Compositions and methods for therapeutic agent delivery

Abstract

Described herein are compositions for delivering a therapeutic agent. Also described herein are methods of using the compositions described herein for delivering a therapeutic agent.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 271,674, filed October 25, 2021, and U.S. Provisional Application No. 63 / 413,165, filed October 4, 2022, the entireties of which are incorporated by reference herein. Incorporated by reference

[0002] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over any such conflicting material. [Background technology]

[0002]

[0003] Interleukin (IL)-12 has been investigated as a potential immunotherapeutic agent for cancer because it is a T cell stimulant that can activate anti-cancer T cells. IL-12 promotes the development of anti-tumor T cells by inducing the production of certain inflammatory cytokines, such as IFN-γ, IL-2, or TNF-α. Summary of the Invention

[0003]

[0004] Due to the limitations of currently available IL therapeutics, there is still a need for compositions and methods for treating subjects in need of treatment with IL.Therefore, in some embodiments, a vector expressing IL-12 is described herein, which provides advantages over currently available IL therapeutics.Firstly, IL-12 protein is not directly injected into subjects, so it does not cause possible systemic dose-limiting toxicity.In addition, local injection of the vector expressing IL-12 is expected to control the expression level in tumor area.

[0004]

[0005] The present disclosure provides a recombinant retroviral vector that contains polynucleotide sequences that separately code for the P40 and P35 subunits of IL-12, and additional polynucleotide sequences therebetween. The P40 and P35 subunits are expressed individually, and then together form a heterodimer linked by disulfide bridges to become functional. In some embodiments, the additional polynucleotide sequence includes a cleavage site between the P40 and P35 subunits. Expected cleavage sites include a furin cleavage site, a self-cleaving peptide (e.g., T2A, P2A, E2A, F2A, or any combination), or both. In some other embodiments, the vector includes only one polynucleotide sequence that codes for an interleukin or a subunit of an interleukin. The present disclosure also provides a method for treating cancer or other disease or condition (e.g., inflammation or infection) in a subject by administering to the subject any of the retroviral vectors disclosed herein.

[0005]

[0006] In some aspects, a recombinant retroviral vector is described herein, comprising a nucleic acid construct comprising a first polynucleotide sequence encoding a P40 subunit of IL-12, a second polynucleotide sequence encoding a P35 subunit of IL-12, and a third polynucleotide sequence between the first and second polynucleotide sequences, wherein the third polynucleotide sequence encodes a cleavage site that facilitates cleavage between the P40 subunit and the P35 subunit. In some embodiments, the cleavage site comprises a furin cleavage site. In some embodiments, the furin cleavage site comprises the amino acid sequence RRKR. In some embodiments, the nucleic acid construct further comprises a fourth polynucleotide sequence between the first and second polynucleotide sequences, wherein the fourth polynucleotide sequence encodes a self-cleaving peptide. In some embodiments, the self-cleaving peptide comprises a T2A peptide, a P2A peptide, an E2A peptide, an F2A peptide, or a combination thereof. In some embodiments, the T2A peptide comprises the amino acid sequence EGRGSLLTCGDVEENPGP. In some embodiments, the self-cleaving peptide comprises a T2A peptide comprising the amino acid sequence GSGEGRGSLTCGDVEENPGP. In some embodiments, the P2A peptide comprises the amino acid sequence ATNFSLLKQAGDVEENPGP. In some embodiments, the P2A peptide comprises the amino acid sequence GSGATNFSLKQAGDVEENPGP. In some embodiments, the E2A peptide comprises the amino acid sequence QCTNYALLKLAGDVESNPGP. In some embodiments, the E2A peptide comprises the amino acid sequence GSGQCTNYALLKLAGDVESNPGP. In some embodiments, the F2A peptide comprises the amino acid sequence VKQTLNFDLLKLAGDVESNPGP. In some embodiments, the F2A peptide comprises the amino acid sequence GSGVKQTLNFDLLKLAGDVESNPGP. In some embodiments, the third polynucleotide sequence is upstream of the fourth polynucleotide sequence.In some embodiments, the nucleic acid construct further comprises a fifth polynucleotide sequence between the third and fourth polynucleotide sequences, the fifth polynucleotide sequence encoding the amino acid sequence GSG. In some embodiments, the first polynucleotide sequence is upstream of the second polynucleotide sequence. In some embodiments, the nucleic acid construct comprises a first start codon immediately upstream of the first polynucleotide sequence and a second start codon immediately upstream of the second polynucleotide sequence. In some embodiments, the nucleic acid construct further comprises a sixth polynucleotide sequence downstream of the first and second polynucleotide sequences, the sixth polynucleotide sequence encoding a His tag or a Flag-tag. In some embodiments, the nucleic acid construct further comprises a polynucleotide sequence encoding a thymidine kinase. In some embodiments, the thymidine kinase is a mutated form that has increased cell killing activity compared to wild-type thymidine kinase. In some embodiments, the nucleic acid construct further comprises a polynucleotide sequence encoding IL-7.

[0006]

[0007] In some aspects, a method for treating cancer in a subject is described herein, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant retroviral vector described herein. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant retroviral vector comprising a nucleic acid construct comprising a polynucleotide sequence encoding thymidine kinase, and co-administering a nucleoside agent. In some embodiments, the polynucleotide sequence encoding thymidine kinase and the polynucleotide sequence encoding interleukin-12 are in the same recombinant retroviral vector. In some embodiments, the polynucleotide sequence encoding thymidine kinase and the polynucleotide sequence encoding interleukin-12 are in different recombinant retroviral vectors. In some embodiments, the method further comprises monitoring interleukin-12 levels in the subject, and inhibiting interleukin-12 expression in the subject when the interleukin-12 level in the subject reaches a predetermined threshold. In some embodiments, the nucleoside agent is at least one of ganciclovir, valganciclovir, acyclovir, valacyclovir, or penciclovir. In some embodiments, the recombinant retroviral vector encoding thymidine kinase and the recombinant retroviral vector encoding interleukin-12 are administered to the subject at different time points.

[0007]

[0008] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0008] [Figure 1]

[0009] FIG. 1 illustrates a mouse IL-12 (mIL-12) gene insert in a payload vector described herein. [Diagram 2]

[0010] FIG. 2 illustrates exemplary vectors described herein (a vector encoding murine IL-12, mIL-12, top; a vector encoding human IL-12, hIL-12, center; and a vector encoding human IL-7, hIL-7 together with IL-12). [Diagram 3]

[0011] FIG. 3 illustrates mIL-12 expression by Western blotting. [Figure 4]

[0012] FIG. 4 illustrates mIL-12 expression by ELISA. [Diagram 5]

[0013] FIG. 5 illustrates the experimental set-up for testing IFN-γ production upon stimulation with conditioned medium (CM) of A375 cells expressing commercial rIL-12 versus mIL-12. [Figure 6]

[0014] FIG. 6 illustrates the results of an IFN-γ ELISA with various amounts of IL-12 for stimulation. [Figure 7]

[0015] FIG. 7 illustrates the experimental set-up for testing hot oncogene expression after stimulation of splenocytes with commercial rIL-12 versus CM of A375 cells expressing mIL-12. [Figure 8]

[0016] FIG. 8 illustrates IFN-γ and inflammatory hot oncogene expression following stimulation of splenocytes with CM of A375 cells expressing commercial rIL-12 and mIL-12. [Figure 9]

[0017] FIG. 9 illustrates the experimental set-up for testing IFN-γ production and hot tumor gene expression after stimulation of splenocytes with CM of CT26 cells expressing commercial rIL-12 versus mIL-12. [Figure 10]

[0018] FIG. 10 illustrates IFN-γ and inflammatory hot oncogene expression following stimulation of splenocytes with CM of commercial rIL-12 and mIL-12 expressing CT26 cells. [Figure 11]

[0019] FIG. 11 illustrates an illustration of mice subcutaneously implanted with CT26 cells expressing either the luciferase gene alone or the luciferase and mIL-12 genes. [Figure 12]

[0020] FIG. 12 illustrates bioluminescence images of animals implanted with CT26 tumor cells. [Figure 13]

[0021] FIG. 13 illustrates the survival curves of animals implanted with CT26 cells expressing mIL-12 or a control. [Figure 14]

[0022] Figure 14 illustrates anti-CD8 and anti-CD11b staining in the background of Dapi blue staining of cellular DNA in control and mIL-12-expressing tumor tissues. As indicated in the figure legend, the arrows indicate the location of anti-CD8 and anti-CD11b staining, and the brackets indicate areas dominated by Dapi blue staining (indicating cells lacking staining signals from either anti-CD8 or anti-CD11b). As evidenced by the grayscale display of three-color microscopy images, mIL-12-expressing CT26 tumor tissues show a high degree of infiltration by CD8+ cytotoxic T cells and CD11b+ myeloid cells that are absent in control CT26 tumor tissues lacking mIL-12 expression. Isolated cells expressing CD8 and CD11b are observed in control tissues, whereas populations of these cells are dramatically enriched in mIL-12-expressing tissues. [Figure 15]

[0023] FIG. 15 illustrates the expression of inflammatory hot oncogenes from tumor tissue expressing mIL-12. [Figure 16]

[0024] FIG. 16 illustrates the viable cell numbers of CT26 cells following incubation with splenocytes extracted from control or CT26 mIL-12 expressing recipient groups. [Figure 17]

[0025] FIG. 17 illustrates an IFN-γ enzyme-linked immunospot (ELISpot) assay using control or mIL-12 splenocytes. [Figure 18A]

[0026] FIG. 18A illustrates an exemplary retroviral vector encoding the P35 subunit of human IL-12. [Figure 18B]

[0027] FIG. 18B illustrates expression of P35 in A375 cells transduced with a retroviral vector described herein for encoding and expressing the P35 subunit of IL-12 (left); and western blotting showing expression and secretion of the P40 subunit by A375 cells stably expressing the P40 subunit. [Figure 19]

