Interleukin-2 and interleukin-12 for cancer treatment

Abstract

The present disclosure provides a first polynucleotide (e.g., a recombinant or isolated first polynucleotide) encoding an interleukin (IL)-2 polypeptide and a second polynucleotide (e.g., a recombinant or isolated second polynucleotide) encoding an IL-12 polypeptide; recombinant nucleic acids and / or gene delivery vehicles comprising them; compositions, formulations, and medicaments comprising one or more of the polynucleotides, recombinant nucleic acids, and / or gene delivery vehicles; methods of use thereof (e.g., for the treatment of cancer); and articles of manufacture or kits thereof. TIFF2026508358000003.tif85128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 449,533, filed March 2, 2023, which is incorporated herein by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The Sequence Listing associated with this application has been submitted electronically via the Patent Center and is incorporated herein by reference in its entirety. The name of the file containing the Sequence Listing is 2401084.xml. The file size is 70,610 bytes and the file was created on March 1, 2024.

[0003] FIELD OF THE INVENTION The present disclosure relates, in part, to a first recombinant polynucleotide encoding an interleukin (IL)-2 polypeptide and a second recombinant polynucleotide encoding an IL-12 polypeptide, recombinant nucleic acids and / or gene delivery vehicles comprising them, pharmaceutical compositions and formulations thereof, and methods of using them (e.g., for the treatment of cancer). [Background technology]

[0004] background Cancer is one of the leading causes of death worldwide. Despite significant advances in clinical care and treatment methods, there remains a need for more effective cancer treatment options to extend survival and reduce cancer mortality.

[0005] All references cited herein, including patent applications, patent publications, non-patent literature, and NCBI / UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0006] overview To meet these and other needs, provided herein are polynucleotides (e.g., recombinant and / or isolated polynucleotides) for use in recombinant nucleic acids and / or gene delivery vehicles (e.g., viral vectors and / or non-viral vectors, e.g., lipid nanoparticles), specifically a first polynucleotide encoding an interleukin (IL) 2 polypeptide and a second polynucleotide encoding an IL-12 polypeptide; pharmaceutical compositions and / or formulations comprising the polynucleotides, the recombinant nucleic acids, and / or the gene delivery vehicles; medicaments comprising the polynucleotides, the recombinant nucleic acids, the gene delivery vehicles, and / or the pharmaceutical compositions or formulations; and / or methods of use thereof, e.g., for the treatment of cancer in a subject in need thereof.

[0007] Accordingly, certain aspects of the present disclosure relate to compositions or pharmaceutical compositions comprising a first recombinant polynucleotide encoding an IL-2 polypeptide and a second recombinant polynucleotide encoding an IL-12 polypeptide. In some embodiments, the first polynucleotide and the second polynucleotide are contiguous. In some embodiments, the first polynucleotide and the second polynucleotide are discontinuous. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises deoxyribonucleic acid (DNA). In some embodiments, the DNA is linear or circular DNA. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises ribonucleic acid (RNA). In some embodiments, the RNA is self-replicating. In some embodiments, the RNA is self-amplifying. In some embodiments, the RNA is not self-replicating. In some embodiments, the RNA is not self-amplifying. In some embodiments, the RNA is messenger RNA (mRNA) or modified mRNA (mmRNA). In some embodiments, the mRNA and / or mmRNA further comprises a 5' untranslated region (UTR), a 3' UTR, a polyadenylation (poly(A)) tail, and / or a 5' cap analog.

[0008] In some embodiments, the pharmaceutical composition comprises a recombinant nucleic acid comprising one or more of the recombinant polynucleotides described herein.In some embodiments, the recombinant nucleic acid is linear DNA, circular DNA, RNA, mRNA, and / or modified mRNA.In some embodiments, the recombinant nucleic acid is a recombinant virus genome.

[0009] In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises one or more gene delivery vehicles comprising the first polynucleotide and / or the second polynucleotide. In some embodiments, the one or more gene delivery vehicles are one or more of viral gene delivery vehicles and / or non-viral gene delivery vehicles. In some embodiments, the one or more gene delivery vehicles are one or more of viral vectors and / or non-viral vectors.

