of claim 1, wherein the sequence encoding the target molecule encodes a reporter gene, a protein of interest, a chimeric antigen receptor (CAR), an armored CAR, a DAP-12, a cytokine, a siRNA, a shRNA, an antisense molecule, a microRNA, or a gene editing construct (e.g., CRISPR/CAS system).
15. The nucleic acid molecule of claim 14, wherein the reporter gene is a fluorescent protein or an enzyme.
16. The nucleic acid molecule of claim 4, wherein the sequence encoding the linker encodes a glycine-serine linker or a cleavable linker.
17. The nucleic acid molecule of claim 16, wherein the cleavable linker is a P2A, T2A, E2A, or F2A linker.
18. The nucleic acid molecule of claim 16, wherein the glycine linker comprises one more of the repeat of GGGGS, GSG, or any combination thereof.
19. The nucleic acid molecule of claim 4, wherein the sequence encoding the intermediate elements encodes an Internal Ribosome Entry Site (IRES).
20. The nucleic acid molecule of any of claims 1-19, wherein the nucleic acid molecule optionally comprises a safety switch.
21. The nucleic acid molecule of claim 1, wherein the sequence encoding the viral structural protein encodes a gag protein.
22. The nucleic acid molecule of claim 1, wherein the sequence encoding the viral structural protein encodes a partial gag protein.
23. The nucleic acid molecule of claim 22, wherein the sequence encoding the partial gag encodes for the HIV-1 psi element, rev response element (RRE), and a Gp41 peptide.
24. The nucleic acid molecule of claim 1, wherein the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD 19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte; Her2/Neu+ cancer cell; NKG2D+ natural killer cell; SLC1A3+ astrocyte; SLC7A10+ adipocyte.
25. The nucleic acid molecule of claim 1, wherein the binder molecule binds to CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, A glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors; A glycosylated CD43 epitope expressed on non-hematopoietic cancers; A kinase anchor protein 4 (AKAP-4); Adrenoceptor beta 3 (ADRB3); AFP; Anaplastic lymphoma kinase (ALK); Androgen receptor; Angiopoietin-binding cell surface receptor 2 (Tie 2); Auto antibody to desmoglein 1 (Dsgl); Auto antibody to desmoglein 3 (Dsg3); B7H3 (CD276); Biotin; Bone marrow stromal cell antigen 2 (BST2); BST1/CD157; Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la);
Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R; CD276/B7H3; CD300 molecule-like family member f (CD300LF); CDH1-CD324; CDH6; CDH17; CDH19; Chromosome X open reading frame 61 (CXORF61); Claudin 6 (CLDN6); Claudinl8.2 (CLD18A2 or CLDN18A.2); CMV pp65; C-MYC epitope Tag; Cripto; CS1 (also referred to as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24); CSF2RA (GM-CSFR- alpha); C-type lectin domain family 12 member A (CLEC12A); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); Cyclin Bl; Cytochrome P450 IB 1 (CYP1B 1); DLL3; EBV-EBNA3c; EGF-bke module- containing mucin-like hormone receptor-like 2 (EMR2); Elongation factor 2 mutated (ELF2M); Ephrin B2; Ephrin type-A receptor 2 (EphA2); Epidermal growth factor receptor (EGFR); Epidermal growth factor receptor variant III (EGFRviii); Epithelial cell adhesion molecule (EPCAM); ERG; ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Fc fragment of IgA receptor (FCAR or CD89); Fc receptor-like 5 (FCRL5); Fibroblast activation protein alpha (FAP); FITC; Fms Like Tyrosine Kinase 3 (FLT3); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Follicle stimulating hormone receptor (FSHR); Fos-related antigen 1; Fucosyl-GMl; G protein coupled receptor class C group 5 member D (GPRC5D); G protein-coupled receptor 20 (GPR20); GAD; Ganglioside G2 (GD2) ; Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer); Ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l- 4)bDGlcp(l-l)Cer); GD3; GFRalpha4; Glycoprotein 100 (gplOO);