[0028] Figure 19 illustrates Western blotting of IL-12 expression (human IL-12 or hIL-12). IL-12 (P70) expression increased when expression of P35 increased. P70 expression was independent of P40 expression (e.g., P70 expression increased with increasing expression of P35 but remained constant with P40 expression). [Figure 20]

[0029] Figure 20 illustrates an exemplary ELISA assay to quantify hIL-12 expression. Similar to Figure 19, increasing expression of P35 alone was able to increase expression of P70. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009]

[0030] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

[0010] overview

[0031] In some embodiments, vectors and methods of using vectors are described herein for expressing therapeutic agents in subjects in need thereof. In some embodiments, the vectors can be formulated into compositions or pharmaceutical compositions. In some embodiments, the vectors are recombinant retroviral vectors. In some embodiments, the vectors encode at least one interleukin (IL) or subunit of an IL. Figure 2 illustrates an exemplary recombinant retroviral vector (mIL-12 retrovector) and shows a nucleic acid encoding IL-12 (mIL-12) as described herein. In some embodiments, the vector encodes at least two subunits of an IL. In some embodiments, the vector encodes a cleavage site, where upon a cleavage event, at least two subunits of an IL encoded by the vector are cleaved. In some embodiments, the cleavage site comprises an autocleavage site (e.g., a ribozyme site or an autocleavage peptide). In some embodiments, the cleavage site comprises a peptide cleavage site, where at least two subunits of an IL can be cleaved by endogenous or exogenous proteases or by autocleavage. In some embodiments, the truncated IL subunits can complex with each other to form functional IL. In some embodiments, the truncated IL subunits can complex with other endogenous IL subunits to form functional IL. In some embodiments, the IL belongs to the IL-12 family, including IL-12, IL-23, IL-27, IL-35, or IL-39. In some embodiments, the IL is IL-12. In some embodiments, the IL-12 comprises a heterodimer comprising a P40 subunit and a P35 subunit. In some embodiments, the IL is IL-7. In some embodiments, the vector encodes IL-12 either as two IL-12 subunits (which then dimerize to form IL-12) or as a single recombinant IL-12 (Figure 3). In some embodiments, the vector comprises a retroviral vector.In some embodiments, the vector encodes an enzyme that can convert a prodrug nucleoside agent into a cytotoxic drug for treating a disease or condition.

[0011]

[0032] In some embodiments, methods for treating a subject in need thereof are also described herein. In some embodiments, the methods include administering to a subject at least one vector encoding at least one IL subunit. In some embodiments, the methods include administering to a subject at least one vector encoding at least one IL subunit and an enzyme that converts a prodrug nucleoside agent. In some embodiments, the methods include formulating the vector described herein into a composition or pharmaceutical composition to be administered to a subject in need thereof. In some embodiments, the methods described herein include contacting a cell with at least one vector ex vivo to express in the cell at least one IL subunit or an enzyme described herein that can convert a prodrug nucleoside agent into a cytotoxic agent (e.g., thymidine kinase). In some embodiments, the methods include administering to a subject in need thereof a cell contacted with at least one vector, and the cell expresses and delivers at least one IL subunit or an enzyme described herein to the subject, thereby treating the subject with a disease or condition.

[0012]

[0033] In some embodiments, the disease or condition is cancer (e.g., neoplasia, tumor, or lesion). In some embodiments, described herein is a method for treating cancer in a subject by contacting cancer cells or tumors with an interleukin (e.g., IL-12 or IL-7) and / or an enzyme capable of converting a prodrug into a cytotoxic drug, where upon contact with the interleukin and / or enzyme, the tumor is converted from a cold tumor to a hot tumor. In some embodiments, a cold tumor is a tumor or cancer cell that lacks significant immunological activity and may exhibit a relatively high degree of tolerance by the immune system for the presence of the tumor. Such tolerance may negatively impact any cancer treatment modality that would be expected to rely on a robust immune response directed toward such cold tumors or cancer cells. In some embodiments, a hot tumor is a tumor or cancer cell that exhibits an increased level of immunological activity that can support a treatment modality that relies on a relatively low tolerance by the immune system from tumor cells. The methods described herein enhance the ability of the vectors described herein to deliver localized expression of interleukins (e.g., IL-12 or IL-7) with the intent of converting a cold tumor into a hot tumor. Such conversion can render the tumor or cancer cells susceptible to an immune response by a subject being treated with the vectors described herein.

[0013]

[0034] In some embodiments, the disease or condition is inflammation or infection (e.g., bacterial, protozoan, mycobacterial, fungal, or viral infection). In some embodiments, the disease or condition is inflammation caused by an infection.

[0014] vector

[0035] In some aspects, a recombinant retroviral vector is described herein, comprising a nucleic acid construct comprising at least one polynucleotide sequence encoding an interleukin, a subunit of an interleukin, or a combination thereof. In some embodiments, the vector encodes at least one interleukin subunit. In some embodiments, the vector encodes at least two interleukin subunits, and the at least two interleukin subunits are the same or different. In some embodiments, the vector encodes one interleukin subunit. In some embodiments, the vector encodes two interleukin subunits. In some embodiments, the vector encodes two different interleukin subunits. In some embodiments, the vector encodes two or more different interleukin subunits. In some embodiments, the vector comprises at least one start codon for expressing an interleukin, a subunit of an interleukin, or a combination thereof. In some embodiments, the vector comprises at least two start codons for expressing two interleukins, two subunits of an interleukin, or a combination thereof. In some embodiments, the vector comprises two codons for expressing each interleukin subunit. In some embodiments, the vector encodes at least one additional enzyme that is not an interleukin. In some embodiments, the vector is a retroviral vector. In some embodiments, the expression of the interleukin, the subunit of the interleukin, at least one additional enzyme, or a combination thereof encoded by the vector can be carried out in vivo. For example, the interleukin, the subunit of the interleukin, at least one additional enzyme, or a combination thereof can be expressed in vivo in a subject in need thereof by administering the vector to the subject either directly or as a composition (e.g., pharmaceutical composition) described herein.In some embodiments, the vector can first be introduced into a cell ex vivo to express the interleukin, a subunit of an interleukin, at least one additional enzyme, or a combination thereof. In some aspects, the cells containing the vector can then be administered to a subject in need thereof, and the administered cells can express the interleukin, a subunit of an interleukin, at least one additional enzyme, or a combination thereof in vivo in the subject in need thereof.

[0015]

[0036] In some embodiments, the vector comprises at least one polynucleotide encoding an interleukin, a subunit of an interleukin, or a combination thereof. Non-limiting examples of interleukins can include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, or IL-41. In some embodiments, the interleukin comprises IL-7. In some embodiments, the interleukin comprises IL-12. In some embodiments, the vector comprises a first polynucleotide sequence encoding the P40 subunit of interleukin-12 (IL-12) and a second polynucleotide sequence encoding the P35 subunit of IL-12. In some aspects, the vector comprises a third polynucleotide sequence between the first and second polynucleotide sequences, the third polynucleotide sequence encoding a cleavage site that facilitates cleavage between the P40 subunit and the P35 subunit. In some aspects, the third polynucleotide sequence encodes a peptide cleavage site. In some embodiments, the cleavage site can be targeted and cleaved by an endogenous protease. In some embodiments, the endogenous protease comprises furin. In some embodiments, the cleavage site is a furin cleavage site comprising an amino acid sequence of RXYR, where R is arginine and Y may be arginine or lysine. In some embodiments, the cleavage site comprises an amino acid sequence of RRKR. In some embodiments, the cleavage site comprises the amino acid sequence of RKRR.

[0016]

[0037] In some embodiments, the peptide cleavage site is a self-cleaving peptide, such as T2A, P2A, E2A, or F2A. In some embodiments, the peptide cleavage site comprises a T2A peptide comprising the amino acid sequence: EGRGSLLTCGDVEENPGP. In some embodiments, the peptide cleavage site comprises a T2A peptide comprising the amino acid sequence: GSGEGRGSLLTCGDVEENPGP. In some embodiments, the peptide cleavage site comprises a P2A peptide comprising the amino acid sequence: ATNFSLLKQAGDVEENPGP. In some embodiments, the peptide cleavage site comprises a P2A peptide comprising the amino acid sequence: GSGATNFSLLKQAGDVEENPGP. In some embodiments, the peptide cleavage site comprises an E2A peptide comprising the amino acid sequence: QCTNYALLKLAGDVESNPGP. In some embodiments, the peptide cleavage site comprises an E2A peptide comprising the amino acid sequence: GSGQCTNYALLKLAGDVESNPGP. In some embodiments, the peptide cleavage site comprises an F2A peptide comprising the amino acid sequence: VKQTLNFDLLKLAGDVESNPGP. In some embodiments, the peptide cleavage site comprises an F2A peptide comprising the amino acid sequence: GSGVKQTLNFDLLKLAGDVESNPGP.

[0017]

[0038] In some embodiments, the vector comprises only one polynucleotide sequence encoding an interleukin or a subunit of an interleukin. For example, the vector may encode only an interleukin subunit, such as IL-12. As shown in Figures 18-20, a retroviral vector encoding the P35 subunit of IL-12 was sufficient to induce or increase the expression of IL-12. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy due to expression of the subunit of the interleukin. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy without reducing the therapeutic efficacy of the interleukin. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy by modulating the expression or abundance of the interleukin. For example, vectors encoding interleukins or subunits of interleukins can reduce toxicity associated with interleukin therapeutic agents by modulating the expression or abundance of interleukins in vivo in a subject in need of treatment with interleukin therapeutic agents.