[0010] In some embodiments, the one or more gene delivery vehicles are one or more viral vectors. In some embodiments, the viral vectors are pseudotyped. In some embodiments, the viral vectors comprise a recombinant viral genome. In some embodiments, the recombinant viral genome comprises a first polynucleotide and / or a second polynucleotide. In some embodiments, the recombinant viral genome is replication-competent. In some embodiments, the recombinant viral genome is replication-defective. In some embodiments, the recombinant viral genome is a recombinant oncolytic viral genome. In some embodiments, the recombinant viral genome is not a recombinant oncolytic viral genome. In some embodiments, the recombinant viral genome is selected from a recombinant adenovirus genome, a recombinant retrovirus genome, a recombinant adeno-associated virus (AAV) genome, a recombinant herpesvirus genome, a recombinant poxvirus genome, a recombinant bacteriophage genome, a recombinant alphavirus genome, a recombinant picornavirus genome, a recombinant iridovirus genome, a recombinant Newcastle disease virus genome, a recombinant baculovirus genome, a recombinant geminivirus genome, a recombinant caulimovirus genome, and any combination and / or derivative thereof.

[0011] In some embodiments, one or more gene delivery vehicles are one or more viral vectors. In some embodiments, the viral vector is pseudotyped. In some embodiments, the viral vector comprises a first polynucleotide and / or a second polynucleotide. In some embodiments, the viral vector is replication-competent. In some embodiments, the viral vector is replication-defective. In some embodiments, the viral vector is an oncolytic virus. In some embodiments, the viral vector is not an oncolytic virus. In some embodiments, the viral vector is selected from adenovirus, retrovirus, AAV, herpesvirus, poxvirus, bacteriophage, alphavirus, picornavirus, iridovirus, Newcastle disease virus, baculovirus, geminivirus, caulimovirus, and any combination and / or derivative thereof.

[0012] In some embodiments, the one or more gene delivery vehicles are one or more non-viral vectors. In some embodiments, the non-viral vector is a chemical compound, a bacterium, a mammalian cell, or a physical delivery system. In some embodiments, the chemical compound is selected from a polymeric compound, a lipidic compound, an inorganic compound, and any combination and / or derivative thereof. In some embodiments, the polymeric compound is a natural polymer, a synthetic polymer, a biopolymer, a biodegradable polymer, a cationic polymer, a protein polymer, a polysaccharide polymer, or any combination and / or derivative thereof. In some embodiments, the lipidic compound is selected from a nanoparticle, a lipidic nanoparticle, a liposome, a cationic liposome, a solid lipidic nanoparticle, a lipidic emulsion, a lipidoid, a cytofectin, a lipidic emulsion, a surfactant, a gemini surfactant, and any combination and / or derivative thereof. In some embodiments, the inorganic compound is selected from a nanocarrier, a DNA nanclew, a gold nanoparticle, a carbon nanotube, a graphene, a quantum dot, an upconversion nanoparticle, a silica nanoparticle, an iron oxide, a ferritin, and any combination and / or derivative thereof. In some embodiments, the physical delivery system is selected from electroporation, a gene gun, a jet gun, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, ballistic DNA injection, sonoporation, photoporation, laser pulses, magnetofection, magnetoporation, magnetic particles, hydroporation, and any combination and / or derivative thereof.

[0013] Other aspects of the present disclosure relate to the use of any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles comprising the recombinant polynucleotides, and / or pharmaceutical compositions described herein as a medicament.

[0014] Other aspects of the present disclosure relate to therapeutic uses of any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles comprising the recombinant polynucleotides, and / or pharmaceutical compositions described herein.

[0015] Another aspect of the present disclosure relates to the use of any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles comprising the recombinant polynucleotides, and / or pharmaceutical compositions described herein in the preparation of a medicament for the treatment of cancer.

[0016] Another aspect of the present disclosure relates to a method of expressing, enhancing, increasing, augmenting, and / or supplementing the level of an IL-2 polypeptide and / or an IL-12 polypeptide in one or more cells of a subject, the method comprising administering to the subject an effective amount of any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles comprising the recombinant polynucleotides, pharmaceutical compositions, and / or medicaments described herein. In some embodiments, the one or more cells are one or more cells of the respiratory tract, airway epithelium, and / or lung. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered to the subject topically, transdermally, subcutaneously, epidermally, intradermally, orally, sublingually, bucally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, epichoroidally, intracranially, intrathecally, intraventricularly, intraarticularly, periarticularly, intratumorally, locally, or via inhalation. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered to the subject intranasally or via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered to the subject via inhalation.In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0017] Another aspect of the present disclosure relates to a method for providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles comprising the recombinant polynucleotides, pharmaceutical compositions, and / or medicaments described herein. In some embodiments, the cancer is selected from solid tumors, blood cancers, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer. In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is osteosarcoma. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered to the subject topically, transdermally, subcutaneously, epidermally, intradermally, orally, sublingually, bucally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, epichoroidally, intracranially, intrathecally, intraventricularly, intraarticularly, periarticularly, intratumorally, locally, or via inhalation. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament is administered to the subject orally, intranasally, intratracheally, or via inhalation.In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered to the subject intranasally or via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered to the subject via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0018] Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles, pharmaceutical compositions, and / or medicaments comprising the recombinant polynucleotides described herein. In some embodiments, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies. In some embodiments, the cancer is selected from solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial carcinoma, and thyroid cancer. In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is osteosarcoma. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, pharmaceutical composition, and / or medicament is administered to the subject topically, transdermally, subcutaneously, epidermally, intradermally, orally, sublingually, bucally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, epichoroidally, intracranially, intrathecally, intraventricularly, intraarticularly, periarticularly, intratumorally, locally, or via inhalation. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polynucleotide, recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, pharmaceutical composition, and / or medicament is administered to the subject orally, intranasally, intratracheally, or via inhalation.In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered to a subject intranasally or via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered to a subject via inhalation. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the recombinant polynucleotide, the recombinant nucleic acid and / or gene delivery vehicle comprising the recombinant polynucleotide, the pharmaceutical composition, and / or the medicament are administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0019] Another aspect of the present disclosure relates to an article of manufacture or kit comprising any of the recombinant polynucleotides, recombinant nucleic acids and / or gene delivery vehicles comprising the recombinant polynucleotides, pharmaceutical compositions, and / or medicaments described herein, together with instructions for their administration. [Brief explanation of the drawings]