Glypican-3 (GPC3); Gonadotropin Hormone receptor (CGHR or GR); GpA33; GpNMB; GPRC5D; Guanylyl cyclase C (GCC); Heat shock protein 70-2 mutated (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexasaccharide portion of globoH glycoceramide (GloboH); High molecular weight-melanoma associated antigen (HMWMAA); HIV1 envelope glycoprotein; HLA; HLA-DOA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DM; HLA-DOB; HLA-DP; HLA-DQ; HLA-DR; HLA-G; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human Telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; IL1 IRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza A hemagglutinin (HA); Insulin-like growth factor 1 receptor (IGF -I receptor); Interleukin 11 receptor alpha (IL-llRa); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Intestinal carboxyl esterase; KIT (CD117); KSHV K8.1; KSHV-gH; LAMP1 ; Legumain; Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1); Leutenizing hormone receptor (LHR); Lewis(Y) antigen; Lews Ag; Livl; Locus K 9 (LY6K); Low conductance chloride channel; Lymphocyte antigen 6 complex; Lymphocyte antigen 75 (LY75); Lymphocyte-specific protein tyrosine kinase (LCK); Mammary gland differentiation antigen (NY-BR-1); Melanoma antigen recognized by T cells 1 (MelanA or MARTI); Melanoma- associated antigen 1 (MAGE-A1); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT- 2); Melanoma inhibitor of apoptosis (ML-IAP); Mesothelin; MPL; Mucin 1 cell surface associated (MUC1); N-Acetyl glucosaminyl-transf erase V (NA 17); Nectin-4; Neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-acetyl-GD2 ganglioside (OAcGD2); Olfactory receptor 51E2 (OR51E2); Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); P53 mutant; Paired box protein Pax-3 (PAX3); Paired box protein Pax-5 (PAX5); Pannexin 3 (PANX3); PDL1; P-glycoprotein; Placenta-specific 1 (PLAC1); Platelet-derived growth factor receptor beta (PDGFR-beta); Polysialic acid; Proacrosin binding protein sp32 (OY-TES1);
Prostase; Prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Prostatic acid phosphatase (PAP); Prostein; Protease Serine 21 (Testisin or PRSS21); Proteasome (Prosome Macropain) Subunit Beta Type 9 (LMP2); PTK7; Ras G12V; Ras Homolog Family Member C (RhoC); Rat sarcoma (Ras) mutant; Receptor for Advanced Gly cation Endproducts (RAGE-1); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Receptor tyrosine-protein kinase ERBB2 or Her-22/neu; Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); Sarcoma translocation breakpoints; Serine 2 (TMPRSS2) ETS fusion gene; Sialyl Lewis adhesion molecule (sLe); SLAMF4; SLAMF6; Slea (CAI 9.9 or Sialyl Lewis Antigen); Sperm protein 17 (SPA17); Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Stage-specific embryonic antigen-4 (SSEA-4); STEAP1; Survivin; Synovial sarcoma X breakpoint 2 (SSX2); TCR Gamma Alternate Reading Frame Protein (TARP); TCR-betal chain; TCR-beta2 chain; TCR-delta chain; TCR-gamma chain; TCRgamma-delta; Telomerase; TGFbetaR2; The antigen recognized by TNT antibody; Thyroid stimulating hormone receptor (TSHR); Timl-/HVCR1; Tissue Factor 1 (TF1); Tn ag; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); TNF receptor family member B cell maturation (BCMA); Transglutaminase 5 (TGS5); Transmembrane protease; TROP2; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7-related (TEM7R); Tumor protein p53 (p53); Tumor-associated glycoprotein 72 (TAG72); Tyrosinase; Tyrosinase-related protein 2 (TRP-2); Uroplakin 2 (UPK2); Vascular endothelial growth factor receptor 2 (VEGFR2); V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Wilms tumor protein (WT1); or X Antigen Family Member 1 A (XAGE1).