[0018]

[0039] In some embodiments, the vector comprises at least one promoter for expressing at least one polynucleotide. For example, the vector comprises a CMV promoter for expressing at least one polynucleotide encoding an interleukin (e.g., P40 subunit and P35 subunit) as described herein. Other examples of promoters can include retroviral LTR; SV40 promoter; Rous sarcoma virus (RSV) promoter; histone promoter; polIII promoter, β-actin promoter; inducible promoter, such as MMTV promoter, metallothionein promoter; heat shock promoter; adenovirus promoter; albumin promoter; ApoAI promoter; B19 parvovirus promoter; human globin promoter; viral thymidine kinase promoter, such as herpes simplex virus thymidine kinase promoter; retroviral LTR; human growth hormone promoter, and MxIFN inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the tissue specific promoter is selected from the group including tyrosinase-related promoters (TRP-1 and TRP-2), DF3 enhancer (for breast cells), SLPI promoter (secretory leukoprotease inhibitor, expressed in many types of carcinomas), TRS (tissue specific regulatory sequence), alpha-fetoprotein promoter (specific for normal and transformed hepatocytes, respectively), carcinoembryonic antigen promoter (for use in transformed cells of the gastrointestinal tract, lung, breast and other tissues), tyrosine hydroxylase promoter (for melanocytes), choline acetyltransferase or neuron specific enolase promoter for use in neuroblastoma, gliofibroblastoma regulatory sequence, tyrosine hydroxylase promoter, c-erb B-2 promoter, PGK promoter, PEPCK promoter, whey acidic promoter (breast tissue), and casein promoter (breast tissue) and adipocyte P2 promoter.In some embodiments, the promoter is a viral specific promoter (e.g., a retroviral promoter, as well as other promoters such as the HIV promoter), hepatitis, herpes (e.g., EBV). In some embodiments, the promoter is the native HSV-TK promoter. In some embodiments, the promoter is a bacterial, fungal, or parasite (e.g., malaria) specific promoter and is utilized to target specific cells or tissues infected with a virus, bacteria, fungus, or parasite.

[0019]

[0040] In some embodiments, the vector comprises two or more promoters to express the interleukin or interleukin subunit separately in the cell. In some embodiments, the vector comprises a nucleic acid sequence encoding a tag, such as a His tag or a Flag tag, for purification, imaging, or expression control purposes.

[0020]

[0041] In some aspects, the vector is a viral vector, such as a retroviral vector. Viral vectors, particularly retroviral vectors, are becoming the most widely used method for inserting genes into mammalian cells, such as human cells. In some embodiments, other viral vectors are derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, or sindbis virus. Non-limiting examples of viral vectors can include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some cases, retroviral vectors include gamma retroviral vectors, such as vectors derived from Moloney murine leukemia virus (MoMLV, MMLV, MuLV, or MLV) or murine stem cell virus (MSCV) genomes. In some cases, retroviral vectors also include lentiviral vectors, such as lentiviral vectors derived from human immunodeficiency virus (HIV) genomes. In some cases, the AAV vector comprises AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 serotype.In some cases, the viral vector is a chimeric viral vector that comprises viral parts from two or more viruses.In additional examples, the viral vector is a recombinant viral vector.

[0021]

[0042] In some embodiments, the vector encodes an enzyme that is not an interleukin. In some aspects, the vector encodes an enzyme that can convert a nucleoside agent into a cytotoxic agent to kill cells associated with a disease or condition described herein. In some embodiments, the enzyme comprises a kinase that has a nucleic acid nucleotide as a substrate. In some embodiments, the kinase is a thymidine kinase, where the thymidine kinase is a salvage pathway enzyme that phosphorylates nucleoside analogs in addition to natural nucleoside substrates. In general, viral thymidine kinases can be therapeutically exploited by administering nucleoside analogs, such as ganciclovir or acyclovir, to cells expressing the viral thymidine kinase, where the viral thymidine kinase phosphorylates the nucleoside analogs to create a toxic product that can kill the cell. The viral thymidine kinases of the present disclosure can be prepared from a wide variety of viral thymidine kinases. In some embodiments, the viral thymidine kinase mutant is derived from a thymidine kinase from the Herpesviridae family, which includes both primate and non-primate herpesviruses, such as avian herpesviruses. Representative examples of suitable herpesviruses include, for example, Herpes Simplex Virus (HSV) type 1, Herpes Simplex Virus type 2, Varicella-Zoster Virus, Marmoset Herpesvirus, Feline Herpesvirus type 1, Pseudorabies Virus, Equine Herpesvirus type 1, Bovine Herpesvirus type 1, Turkey Herpesvirus, Marek's Disease Virus, Herpesvirus Saimiri, or Epstein-Barr Virus.

[0022]

[0043] In some embodiments, the thymidine kinase described herein may be a mutant thymidine kinase, where the mutant thymidine kinase comprises at least one amino acid mutation. In some embodiments, the mutant thymidine kinase is a mutant herpes simplex virus type 1 thymidine kinase (HSV1-TK) comprising at least one amino acid mutation compared to the wild-type amino acid sequence of HSV1-TK: MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLR (SEQ ID NO: 1). In some embodiments, the mutant HSV1-TK comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the HSV1-TK amino acid sequence (e.g., SEQ ID NO: 1). In some embodiments, the mutant HSV-1-TK comprises a nuclear export sequence (NES). In some embodiments, the NES comprises the amino acid sequence of LQKKLEELELDG (SEQ ID NO: 2).

[0023]

[0044] In some embodiments, the mutant HSV1-TK comprises at least one amino acid mutation at amino acid residues 25, 26, 32, 33, 167, 168, or a combination thereof, compared to the wild-type amino acid sequence of HSV1-TK (SEQ ID NO: 1). In some embodiments, the mutation comprises substituting a wild-type amino acid with a polar, non-polar, basic, or acidic amino acid. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167. In another example, the sequence is mutated at amino acid residue 168. In another example, the sequence is mutated at amino acid residues 167 and 168. Amino acid residue 167 may be mutated to histidine, lysine, cysteine, serine, and phenylalanine. Amino acid residue 168 may be mutated to histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26. The amino acid residues 25 and / or 26 may be mutated to an amino acid selected from the group consisting of glycine, serine, and glutamic acid. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33. The amino acid residues 32 and / or 33 may be mutated to an amino acid selected from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and / or 33. The amino acid residues 25, 26, 32, and / or 33 may be mutated to an amino acid selected from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid.

[0024]

[0045] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26; and 167, where the mutation at amino acid residues 25 and / or 26 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26; and 168, where the mutation at amino acid residues 25 and / or 26 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26; and 167 and / or 168, wherein the mutation at amino acid residues 25 and / or 26 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residues 167 and / or 168 comprises histidine, lysine, cysteine, serine, or phenylalanine.

[0025]

[0046] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33; and 167, where the mutation at amino acid residues 32 and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33; and 168, where the mutation at amino acid residues 32 and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33; and 167 and / or 168, where the mutation at amino acid residues 32 and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residues 167 and / or 168 comprises histidine, lysine, cysteine, serine, or phenylalanine.

[0026]

[0047] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and 33; and 167, where the mutations at amino acid residues 25, 26, 32, and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and 33; and 168, where the mutations at amino acid residues 25, 26, 32, and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and 33; and 167 and / or 168, where the mutations at amino acid residues 25, 26, 32, and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutations at amino acid residues 167 and / or 168 include histidine, lysine, cysteine, serine, or phenylalanine.

[0027]

[0048] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and 26 or 32 and 33; and 167, where the mutations at amino acid residues 25 and 26 or 32 and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and 26 or 32 and 33; and 168, where the mutations at amino acid residues 25 and 26 or 32 and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and 26 or 32 and 33; and 167 and / or 168, where the mutations at amino acid residues 25 and 26 or 32 and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutations at amino acid residues 167 and / or 168 include histidine, lysine, cysteine, serine, or phenylalanine.

[0028]

[0049] In some embodiments, the mutant HSV1-TK is mutated at any one or more of amino acid residues 25, 26, 32, and / or 33; and 167, wherein the mutation at any one or more of amino acid residues 25, 26, 32, and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at any one or more of amino acid residues 25, 26, 32, and / or 33; and 168, wherein the mutation at any one or more of amino acid residues 25, 26, 32, and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at any one or more of amino acid residues 25, 26, 32 and / or 33; and 167 and / or 168, wherein the mutation at any one or more of amino acid residues 25, 26, 32 and / or 33 comprises a glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residues 167 and / or 168 comprises a histidine, lysine, cysteine, serine, or phenylalanine.

[0029]

[0050] In some embodiments, the vector, in addition to encoding mutant HSV1-TK, may also encode PiT-2, PiT-1, mCat-1 (mouse cationic receptor-1; target of ecotropic Moloney MLV), or other receptors used by gammaretroviruses.

[0030]

[0051] In some embodiments, the mutant HSV1-TK comprises an increased enzymatic activity that converts a nucleoside agent to a cytotoxic agent compared to wild-type HSV1-TK. In some embodiments, the mutant HSV1-TK increases the enzymatic activity that converts a nucleoside agent to a cytotoxic agent by at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or more compared to the enzymatic activity of wild-type HSV1-TK that converts the same nucleoside agent (e.g., a prodrug) to a cytotoxic agent.

[0031]

[0052] In some embodiments, mutant HSV1-TK increases the bystander effect to kill cells related to disease or condition. "Bystander effect" as used herein refers to the phenomenon that HSV1-TK positive cells (e.g., cells contacted with vectors described herein) exert lethal effects on neighboring HSV1-TK negative cells after HSV1-TK expression is induced in HSV1-TK positive cells. In some embodiments, mutant HSV1-TK increases the bystander effect by at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or higher, compared with the bystander effect induced by wild-type HSV1-TK positive cells.

[0032] method

[0053] In some embodiments, methods of using the vectors described herein are disclosed herein. In some embodiments, the methods include treating a disease or condition in a subject in need thereof by administering to the subject a vector or a pharmaceutical composition comprising the vector described herein. In some embodiments, the methods include contacting a cell with the vector, followed by administering the cell to the subject. In some embodiments, the cell contacted with the vector is an autologous cell. For example, the cell may first be isolated from the subject, and optionally cultured or expanded before contacting with the vector. In some embodiments, expression of an interleukin (e.g., P40 or P35 of IL-12 or IL-7) or HSV1-TK encoded by the vector can be verified in the cell prior to administering the cell to the subject. Figures 11-17 illustrate an in vivo example, in which cells contacted with a vector described herein can result in the death of cancer cells after translation to a tumor-bearing mouse.