26. The nucleic acid of any one of claims 1-25, wherein the nucleic acid molecule comprises an antibiotic resistance gene.
27. The nucleic acid of claim 26, wherein the antibiotic resistance gene confers resistance to an antibiotic selected from the group comprising kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.
28. A nucleic acid molecule sequentially comprising in a 5’ to 3’ direction: a. a polynucleotide encoding a RSV promoter; b. a polynucleotide encoding a 5’ LTR; c. a polynucleotide encoding a partial gag, wherein the partial gag further comprises
HIV-1 psi element, rev response element (RRE), and a Gp41 peptide; d. a polynucleotide encoding a central polypurine tract (cPPT); e. a polynucleotide encoding an EF-1 alpha promoter; f. a polynucleotide encoding a first target molecule; g. optionally a polynucleotide encoding a first linker or first intermediate element; h. optionally a polynucleotide encoding a second target molecule; i. optionally a polynucleotide encoding a second or more (e.g., third, fourth, fifth) linker or second or more (e.g., third, fourth, fifth) intermediate element; j. optionally a polynucleotide encoding a third or more (e.g., fourth, fifth, sixth) target molecule; k. a polynucleotide encoding a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE); and l. a polynucleotide encoding a 3’ LTR; wherein the sequences are operatively connected to one another, and optionally wherein g)-j) repeat n times, wherein n is any integer (e.g., 0, 1, 2, 3, 4).
29. The nucleic acid molecule of claim 28, wherein the nucleic acid molecule comprises one or more polynucleotides encoding one or more target molecules (e.g., n is 1, 2, 3, 4).
30. The nucleic acid molecule of claim 29, wherein the polynucleotide encoding one or more target molecules is a polycistronic nucleotide sequence encoding more than one target molecule.
31. The nucleic acid molecule of any one of claims 29-30, wherein the polycistronic nucleotide sequence comprises one more or more linkers or intermediate elements separating the more than one target molecules.
32. The nucleic acid molecule of any one of claims 29-31, wherein the polycistronic nucleotide sequence encodes 2, 3, 4, or 5 target molecules, wherein each target molecule is separated by a linker or intermediate element, wherein the sequences encoding the target molecules are operatively connected to one another.
33. The nucleic acid molecule of any one of claims 29-32, wherein the nucleic acid molecule comprises encoding the target molecule is a polycistronic (e.g., bicistronic, tricistronic, quadci str onic) expression system.
34. The nucleic acid molecule of any one of claims 28-33, wherein the sequence encoding the first, second, or more target molecule encodes a reporter gene, a protein of interest, a chimeric antigen receptor (CAR), an armored CAR, a DAP- 12, a cytokine, a binder molecule, a siRNA, a shRNA, an antisense molecule, a microRNA, or a gene editing construct (e.g., CRISPR/CAS system).
35. The nucleic acid molecule of claim 34, wherein the reporter gene is a fluorescent protein or an enzyme.
36. The nucleic acid molecule of claim 34, wherein at least one of the first, second, or more target molecules encodes a chimeric antigen receptor (CAR).
37. The nucleic acid molecule of claim 34, wherein the binder molecule binds to an immune cell, such as a T cell, B cell; NK cell, dendritic cell, neutrophils, macrophages, a cancer cell; or, for example, CD3+ T cell; CD4+ T cell; CD7+ T cell, CD8+ T cell; CD19+ B cell; CD 19+ cancer cell; CD20+ B cell; CD20+ cancer cell, CD30+ lung epithelial cell; CD34+ haematopoietic stem cell; CD105+ endothelial cell; CD105+ haematopoietic stem cell; CD117+ haematopoietic stem cell; CD133+ cancer cell; EpCAM+ cancer cell; GluA2+ neuron; GluA4+ neuron; Haematopoietic stem cell; Hepatocyte; Her2/Neu+ cancer cell; NKG2D+ natural killer cell; SLC1A3+ astrocyte; SLC7A10+ adipocyte.