[0033]

[0054] In some embodiments, the method includes administering two or more vectors to a subject, where a first of the two or more vectors encodes an interleukin (e.g., P40 or P35 of IL-12 or IL-7) as described herein, and a second of the two or more vectors encodes a thymidine kinase (e.g., mutated HSV1-TK) as described herein. In some embodiments, the method includes first contacting a cell with two or more vectors, followed by administering the cell to a subject. In embodiments, the interleukin (e.g., P40 or P35 of IL-12 or IL-7) and the thymidine kinase (e.g., mutated HSV1-TK) are encoded by the same vector. In some embodiments, the administration is by any suitable mode of administration systemically (e.g., intravenously, inhalation, etc.). In some embodiments, the subject is a human. In some embodiments, the disease or condition is a cancer or a lesion. In some embodiments, the two or more vectors may be co-administered. In some embodiments, two or more vectors may be co-administered at the same time.In some embodiments, two or more vectors may be co-administered at different time points.For example, a first vector that encodes IL-12 or IL-7 can be administered to a subject, followed by a second vector that encodes mutant thymidine kinase at different time points.

[0034]

[0055] In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition delivers interleukin to cells or microenvironments associated with a disease or condition. In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition that delivers interleukin reduces toxicity in a subject (e.g., as determined by reduced cell death or reduced expression of hot tumor genes in cells not associated with a disease or condition) compared to direct administration of interleukin to a subject. In some embodiments, the toxicity of delivering interleukin by the vector, the cell comprising the vector, or the pharmaceutical composition described herein is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more compared to the toxicity induced by direct administration of interleukin to a subject.

[0035]

[0056] In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition delivers IL-12 or IL-7 to cells or microenvironments associated with a disease or condition. In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition delivering IL-12 (either as P40 and P35 subunits or recombinant IL-12) or IL-7 reduces toxicity in a subject (e.g., as determined by reduced cell death of cells not associated with a disease or condition, or reduced expression of hot tumor genes) compared to direct administration of IL-12 or IL-7 to a subject. In some embodiments, the toxicity of delivering IL-12 or IL-7 by the vector, the cell comprising the vector, or the pharmaceutical composition described herein is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more compared to the toxicity induced by direct administration of IL-12 or IL-7 to a subject.

[0036]

[0057] In some embodiments, the IL-12 or IL-7 encoded by the vector is expressed and secreted by the cells. In some embodiments, the IL-12 or IL-7 expressed or secreted by the cells can stimulate innate immune signaling or responses in the subject. In some embodiments, the method includes stimulating the production of an endogenous cytokine (e.g., IFN-γ) with the expressed or secreted interleukin (e.g., P40 or P35 of IL-12 or IL-7) to treat a disease or condition. As shown in Figures 5-10, the IL-12 encoded by the recombinant retroviral vector can stimulate cytokine or hot oncogene expression (e.g., IFN-γ, T-bet, IL-2, IL-15, and TNFα) in splenocytes. Hot oncogene expression can refer to the expression of gene products, such as cytokines described herein, that initiate an endogenous immune response. Thus, the expression of a hot oncogene can cause the death of cells associated with a disease or condition (e.g., cancer or tumor cells) by an endogenous immune response.

[0037]

[0058] In some embodiments, the vector comprises only one polynucleotide sequence encoding an interleukin or a subunit of an interleukin. For example, the vector may only encode an interleukin subunit, such as IL-12. As shown in Figures 18-20, a retroviral vector encoding the P35 subunit of IL-12 was sufficient to induce or increase expression of IL-12 when P40 was present. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy due to expression of the subunit of an interleukin. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy without reducing the therapeutic efficacy of the interleukin. In some embodiments, the vector encoding an interleukin or a subunit of an interleukin can reduce toxicity associated with an interleukin therapy by modulating the expression or abundance of the interleukin. For example, vectors encoding interleukins or subunits of interleukins can reduce toxicity associated with interleukin therapeutic agents by modulating the expression or abundance of interleukins in vivo in a subject in need of treatment with interleukin therapeutic agents.

[0038]

[0059] In some embodiments, the vector, cell comprising the vector, or pharmaceutical composition that delivers an interleukin has increased efficacy for treating a disease or condition in a subject (e.g., as determined by increased cell death of tumor cells or increased expression of hot tumor genes) compared to administering the interleukin directly to the subject. In some embodiments, the efficacy for treating a disease or condition by delivering an interleukin with a vector, cell comprising the vector, or pharmaceutical composition described herein is at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more reduced compared to the efficacy of treating the disease or condition by administering the interleukin directly to a subject.

[0039]

[0060] In some embodiments, a vector, cell comprising a vector, or pharmaceutical composition delivering IL-12 (either as the P40 and P35 subunits or recombinant IL-12) or IL-7 has increased efficacy for treating a disease or condition in a subject (e.g., as determined by increased cell death of tumor cells or increased expression of hot tumor genes) compared to direct administration of IL-12 or IL-7 to the subject. In some embodiments, the efficacy for treating a disease or condition by delivering IL-12 or IL-7 by a vector, cell comprising a vector, or pharmaceutical composition described herein is at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more reduced compared to the efficacy of treating a disease or condition by direct administration of IL-12 or IL-7 to a subject.

[0040]

[0061] In some embodiments, the vector, cell comprising the vector, or pharmaceutical composition is administered at least once during the period (e.g., every 2 days, twice a week, once a week, every week, 3 times a month, twice a month, once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is administered twice or more during the period (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times).

[0041]

[0062] In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition is administered in a therapeutically effective amount by various forms and routes, such as oral or topical administration. In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition may be administered intratumorally, parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, subarachnoidally, intraocularly, intrasternally, ocularly, endothelially, topically, intranasally, intrapulmonary, rectally, intraarterially, intrathecally, by inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal administration, such as by injection or infusion. In some embodiments, the composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa administration). In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition is delivered via multiple routes of administration.

[0042]

[0063] In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition is administered by intravenous infusion. In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition comprising the vector is administered by slow continuous infusion over a long period of time, for example, over a period of time longer than 24 hours. In some aspects, the vector, the cell comprising the vector, or the pharmaceutical composition may be administered in a localized manner, for example, via injection of the drug directly into an organ, and may optionally be administered in a depot or sustained release formulation or implant.

[0043]

[0064] In some embodiments, the method includes monitoring expression of an interleukin, such as IL-12 or IL-7, in the subject after the subject has been treated. In some aspects, the method includes monitoring expression levels of IL-12 or IL-7, and when IL-12 or IL-7 expression in the subject reaches a predetermined threshold, an interleukin inhibitor can be administered to the subject.

[0044]

[0065] In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition provided herein may be administered with at least one additional therapeutic agent, such as an antiviral therapy, a chemotherapeutic agent, an antibiotic, a cell therapy, a cytokine therapy, or an anti-inflammatory agent. In some embodiments, the at least one additional therapeutic agent comprises a nucleoside agent (e.g., a prodrug). Non-limiting examples of prodrugs include, for thymidine kinase, FHBG (9-[4-fluoro-3-(hydroxymethyl)butyl]guanine), FHPG (9-([3-fluoro-1-hydroxy-2-propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil, and the like. ), FEAU (fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil), FMAU (fluoro-5-methyl-1-beta-D-arabinofuranosyluracil), FHOMP (6-((1-fluoro-3-hydroxypropan-2-yloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione), ganciclovir, valganciclovir, acyclovir, valaciclovir, Examples of such compounds include valacivlovir, penciclovir, radiolabeled pyrimidines with a 4-hydroxy-3-(hydroxymethyl)butyl side chain at N-1 (HHG-5-FEP), or 5-(2-)hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine derivatives with 2,3-dihydroxypropyl, acyclovir, ganciclovir and penciclovir-like side chains; ifosfamide for oxidoreductase; 6-methoxypurine arabinoside for VZV-TK; 5-fluorocytosine for cytosine deaminase; doxorubicin for beta-glucuronidase; CB1954 and nitrofurazone for nitroreductase; and N-(cyanoacetyl)-L-phenylalanine or N-(3-chloropropionyl)-L-phenylalanine for carboxypeptidase A.In some embodiments, the nucleoside agent comprises ganciclovir, valganciclovir, acyclovir, valacyclovir, or penciclovir.

[0045]

[0066] In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition provided herein can be administered before, during, or after the appearance of a disease or condition. In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition can be used as a prophylactic and can be administered continuously to a subject. In some embodiments, the vector, the cell comprising the vector, or the pharmaceutical composition can be administered to a subject before the onset of symptoms associated with a disease or condition.

[0046]

[0067] The actual dosage level of the agent (e.g., vector, cell containing vector, or pharmaceutical composition) of the present disclosure can be varied to obtain an amount of agent that achieves the desired therapeutic response for a particular subject, composition, and mode of administration without causing toxicity to the subject (e.g., subject for immunization or subject for treatment). The selected dosage level may depend on various pharmacokinetic factors, such as the activity of the particular composition described herein, the route of administration, the time of administration, the frequency of excretion, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0047]

[0068] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Dosage unit form, as used herein, refers to physically discrete units suitable as a combined dosage for a subject (e.g., a subject for immunization or a subject for treatment); each unit contains a predetermined amount of active agent calculated to produce a desired therapeutic effect depending on the required pharmaceutical carrier. The details regarding dosage unit forms of the present disclosure can be determined by and directly depend on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art in compounding such active agents for the treatment of susceptibility in an individual. Dosage can be determined by reference to the plasma or local concentration of the circular polyribonucleotide or antibody or antigen-binding fragment thereof. Dosage can be determined by reference to the plasma or local concentration of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.

[0048]

[0069] In some embodiments, the vectors, cells containing the vectors, or pharmaceutical compositions described herein may be in unit dosage form suitable for single administration of precise dosages. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of the composition. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosage form may be in the form of a package containing discrete amounts of the formulation. Non-limiting examples are packaged injectables, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with or without preservatives. The formulations for injection disclosed herein may be in unit dosage form, for example, in ampoules containing preservatives or in multi-dose containers.

[0049]

[0070] The dosage of the vector, the cell containing the vector, or the pharmaceutical composition is preferably within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. The therapeutically effective dose can be initially estimated from cell culture assays. The dose can be provided in an animal model that achieves a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal infection or half-maximal inhibition) when determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by RT-qPCR or ddPCR methods.

[0050]

[0071] The effective amount or therapeutically effective amount of the vector, cell containing the vector, or pharmaceutical composition disclosed herein to be administered to a subject in need of treatment can be determined in various ways.As an example, the amount can be based on the titer or efficacy of the virus in an animal model.Alternatively, the dosage regimen used in clinical trials can be used as a general guideline.

[0051]

[0072] In some embodiments, the daily dose may be administered in a single dose or in several doses at various times throughout the day. In some embodiments, higher dosages may be required and may be reduced over time once an optimal initial response is obtained. In some embodiments, treatment may be continuous for days, weeks, or years, or may be spaced apart with intervening rest periods. In some embodiments, dosage is modified according to other treatments that the individual may have undergone. However, the method of treatment is in no way limited to a particular concentration or range of retroviral particles, which may vary for each individual being treated and each derivative used. Individualization of dosage may be required to achieve maximum effect for a given individual. In some embodiments, the dosage administered to the individual being treated varies depending on the individual's age, severity or stage of the disease, and response to treatment courses. In some embodiments, clinical parameters for determining dosage include, but are not limited to, tumor size, changes in levels of tumor markers used in clinical trials for a particular malignancy. In some embodiments, the treating physician determines the therapeutically effective amount to be used for a given individual. In some embodiments, the therapies disclosed herein are administered as frequently as needed, for as long as deemed necessary by the treating physician.

[0052]

[0073] In some embodiments, multiple courses of therapeutic agents (e.g., a first and a second course of therapeutic agents) are administered to a subject in need of treatment. In some embodiments, the first and / or second course of therapeutic agents are administered intravenously. In other embodiments, the first and / or second course of therapeutic agents are administered via intra-arterial infusion, including but not limited to infusion via the hepatic artery, cerebral artery, coronary artery, pulmonary artery, iliac artery, celiac artery, gastric artery, splenic artery, renal artery, gonadal artery, subclavian artery, vertebral artery, axillary artery, brachial artery, radial artery, ulnar artery, carotid artery, femoral artery, inferior mesenteric artery, and / or superior mesenteric artery. Intra-arterial infusion may be achieved using an endovascular procedure, a percutaneous procedure, or a surgical approach involving an incision. In some embodiments, the first and second courses of therapeutic agents may be administered sequentially. In yet other embodiments, the first and second courses of therapeutic agents may be administered simultaneously. In yet other embodiments, the optional third course of therapeutic agent may be administered sequentially or simultaneously with the courses of the first and second therapeutic agents.

[0053]

[0074] In some embodiments, the vector, cell comprising the vector, or pharmaceutical composition disclosed herein may be administered with a course of therapeutic agent administered sequentially or simultaneously in high doses based on accumulation. For example, in some embodiments, the course of therapeutic agent may be administered systemically, e.g., intravenously, to a patient in need thereof based on accumulation. The course of the first therapeutic agent may be administered systemically. Alternatively, the course of the first therapeutic agent may be administered in a localized manner, e.g., intra-arterially, to a patient in need thereof via intra-arterial infusion based on accumulation.

[0054]

[0075] In yet other embodiments, a subject in need thereof may receive a combination of systemic and intra-arterial infusion administration of high doses of vectors, cells comprising the vector, or pharmaceutical compositions, either sequentially or simultaneously. For example, a patient in need thereof may first be administered a systemic dose of vectors, cells comprising the vector, or pharmaceutical compositions on a depot basis, followed by an additional course of therapeutic agent administered via intra-arterial infusion, e.g., hepatic artery infusion, as a depot-based delivery.

[0055]

[0076] The subject in need of treatment can also be administered a course of therapeutic agent that delivers vector, cells containing vector, or pharmaceutical composition over a period of time, either systemically or locally (e.g., intra-arterial infusion, e.g., hepatic artery infusion). In some embodiments, the period can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. Administration can also be performed in a chronic manner, i.e., for an indefinite period of time or indefinitely.

[0056]

[0077] The administration of the vector, the cell comprising the vector, or the pharmaceutical composition may also be performed in a regular manner, for example, at least once a day, at least twice a day, at least three times a day, at least four times a day, or at least five times a day. The regular administration of the delivery of the vector, the cell comprising the vector, or the pharmaceutical composition may depend on the mode of administration in addition to the time of delivery. For example, parenteral administration may be performed only once a day for a long period of time, whereas the oral administration of the delivery of the vector, the cell comprising the vector, or the pharmaceutical composition may be performed more than once a day, in which case the administration of the delivery of the vector, the cell comprising the vector, or the pharmaceutical composition is performed for a shorter period of time.

[0057]

[0078] In one embodiment, the subject is allowed to rest for 1-2 days between the first and second therapeutic courses. In some embodiments, the subject is allowed to rest for 2-4 days between the first and second therapeutic courses. In other embodiments, the subject is allowed to rest for at least 2 days between the first and second therapeutic courses. In still other embodiments, the subject is allowed to rest for at least 4 days between the first and second therapeutic courses. In still other embodiments, the subject is allowed to rest for at least 6 days between the first and second therapeutic courses. In some embodiments, the subject is allowed to rest for at least 1 week between the first and second therapeutic courses. In still other embodiments, the subject is allowed to rest for at least 2 weeks between the first and second therapeutic courses. In one embodiment, the subject is allowed to rest for at least 1 month between the first and second therapeutic courses. In some embodiments, the subject is allowed to rest for at least 1-7 days between the course of the second therapeutic agent and the optional course of the third therapeutic agent, hi yet other embodiments, the subject is allowed to rest for at least 1-2 weeks between the course of the second therapeutic agent and the optional course of the third therapeutic agent.

[0058]

[0079] In some embodiments, the vector, cell comprising the vector, or pharmaceutical composition is administered to increase the local concentration of interleukins (e.g., P40 or P35 of IL-12 or IL-7) and thymidine kinases (e.g., mutated HSV1-TK) in cells or microenvironments (e.g., cancer or lesions) associated with a disease or condition described herein. In some embodiments, the vector, cell comprising the vector, or pharmaceutical composition is administered via intra-arterial infusion, thereby increasing the local concentration of the therapeutic vector to a specific organ system. In yet other embodiments, the vector, cell comprising the vector, or pharmaceutical composition is administered intratumorally. In some embodiments, depending on the location of the target lesion, catheterization of the hepatic artery is followed by infusion into the pancreaticoduodenal artery, right hepatic artery, and middle hepatic artery, respectively, to locally target liver lesions. In some embodiments, local distribution of the peptide or delivery vector to other organ systems, such as the lungs, gastrointestinal, brain, reproductive tract, spleen, or other defined organ systems, is achieved via catheterization or other local delivery systems. In some embodiments, intra-arterial infusion is accomplished via any other available arterial source, including, but not limited to, infusion via the hepatic, cerebral, coronary, pulmonary, iliac, celiac, gastric, splenic, renal, gonadal, subclavian, vertebral, axillary, brachial, radial, ulnar, carotid, femoral, inferior mesenteric and / or superior mesenteric arteries. In some embodiments, intra-arterial infusion is accomplished using an endovascular procedure, a percutaneous procedure or a surgical approach involving an incision.

[0059] Pharmaceutical Compositions

[0080] Described herein is a pharmaceutical composition comprising a therapeutic agent (e.g., a vector or a cell comprising a vector as described herein). In some embodiments, the cell contacted with the vector as described herein expresses an interleukin (e.g., P40 or P35 of IL-12 or IL-7) or a thymidine kinase (e.g., a mutated HSV1-TK) in vivo or in vitro. In some embodiments, the cell is obtained from a subject; expanded in an in vitro environment; and administered back to the subject to treat a disease or condition in the subject. In some embodiments, the cell is obtained from a source that is not from the subject. In some embodiments, the cell is obtained from a cell line. In some embodiments, the cell is formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a nucleoside agent as described herein.

[0060]

[0081] In some embodiments, the pharmaceutical composition comprises pharma- ceutical acceptable carrier, excipient, or diluent.In some embodiments, the pharmaceutical composition described herein comprises at least one additional active agent other than the cells described herein.In some embodiments, the at least one additional active agent is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an antiangiogenic agent, or a checkpoint inhibitor.

[0061]

[0082] In carrying out the methods of treatment or use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease or condition, such as cancer or a lesion, to be treated.In some embodiments, the mammal is a human.The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors.The therapeutic agent described herein, in some cases the composition, can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0062]

[0083] The pharmaceutical compositions described herein can be administered to a subject by suitable administration routes, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes.Compositions described herein include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0063]

[0084] Pharmaceutical compositions containing a therapeutic agent can be manufactured in a conventional manner, e.g., by way of example only, conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping or compressing processes.

[0064]

[0085] Pharmaceutical compositions may contain at least exogenous therapeutic agents as active ingredients in free acid or free base form, or in pharmaceutical acceptable salt form.In addition, the methods and compositions described herein include the use of N-oxides (optionally), crystalline forms, amorphous phases, as well as active metabolites of these compounds with the same type of activity.In some embodiments, therapeutic agents are present in unsolvated form or in solvated form with pharmaceutical acceptable solvents, such as water, ethanol, etc.Solvated forms of therapeutic agents are also considered to be disclosed herein.