38. The nucleic acid molecule of claim 34, wherein the binder molecule binds to CD7, CD8, cKit (CD117), CD4, CD3, CD5, CD6, CD2, TCR alpha, TCR beta, TCR gamma, TCR delta, CD10, CD34, CD110, CD33, CD14, CD68, CCR7, CD62L, CD25, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CXCR3, A glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors; A glycosylated CD43 epitope expressed on non-hematopoietic cancers; A kinase anchor protein 4 (AKAP-4); Adrenoceptor beta 3 (ADRB3); AFP; Anaplastic lymphoma kinase (ALK); Androgen receptor; Angiopoietin-binding cell surface receptor 2 (Tie 2); Auto antibody to desmoglein 1 (Dsgl); Auto antibody to desmoglein 3 (Dsg3); B7H3 (CD276); Biotin; Bone marrow stromal cell antigen 2 (BST2); BST1/CD157; Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la); Carbonic anhydrase IX (CA1X); Carcinoembryonic antigen (CEA); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites); CCR4; CD5; CD19; CD20; CD22; CD24; CD30; CD32 (FCGR2A); CD33; CD34; CD38; CD44v6; CD72; CD79a; CD79b; CD97; CD99; CD123; CD171; CD179a; CD179b-IGLll; CD200R; CD276/B7H3; CD300 molecule-like family member f (CD300LF); CDH1-CD324; CDH6; CDH17; CDH19; Chromosome X open reading frame 61 (CXORF61); Claudin 6 (CLDN6); Claudinl8.2 (CLD18A2 or CLDN18A.2); CMV pp65; C-MYC epitope Tag; Cripto; CS1 (also referred to as CD2 subset 1 or CRACC or SLAMF7 or CD319 or 19A24); CSF2RA (GM-CSFR- alpha); C-type lectin domain family 12 member A (CLEC12A); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); Cyclin Bl; Cytochrome P450 IB 1 (CYP1B 1); DLL3; EBV-EBNA3c; EGF-bke module- containing mucin-like hormone receptor-like 2 (EMR2); Elongation factor 2 mutated (ELF2M); Ephrin B2; Ephrin type-A receptor 2 (EphA2); Epidermal growth factor receptor (EGFR); Epidermal growth factor receptor variant III (EGFRviii); Epithelial cell adhesion molecule (EPCAM); ERG; ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML); Fc fragment of IgA receptor (FCAR or CD89); Fc receptor-like 5 (FCRL5); Fibroblast activation protein alpha (FAP); FITC; Fms Like Tyrosine Kinase 3 (FLT3); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Follicle stimulating hormone receptor (FSHR); Fos-related antigen 1; Fucosyl-GMl; G protein coupled receptor class C group 5 member D (GPRC5D); G protein-coupled receptor 20 (GPR20); GAD; Ganglioside G2 (GD2) ; Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer); Ganglioside GM3 (aNeu5Ac(2-3)bDClalp(l- 4)bDGlcp(l-l)Cer); GD3; GFRalpha4; Glycoprotein 100 (gplOO); Glypican-3 (GPC3); Gonadotropin Hormone receptor (CGHR or GR); GpA33; GpNMB;
GPRC5D; Guanylyl cyclase C (GCC); Heat shock protein 70-2 mutated (mut hsp70-2); Hepatitis A virus cellular receptor 1 (HAVCR1); Hexasaccharide portion of globoH glycoceramide (GloboH); High molecular weight-melanoma associated antigen (HMWMAA); HIV1 envelope glycoprotein; HLA; HLA-DOA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DM; HLA-DOB; HLA-DP; HLA-DQ; HLA-DR; HLA-G; HTLVl-Tax; Human papilloma virus E6 (HPV E6); Human papilloma virus E7 (HPV E7); Human Telomerase reverse transcriptase (hTERT); IgE; IL13Ra2; IL1 IRa; Immunoglobulin lambda-like polypeptide 1 (IGLL1); Influenza A hemagglutinin (HA); Insulin-like growth factor 1 receptor (IGF -I receptor); Interleukin 11 receptor alpha (IL-llRa); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Intestinal carboxyl esterase; KIT (CD117); KSHV K8.1; KSHV-gH; LAMP1 ; Legumain; Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1); Leutenizing hormone receptor (LHR); Lewis(Y) antigen; Lews Ag; Livl; Locus K 9 (LY6K); Low conductance chloride channel; Lymphocyte antigen 6 complex; Lymphocyte antigen 75 (LY75); Lymphocyte-specific protein tyrosine kinase (LCK); Mammary gland differentiation antigen (NY-BR-1); Melanoma antigen recognized by T cells 1 (MelanA or MARTI); Melanoma- associated antigen 1 (MAGE-A1); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT- 2); Melanoma inhibitor of apoptosis (ML-IAP); Mesothelin; MPL; Mucin 1 cell surface associated (MUC1); N-Acetyl glucosaminyl-transf erase V (NA 17); Nectin-4; Neural cell adhesion molecule (NCAM); NKG2D; NYBR1; O-acetyl-GD2 ganglioside (OAcGD2); Olfactory receptor 51E2 (OR51E2); Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); P53 mutant; Paired box protein Pax-3 (PAX3); Paired box protein Pax-5 (PAX5); Pannexin 3 (PANX3); PDL1; P-glycoprotein; Placenta-specific 1 (PLAC1); Platelet-derived growth factor receptor beta (PDGFR-beta); Polysialic acid; Proacrosin binding protein sp32 (OY-TES1); Prostase; Prostate carcinoma tumor antigen- 1 (PCT A-l or Galectin 8); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Prostatic acid phosphatase (PAP); Prostein; Protease Serine 21 (Testisin or PRSS21); Proteasome (Prosome Macropain) Subunit Beta Type 9 (LMP2); PTK7; Ras G12V; Ras Homolog Family Member C (RhoC); Rat sarcoma (Ras) mutant; Receptor for Advanced Gly cation Endproducts (RAGE-1); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Receptor tyrosine-protein kinase ERBB2 or Her-22/neu; Renal ubiquitous 1 (RU1); Renal ubiquitous 2 (RU2); Sarcoma translocation breakpoints; Serine 70
2 (TMPRSS2) ETS fusion gene; Sialyl Lewis adhesion molecule (sLe); SLAMF4; SLAMF6; Slea (CAI 9.9 or Sialyl Lewis Antigen); Sperm protein 17 (SPA17); Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Stage-specific embryonic antigen-4 (SSEA-4); STEAP1; Survivin; Synovial sarcoma X breakpoint 2 (SSX2); TCR Gamma Alternate Reading Frame Protein (TARP); TCR-betal chain; TCR-beta2 chain; TCR-delta chain; TCR-gamma chain; TCRgamma-delta; Telomerase; TGFbetaR2; The antigen recognized by TNT antibody; Thyroid stimulating hormone receptor (TSHR); Timl-/HVCR1; Tissue Factor 1 (TF1); Tn ag; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); TNF receptor family member B cell maturation (BCMA); Transglutaminase 5 (TGS5); Transmembrane protease; TROP2; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7-related (TEM7R); Tumor protein p53 (p53); Tumor-associated glycoprotein 72 (TAG72); Tyrosinase; Tyrosinase-related protein 2 (TRP-2); Uroplakin 2 (UPK2); Vascular endothelial growth factor receptor 2 (VEGFR2); V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Wilms tumor protein (WT1); or X Antigen Family Member 1 A (XAGE1).
39. The nucleic acid molecule of any one of claims 28-38, wherein the sequence encoding the first, second or more linker encodes a glycine-serine linker or a cleavable linker.
40. The nucleic acid molecule of any one of claims 28-39, wherein the cleavable linker is a P2A, T2A, E2A, or F2A linker.