[0065]

[0086] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more preservatives for inhibiting microbial activity.Suitable preservatives include mercury-containing substances, such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0066]

[0087] In some embodiments, the pharmaceutical compositions described herein benefit from antioxidants, metal chelators, thiol-containing compounds and other general stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfates and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0067]

[0088] The pharmaceutical compositions described herein can be formulated into any suitable dosage form, including but not limited to aqueous oral dispersion, liquid, gel, syrup, elixir, slurry, suspension, solid oral dosage form, aerosol form, controlled release formulation, fast dissolving formulation, effervescent formulation, lyophilized formulation, tablet, powder, pill, dragee, capsule, delayed release formulation, sustained release formulation, pulsed release formulation, multiparticulate formulation, and mixed immediate release and controlled release formulation.In one embodiment, the therapeutic agent discussed herein, for example, therapeutic agent, is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous or intravenous injection.In one embodiment, the formulation suitable for intramuscular, subcutaneous or intravenous injection includes physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powder for rehydration into sterile injectable solution or dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene-glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispensing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of substances that delay absorption, such as aluminum monostearate and gelatin.

[0068]

[0089] For intravenous injection or drip or infusion, the pharmaceutical compositions described herein are formulated in the form of an aqueous solution, preferably in the form of a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiologically buffered saline.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.

[0069]

[0090] Parenteral injections may include bolus injections or continuous infusions. Pharmaceutical compositions for injection may be in unit dosage form, for example in the form of ampoules or multi-dose containers with added preservatives. The pharmaceutical compositions described herein may be in a form suitable for parenteral injections, as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, or may contain forming agents such as suspending, stabilizing and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable vehicle, for example sterile pyrogen-free water, before use.

[0070]

[0091] For administration by inhalation, the therapeutic agent is formulated for use as an aerosol, mist or powder. The pharmaceutical composition described herein is conveniently delivered in the form of an aerosol spray supply from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, such as, by way of example only, gelatin capsules and cartridges, may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base, such as lactose or starch, for use in an inhaler or insufflator. The formulation containing the composition is prepared as a solution in saline employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Preferably, these compositions and formulations are prepared using suitable non-toxic pharma-ceutically acceptable ingredients. The selection of suitable carrier depends on the exact nature of the desired nasal dosage form, for example, solution, suspension, ointment or gel.Nasal dosage forms generally contain a large amount of water in addition to active ingredient.A small amount of other ingredients, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffering agents and other stabilizers and solubilizers, are optionally present.Preferably, nasal dosage forms should be isotonic with nasal secretions.

[0071]

[0092] In another embodiment, the dosage form includes microencapsulated formulation.In some embodiments, one or more other compatible materials are present in the microencapsulated material.Non-limiting examples of materials include pH adjuster, erosion promoter, antifoaming agent, antioxidant, flavoring agent, and carrier material, such as binder, suspending agent, disintegrant, filler, surfactant, solubilizer, stabilizer, lubricant, wetting agent, and diluent.

[0072]

[0093] The liquid formulation for oral administration is optionally an aqueous suspension selected from the group consisting of, but not limited to, pharma-ceutically acceptable aqueous oral dispersion, emulsion, solution, elixir, gel, and syrup.In addition to the therapeutic agent, the liquid formulation optionally comprises additives, such as (a) disintegrant; (b) dispersant; (c) wetting agent; (d) at least one preservative, (e) viscosity enhancing agent, (f) at least one sweetener, and (g) at least one flavoring agent.In some embodiments, the aqueous dispersion further comprises a crystal formation inhibitor.

[0073]

[0094] In some embodiments, the pharmaceutical compositions described herein may be self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by forceful mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to disperse the droplets throughout the solution. In addition, water or aqueous phase is optionally added immediately prior to administration, thereby ensuring the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides an improvement in the bioavailability of hydrophobic active ingredients.

[0074]

[0095] Oral formulations are administered using various formulations known in the art.In addition, the oral dosage forms described herein may further comprise a biodegradable (hydrolyzable) polymer carrier, which also helps the dosage form to adhere to oral mucosa.For oral or sublingual administration, the composition may take the form of tablets, lozenges, or gels that are formulated in a conventional manner.

[0075]

[0096] For intravenous injection, the pharmaceutical composition is optionally formulated in the form of an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiologically buffered saline.For transmucosal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.

[0076]

[0097] Parenteral injections optionally include bolus injections or continuous infusions. Preparations for injections are optionally in unit dosage form, for example in ampoules or multi-dose containers with added preservatives. In some embodiments, the compositions described herein are in a form suitable for parenteral injections as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain forming agents such as suspending agents, stabilizing agents and / or dispersing agents. Compositions for parenteral administration include aqueous solutions of the agent that modulates the activity of the carotid body in water-soluble form. In addition, suspensions of the agent that modulates the activity of the carotid body, for example oily suspension injections, are optionally prepared as needed.

[0077]

[0098] Traditional formulation techniques include, for example, one or a combination of the following methods: (1) dry blending, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Wurster coating), tangential coating, top spraying, tableting, extrusion, and the like.

[0078]

[0099] In some embodiments, a pharmaceutical composition may be provided that includes particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granule for suspension, which when mixed with water, provides a substantially uniform suspension.

[0079]

[0100] In addition, the pharmaceutical composition optionally contains one or more pH adjusting or buffering agents, examples of which include acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citric acid / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0080]

[0101] In addition, the pharmaceutical composition optionally contains one or more salts in an amount necessary to bring the osmolality of the pharmaceutical composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0081]

[0102] In one embodiment, the aqueous suspension and dispersion described herein remain homogeneous for at least 4 hours.In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical stirring lasting less than 1 minute.In yet another embodiment, stirring is not required to maintain a homogeneous aqueous dispersion.

[0082]

[0103] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal passages in the form of drops or sprays. Nasal solutions may resemble nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range may also be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0083]

[0104] Aerosol formulations for inhalation and inhalation can be designed to deliver the drug or drug combination to the subject's respiratory tree when administered via the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer. Inhalation or insufflation containing micronized or liquid drugs may be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the drug or drug combination in a propellant, for example, to aid in distribution. The propellant may be a liquefied gas, examples of which include halocarbons, e.g., fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers. The aerosol formulation may also contain other ingredients, such as ethanol, isopropanol, propylene glycol, as well as surfactants or other ingredients such as oils and detergents. These ingredients may help stabilize the formulation and / or lubricate the valve components.

[0084] kit

[0105] In some embodiments, kits are described herein for using the vectors described herein. In some embodiments, the kits can be used to treat a disease or condition in a subject. In some embodiments, the kits include a group of materials or components apart from the vector or cells containing the vector. In some embodiments, the kits include components for assaying the number of units of a biomolecule (e.g., a vector, a cell, a therapeutic agent including IL-12 or IL-7, a mutant HSV1-TK, or a combination thereof) synthesized and / or released or expressed by the cells described herein. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, the kit may be designed for the purpose of treating a disease or condition (e.g., cancer or lesion) disclosed herein in a subject. In some embodiments, the kits are specifically designed for the purpose of treating a mammalian subject. In some embodiments, the kits are specifically designed for the purpose of treating a human subject.

[0085]

[0106] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the vector, cell, or pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the kit includes instructions for further engineering the vector or cell to express a biomolecule (e.g., a therapeutic agent including IL-12 or IL-7, and mutant HSV1-TK). In some embodiments, the kit includes instructions for thawing or otherwise restoring biological activity of cells that may have been preserved during storage or transportation. In some embodiments, the kit includes instructions for measuring the viability of preserved cells to confirm efficacy for its intended purpose (e.g., therapeutic efficacy when used to treat a subject).

[0086]

[0107] Optionally, the kit also contains other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful equipment. The assembled materials or components in the kit may be provided to the physician, stored in any convenient and suitable manner that preserves operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form, and they may be provided at room temperature, refrigerated temperature, or frozen temperature. The components are typically contained in suitable packaging materials.

[0087]

[0108] The use of independent or consecutive terms, such as "will", "will not be", "shall be", "shall not be", "must", "do not have to be", "initialy", "initially", "next", "consequently", "before", "after", "finally", and "finally" are not meant to be limiting on the scope of the embodiments of the invention disclosed herein, but are meant to be exemplary.

[0088]

[0109] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Additionally, the terms "including," "including," "having," "having," "with," or derivatives thereof are intended to be inclusive in a manner similar to the term "comprising" to the extent such terms are used either in the detailed description and / or claims.

[0089]

[0110] The phrases "at least one," "one or more," and "and / or," as used herein, are open-ended expressions that are valid both conjunctively and disjunctively. For example, each of the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0090]

[0111] "Or," as used herein, can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can also refer to "A or B," "A but not B," "A but not B," and "A and B." In some cases, context may have a particular meaning.

[0091]

[0112] Any of the systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily indicate a priority, order of importance, or order of execution.

[0092]

[0113] The term "about," when used in reference to a number or numerical range, means that the stated number or numerical range is approximate within experimental variation (or within statistical experimental error), and that the number or numerical range may vary, for example, from 1% to 15% of the stated number or numerical range. In embodiments, the term "about" refers to ±10% of the stated number or value.

[0093]

[0114] The term "increased", "increasing" or "increase" is generally used herein to mean an increase in a statically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, for example, an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to 100%, including 100%, or any increase between 10 and 100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more compared to a reference level.

[0094]

[0115] The term "reduced", "reducing" or "reducing" is generally used herein to mean a statistically significant reduction. In some embodiments, "reduced" or "reducing" means a reduction of at least 10% compared to a reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a reduction of up to 100%, including 100% (for example, non-existent or undetectable compared to a reference level), or any reduction between 10 and 100%. In the context of a marker or symptom, these terms mean a statistically significant reduction of such a level. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably a reduction to a level that is accepted as being within the normal range for an individual without a given disease.

[0095]

[0116] "Nucleic acid" as used herein refers to a polynucleotide that contains at least two covalently linked nucleotide or nucleotide analog subunits. Nucleic acid is generally deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA. Nucleic acid is generally single-stranded, double-stranded, or a mixture thereof. For the purposes herein, unless otherwise specified, or clear from the context, nucleic acid is double-stranded.

[0096]

[0117] "DNA," as used herein, is meant to include DNA molecules of all types and sizes, including, for example, cDNA, plasmids, and DNA containing modified nucleotides and nucleotide analogs.

[0097]

[0118] "Nucleotide," as used herein, includes nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides, such as, but not limited to, phosphorothioate nucleotides and deazapurine nucleotides, as well as other nucleotide analogs.

[0098]

[0119] The term "polynucleotide" as used herein refers to a polymeric form of nucleotide of any length, examples of which include ribonucleotides and deoxyribonucleotides.Such terms also include single-stranded and double-stranded DNA, as well as single-stranded and double-stranded RNA.This term also includes modified polynucleotides, such as methylated or capped polynucleotides.

[0099]

[0120] The term "subject" as used herein refers to the animal, plant, insect and bird into which large DNA molecule is introduced.It also includes higher organisms such as mammals and birds, for example, humans, primates, rodents, cows, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chickens, etc.Subjects may or may not have a disease or condition.

[0100]

[0121] "Administering to a subject," as used herein, is a procedure in which one or more delivery agents and / or large nucleic acid molecules, together or separately, are introduced into or applied onto a subject such that target cells present in the subject ultimately come into contact with the agents and / or large nucleic acid molecules.

[0101]

[0122] "Delivery vector" or "delivery vehicle" or "therapeutic vector" or "therapeutic system" as used herein refers to both viral and non-viral particles that encapsulate and transport exogenous nucleic acid molecules to target cells or tissues. Viral vehicles include, but are not limited to, retroviruses, adenoviruses, lentiviruses, herpes viruses, and adeno-associated viruses. Non-viral vehicles include, but are not limited to, microparticles, nanoparticles, virosomes, and liposomes. "Targeting" as used herein refers to the use of ligands that associate with the delivery vehicle and target the vehicle to cells or tissues. Ligands include, but are not limited to, antibodies, receptors, and collagen binding domains.

[0102]

[0123] "Delivery" is used synonymously with "transduction" and as used herein refers to the process by which an exogenous nucleic acid molecule is transferred to a cell so that it is located inside the cell. Delivery of a nucleic acid is a process separate from expression of the nucleic acid.

[0103]

[0124] "Expression" as used herein refers to the process that nucleic acid is translated into peptide or transcribed into RNA that can be translated into, for example, peptide, polypeptide or protein.If nucleic acid is derived from genomic DNA, expression includes splicing of mRNA if appropriate eukaryotic host cell or organism is selected.When expressing heterologous nucleic acid in host cell, heterologous nucleic acid must first be delivered to cell, and then, once inside cell, it must be finally present in nucleus.

[0104]

[0125] "Course of therapeutic agent" as used herein refers to regular or timed administration of the vector disclosed herein within a given period of time. Such period may be at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 6 months. Administration may also be in a chronic manner, i.e., for an indefinite period of time. Regular or timed administration includes administration once a day, twice a day, three times a day, or other set timed administration.

[0105]

[0126] The terms "co-administered," "administered in combination with," and their grammatical equivalents, as used herein, are meant to encompass administration of a selected therapeutic agent to a single patient and are intended to include treatment regimens in which the agents are administered by the same or different routes of administration or at the same or different times. In some embodiments, the therapeutic agents disclosed in the present application are expected to be co-administered with other agents. These terms encompass administration of two or more agents to an animal such that both agents and / or their metabolites are present in the animal at the same time. Examples include co-administration in separate compositions, administration at different times in separate compositions, and / or administration in a composition in which both agents are present. Thus, in some embodiments, the therapeutic agent and the other agent are administered in the form of a single composition. In some embodiments, the therapeutic agent and the other agent are admixed in a composition. In further embodiments, the therapeutic agent and the other agent are administered in separate doses at separate times.

[0106]

[0127] The term "mutant thymidine kinase," as used herein, refers not only to the specific proteins described herein (as well as the nucleic acid sequences encoding these proteins), but also to derivatives thereof, which may include various structural forms of the primary protein that retain biological activity.

[0107]

[0128] The term "mutated" or "replaced with another nucleotide" as used herein means that a nucleotide at a particular position is replaced with a nucleotide other than the one present at that position in the unmutated or previously mutated sequence. That is, in some cases, a particular modification may be made with a different nucleotide. In some embodiments, the replacement is made such that the relevant splice donor and / or acceptor site is no longer present in the gene.

[0108]

[0129] "Polar amino acid," as used herein, refers to the amino acid residues Asn (N), Cys (C), Gln (Q), Gly (G), Ser (S), Thr (T) or Tyr (Y).

[0109]

[0130] "Nonpolar amino acid," as used herein, refers to the amino acid residues Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), or Val (V).

[0110]

[0131] "Basic amino acid," as used herein, refers to the amino acid residues Arg (R), His (H), or Lys (K).

[0132] "Acidic amino acid," as used herein, refers to the amino acid residues Asp (D) or Glu (E).

[0111]

[0133] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific depictions, arrangements, or relative proportions described herein, which depend upon various conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein can be employed in the practice of the present invention. It is therefore anticipated that the present invention shall cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are thereby covered. EXAMPLES

[0112]

[0134] The following illustrative examples are representative of embodiments of the stimuli, systems, and methods described herein and are not meant to be limiting in any way. Example 1. Generation of mouse IL-12 and human IL-12 vectors Generation of mouse IL-12 vector

[0135] Mouse IL-12 (mIL-12) was first engineered into a vector as shown in Figure 1 (mouse_IL-12, 1740bp; AgeI_Start_P40_furin site_GSG_T2A_Start_P35_HIS_Stop_BamHI).

[0113]

[0136] P40 and P35 (peptide sequences shown in Table 1) were linked by a T2A linker (GSGEGRGSLLTCGDVEENPGP) in the payload vector. To increase protein expression by ensuring cleavage between P40 and P35, a furin site (RRKR) followed by the amino acid GSG was added to the end of the P40 subunit (Figure 2). This resulted in the creation of a P40 protein with two additional amino acids (RR) only at the end of the protein sequence, instead of the extra 23 amino acids without a furin site. A HIS tag was added after P35 for detection as well. The mIL-12 gene was synthesized, amplified, and inserted into the payload vector at the AgeI and BamHI sites with Genscript (Piscataway, NJ) (Figure 2).

[0114] [Table 1]

[0115]

[0137] A375 melanoma test cell line was transduced with mIL-12 vector (e.g., retroviral vector) and cloned as a single clonal cell line. Expression of secreted protein from the clonal cell line was tested by mIL-12 ELISA and Western blotting (Figure 3). In the Western blot, the reduced sample clearly showed two subunits, P40 and P35, whereas the non-reduced sample showed a protein size of 70 kDa, indicating the presence of a heterodimer containing P40 and P35. Conditioned medium (CM) from A375 cells transduced with mIL-12 retroviral vector was used to quantitate the amount of secreted mIL-12 by ELISA. Clone 2-E2 was selected for further in vitro studies (Figure 4).

[0116] Generation of human IL-12 vector

[0138] Human IL-12 (Table 2) was also incorporated into a retroviral vector as follows: human_IL-12, 1740bp; AgeI_Kozak_Start_P40_Furin site_GSG_T2A_Start_P35_Stop_BamHI. For human IL-12, the same integration method was applied as for the production of mIL-12, except for the HIS tag. The HIS tag in the hIL-12 gene was removed.

[0117] [Table 2]

[0118]

[0139] P40 and P35 (peptide sequences shown in Table 2) were linked by a T2A linker (GSGEGRGSLLTCGDVEENPGP) in the payload vector. To increase protein expression by ensuring cleavage between P40 and P35, a furin site (RRKR) followed by the amino acid GSG was added to the end of the P40 subunit (Figure 2). This resulted in the creation of a P40 protein with two additional amino acids (RR) only at the end of the protein sequence, instead of the extra 23 amino acids without the furin site. The hIL-12 gene was synthesized, amplified, and inserted into the payload vector at the AgeI and BamHI sites with Genscript (Piscataway, NJ).

[0119] Efficacy of mIL-12 in vitro

[0140] The functionality of mIL-12 was examined by incubating mouse splenocytes with mIL-12 and measuring INF-γ. 6 Mouse splenocytes were stimulated for 48 hours with either commercial recombinant mouse IL-12 (rIL-12) or CM from A375 cells transduced with and stably expressing mIL-12 retroviral vector. After stimulation, CM from each culture was collected and measured for IFN-γ production by ELISA (Figure 5). Results showed that CM from A375 cells stably expressing mIL-12 dose-dependently induced mouse IFN-γ production in mouse splenocytes and was as effective as rIL-12 (Figure 6).

[0120]

[0141] We further evaluated the activation of inflammatory genes from immune cells in A375 cells stably expressing mIL-12. 6Mouse splenocytes were stimulated with either commercial rIL-12 or CM from A375 stably expressing mIL-12 for 48 hours at an IL-12 concentration of 2ng / mL. After stimulation, cells from each group were collected and analyzed for expression of inflammatory genes by qPCR. Results demonstrated that CM from A375 stably expressing mIL-12 induced IFN-γ and other inflammatory genes just as effectively as commercial rIL-12 (Figures 7 and 8).