41. The nucleic acid molecule of any one of claims 28-40, wherein the glycine linker comprises one more of the repeat of GGGGS, GSG, or any combination thereof.
42. The nucleic acid molecule of any one of claims 28-41, wherein the sequence encoding the first, second or more intermediate element encodes an Internal Ribosome Entry Site (IRES).
43. The nucleic acid molecule of any one of claims 28-42, wherein the nucleic acid molecule optionally comprises a safety switch.
44. The nucleic acid molecule of claim 28, wherein a)-k) are operatively connected to each other in a 5’ to 3’ direction.
45. The nucleic acid molecule of claim 44, wherein a 3’ end of a) is operatively connected to 5’ end of b); a 3’ end of b) is operatively connected to a 5’ end of c); a 3’ end of c) is operatively connected to a 5’ end of d); a 3’ end of d) is operatively connected to a 5’ end of e); 71 a 3’ end of e) is operatively connected to a 5’ end of f); optionally a 3’ end of f) is operatively connected to a 5’ end of g); optionally a 3’ end of g) is operatively connected to a 5’ end of h); optionally a 3’ end of h) is operatively connected to a 5’ end of i); optionally a 3’ end of i) is operatively connected to a 5’ end of j); a 3’ end of f) or optionally j) is operatively connected to a 5’ end of k); and a 3’ end of k) is operatively connected to a 5’ end of 1).
46. The nucleic acid molecule of any one of claim 1-45, wherein the nucleic acid molecule is a plasmid.
47. The nucleic acid molecule of claim 1-46, wherein the nucleic acid molecule is a nonintegrating plasmid.
48. A host cell comprising the nucleic acid molecules of any one of claims 1-47.
49. A pharmaceutical composition comprising the host cell of claim 48.
50. A pharmaceutical composition comprising the nucleic acid molecules of any one of claims 1-47.
51. A method of delivering a molecule to a cell, the method comprising contacting a cell with a nucleic acid sequence of any one of claims 1-47 into a cell of the subject or into the subject and said nucleic acid sequence is taken up by the cell in the subject.
52. The method of claim 51, wherein the nucleic acid sequence is introduced into the cell or subject by electroporation, injection, sonication, transfection, transduction, gene guns, encompassed by nanoparticles, lipoparticles, or other modes of administration suitable for introducing a nucleic molecule into a subject or cell.
53. A method of preparing a virus comprising a nucleic acid molecule encoding a target molecule, the method comprising contacting a cell with the nucleic acid molecule of any one of claims 1-47 under conditions sufficient to produce the virus comprising the nucleic acid molecule encoding the target molecule.
54. The method of claim 53, wherein the cell is a producer cell (e.g., HEK293, HEK293T, HEK293FT).
55. The method of any one of claims 53 or 54, wherein the virus is a lentivirus.
56. The method of any one of claims 53-55, wherein the virus is a pseudotyped lentivirus. 72
57. The method of claim 56, wherein the pseudotyped lentivirus is pseudotyped with VSV-G, Nipah virus fusion protein (NiV-F), Nipah virus attachment protein (NiV-G), Measles virus hemagglutinin (MV-H), or Measles virus fusion protein (MV-F).
58. The method of claim 56, wherein the pseudotyped lentivirus is pseudotyped with a human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, immunodeficiency virus in simian (SIV), maedi / visna virus, Sindbis virus, influenza virus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, or SARS-CoV virus.
59. A method of treating a disease in a subject, the method comprising administering to the subject the virus prepared according to any one of claims 53-58 to treat the disease.
60. The method of claim 59, wherein the disease is an immune disease, cancer, genetic disease, allergic disease, inflammatory disease, infectious disease, metabolic disease, neurological disease, muscular disease, or any combination thereof.
61. The method of claim 59, wherein the subject is in need of treatment.
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CN117556266B (en) * 2024-01-11 2024-03-22 之江实验室 Signal identification model training method, signal identification method and device

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