[0121]

[0142] We then transduced a murine colorectal cancer cell line, CT26, with the mIL-12 retroviral vector and examined whether the secreted mIL-12 could produce IFN-γ and activate inflammatory genes from immune cells. 6 Mouse splenocytes were stimulated with either commercial rIL-12 or CM from mIL-12-expressing CT26 cells at a concentration of 2ng / mL for 48 hours. After stimulation, supernatants and cells from each group were collected and analyzed for IFN-γ production by ELISA and for inflammatory gene expression by qPCR. The results showed that CM from mIL-12-expressing CT26 cells induced IFN-γ production and other inflammatory genes just as effectively as commercial rIL-12 (Figures 9 and 10). Some preliminary animal experiments were performed with 1.5×10 CT26 cells stably expressing the luciferase gene from retroviral vector cells or 1.5×10 CT26 cells stably expressing the luciferase and mIL-12 genes. 5Imaging was performed by implanting 1000 cells in the lateral part of the right shoulder. For continuous in vivo monitoring of luciferase-expressing CT26 tumors in the lateral part of the shoulder, an IVIS imaging system was used (Figure 11). In Figure 12, bioluminescence images of animals implanted with either luciferase-expressing CT26 or luciferase and mIL-12 tumor cells are shown. Prior to in vivo imaging, tumor-bearing animals received 200 μL of luciferin (15 mg / mL). Survival curves showed 100% survival up to 63 days until the end of the study with mIL-12-expressing CT26, whereas only one animal survived in the control group (Figure 13).

[0122]

[0143] Some CT26 tumor tissues were extracted from the control and mIL-12 groups and stained for the presence of CD8 (cytotoxic T cells) and CD11b (myeloid) (Figure 14). Increased CD8 and CD11b positive immune cells were detected in CT26 mIL-12 expressing tumor tissues. CT26 tumor tissues from the control and mIL-12 groups were collected and analyzed for expression of multiple inflammatory hot tumor genes, including the T cell attracting chemokines CXCL9 and CXCL10, by qPCR (Figure 15). Tumor tissues from three individual CT26 mIL-12 expressing recipients were compared to controls. Approximately 5x10 5 5 x 10 splenocytes 4mIL-12 splenocytes were mixed with native CT26 cells for 72 hours and viable CT26 cells were counted. The data showed that mIL-12 splenocytes were able to kill CT26 target cells more effectively, as shown by light microscopy pictures of the cells (Figure 16). The IFN-γ Enzyme-Linked ImmunoSpot (ELISpot) assay is an immunoassay that measures the frequency of cells secreting IFN-γ at the single cell level. In this assay, immune cells are cultured on a surface coated with a specific IFN-γ capture antibody in the presence of a stimulus (antigen, cancer cells, etc.). To perform this assay, approximately 3 x 10 IFN-γ cells from either control or immunized animals expressing CT26 mIL-12 were ... 5 1 x 10 splenocytes 5 The cells were mixed with native CT26 cancer cells (target stimuli) for 24 hours. Each spot corresponds to a cell secreting an individual cytokine (Figure 17). The mIL-12 retroviral vector transduced both A375 and CT26 cells with high levels of mIL-12. The secreted mIL-12 was functionally as effective as commercial IL-12 in inducing T cell activation in vitro and promoting IFN-γ production.

[0123] mIL-12 promoted survival of tumor-bearing animals (100% of tumor-bearing animals expressed CT26 mIL-12).

[0144] Increased immune cell infiltration and up-regulation of inflammatory hot tumor gene expression were observed in CT26 mIL-12-expressing tumor tissues. Peripheral immune cells from animals expressing CT26 mIL-12 were similarly able to effectively kill native CT26 tumor cells in vitro.

[0124] Example 2. P35 Subunit and P70 Expression

[0145] Interleukin-12 (IL-12) is a heterodimeric proinflammatory cytokine that regulates T cell and natural killer cell responses, induces the production of interferon-γ (IFN-γ), supports the differentiation of T helper 1 (TH1) cells, and is a critical link between natural resistance and adaptive immunity. IL-12 has been shown to be therapeutically effective for treating certain diseases or conditions (e.g., cancer), but systemic administration of IL-12 has been shown to be toxic.

[0125]

[0146] In order to reduce the toxicity of IL-12 for therapeutic purposes, retroviral vectors are designed to encode and express only the P35 subunit. Figure 18A illustrates an exemplary retroviral vector encoding the P35 subunit of IL-12. The expression of the P35 subunit mediated by retroviral vectors can modulate the expression of IL-12, and thus the toxicity associated with IL-12. Figure 18B illustrates the expression of P35 in A375 cells transduced with retroviral vectors described herein for encoding and expressing the P35 subunit of IL-12 (left); and Western blots showing the expression and secretion of the P40 subunit by A375 cells (A375-P40 stable cell line).

[0126]

[0147] A375 cells stably expressing P40 were plated at 12,000 cells / cm on day 1 in 96-well plates in 100 μL of growth medium / 96 well. 2A375-P40 stable cells were seeded at 100 μL / mL. After the cells were allowed to attach, a retroviral vector encoding P35 (3.36e8 vector genomes / mL) was transduced into the A375-P40 stable cells with 8 μg / mL polybrene in serial dilutions in 100 μL of growth medium (Table 3). Fresh medium was replaced for the transduced A375 cells on the day after transduction. Two days after medium replacement, cell conditioned medium was collected and subjected to WB and hIL-12 ELISA. Cells were grown for another 3 days, and cells and conditioned medium were again examined by WB and hIL-12 ELISA. All ELISA samples were tested at a dilution of 1:20. Figure 19 illustrates the Western blotting of IL-12 expression (human IL-12 or hIL-12). IL-12 (P70) expression increased when the expression of P35 increased. P70 expression was independent of P40 expression (e.g., P70 expression increased with increasing expression of P35, whereas P40 expression was constant). Figure 20 illustrates an exemplary ELISA assay to quantify hIL-12 expression. Similar to Figure 19, increasing P35 expression alone can increase the expression of P70.

[0127] [Table 3]

[0128]

[0148] A375-P40 cells were seeded in 96-well plates as described in

[0145] . To avoid the effect of evaporation, rows A and H and rows 1 and 12 were not used but were maintained in cell-free growth medium during the experiment. As cell growth controls, columns 2 and 3 were seeded with A375-P40 cells without P35 retrovector transduction. Conditioned medium and cells from columns B to E were pooled for WB and ELISA tests. Columns F and G (not shown) were maintained in growth medium.

[0129]

[0149] Although the foregoing disclosure has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the present disclosure. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

Claims

1. A murine leukemia virus recombinant retroviral vector for use in treating cancer in a subject, comprising a nucleic acid construct including a first polynucleotide sequence encoding the P40 subunit of interleukin-12 (IL-12), a second polynucleotide sequence encoding the P35 subunit of interleukin-12, and a third polynucleotide sequence between the first and second polynucleotide sequences, wherein the third polynucleotide sequence encodes a cleavage site that facilitates cleavage between the P40 subunit and the P35 subunit.

2. The murine leukemia virus recombinant retroviral vector of claim 1, wherein the administration route is selected from the group consisting of intravenous route, intra-arterial route, and intratumoral route.

3. A murine leukemia virus recombinant retroviral vector as described in claim 1, which induces cell lethal activity in a subject, thereby treating cancer in the subject.

4. The murine leukemia virus recombinant retroviral vector of claim 1 , wherein the cleavage site comprises a furin cleavage site.

5. The murine leukemia virus recombinant retroviral vector of claim 4, wherein the furin cleavage site comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

12.

6. The murine leukemia virus recombinant retroviral vector of claim 1, wherein the nucleic acid construct further comprises a fourth polynucleotide sequence between the first and second polynucleotide sequences, the fourth polynucleotide sequence encoding a self-cleaving peptide.

7. The murine leukemia virus recombinant retroviral vector of claim 6, wherein the self-cleaving peptide comprises any one of the amino acid sequences of SEQ ID NOs: 4 to 11, or a combination thereof.

8. The murine leukemia virus recombinant retroviral vector of claim 1 , wherein the nucleic acid construct further comprises a polynucleotide sequence encoding thymidine kinase.

9. A murine leukemia virus recombinant retroviral vector as described in claim 1 for use in combination with a second murine leukemia virus recombinant retroviral vector administered to a subject in a therapeutically effective amount, the second murine leukemia virus recombinant retroviral vector comprising a nucleic acid construct including a polynucleotide sequence encoding thymidine kinase.

10. A murine leukemia virus recombinant retroviral vector as described in claim 9, wherein the second murine leukemia virus recombinant retroviral vector is co-administered with a nucleoside agent, and the nucleoside agent is at least one of ganciclovir, valganciclovir, acyclovir, valacyclovir, or penciclovir.

11. The murine leukemia virus recombinant retroviral vector of claim 9, wherein the murine leukemia virus recombinant retroviral vector and the second murine leukemia virus recombinant retroviral vector are administered to a subject at different time points.

12. A murine leukemia virus recombinant retroviral vector as described in claim 10, which is administered to a subject one to two days after administration of a second murine leukemia virus recombinant retroviral vector.

13. The murine leukemia virus recombinant retroviral vector according to any one of claims 1 to 12, wherein at least 1 x 10 5 total viral particles of the murine leukemia virus recombinant retroviral vector are cumulatively administered to a subject.

14. A murine leukemia virus recombinant retroviral vector described in any one of claims 1 to 12, wherein administration of the murine leukemia virus recombinant retroviral vector causes a reduction in toxicity in the subject by at least 0.1 times compared to the toxicity induced by direct administration of IL-12 protein in the subject, and / or causes an increase in the effectiveness of cancer treatment in the subject by at least 0.1 times compared to the effectiveness of cancer treatment by direct administration of IL-12 protein in the subject.

15. A murine leukemia virus recombinant retroviral vector described in any one of claims 1 to 12, delivered to a cell or cancer-associated microenvironment in a subject.

16. A murine leukemia virus recombinant retroviral vector described in any one of claims 1 to 12, wherein administration of the murine leukemia virus recombinant retroviral vector reduces toxicity associated with administration of IL-12 protein to a subject without reducing the effectiveness of cancer treatment by administration of IL-12 protein to the subject.

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