[0020] [Figure 1] Figures 1A-1B show the levels of cytokines (IL-12, Figure 1A; IL-2, Figure 1B) secreted by HEK293FT cells 24 hours after infection at a multiplicity of infection (MOI) of 1 with modified herpes simplex viruses encoding transgenes for human IL-12 (Figure 1A) and human IL-2 (Figure 1B). [Figure 2]Figures 2A-2B show in vitro bioactivity assays of HSV-derived IL-12 (Figure 2A) and IL-2 (Figure 2B) compared to commercially available recombinant proteins as a function of IFNγ release from splenocytes (Figure 2A) or HEK-Blue™ IL2 reporter cells (Figure 2B). Data representing cells assayed in triplicate are presented as mean ± standard deviation (SD). [Figure 3A]Figures 3A-3H show the in vivo pharmacokinetic evaluation of HSV-IL12 / HSV-IL2 after intradermal administration in healthy mice. Figures 3A and 3C show the genomic (circles) and transcript (squares) levels in the skin of C57BL / 6 mice at the indicated time points after intradermal administration of HSV-IL12 / HSV-IL2. To measure the genomic and transcript levels of IL12 (Figure 3A) and IL2 (Figure 3C), qPCR (Figure 3A circles and Figure 3C circles) and qRT-PCR (Figure 3A squares and Figure 3C squares), respectively, were performed. Data represent samples performed in duplicate and are presented as the mean ± standard error of the mean (SEM) for n = 2-3 animals per group. Figures 3B and 3D show the protein concentrations of IL-12 (Figure 3B) and IL-2 (Figure 3D) in skin homogenates at the indicated time points after intradermal administration of HSV-IL12 / HSV-IL2. Skin tissue was homogenized and assayed by ELISA in duplicate. Protein concentrations of homogenates were measured by BCA assay, and IL-12 or IL-2 concentrations were normalized to total protein. Data are presented as mean ± SEM for n = 2–3 animals per group. Figures 3E and 3F show the protein concentrations of IL-12 (Figure 3E) and IL-2 (Figure 3F) in serum at the indicated time points after intradermal (ID) administration of HSV-IL12 / HSV-IL2 or intravenous (IV) administration of recombinant proteins. Protein concentrations of serum samples were measured by ELISA. Values ​​indicate the magnitude difference between peak exposures (5 min after IV recombinant protein administration vs. 24 h after ID vector administration). Data are presented as mean ± SEM, and symbols indicate independent animals (n = 2–3 per group). Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc analysis. *** = p < 0.001. Figures 3G and 3H show the protein concentrations of IL-12 (Figure 3G) and IL-2 (Figure 3H) in skin biopsies at the indicated time points after HSV-IL12 / HSV-IL2 ID administration or recombinant protein IV administration. Cytokine concentrations were normalized to total protein. Data are presented as mean ± SEM, and symbols indicate independent animals (n = 2–3 per group). Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc analysis.**=p<0.01;****=p<0.0001. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 3H] See legend to Figure 3A. [Figure 4A]Figures 4A-4I show the in vivo evaluation of HSV-IL12 / HSV-IL2 after intratracheal instillation in healthy mice. Figures 4A-4E show the in vivo evaluation of HSV-IL12 / HSV-IL2 in BALB / c female mice on days 0 and 7 after intratracheal instillation of either vehicle control or a high or medium dose of HSV-IL12 / HSV-IL2 (approximately 10 or 10 total PFU, respectively). On day 8, animals were sacrificed 24 hours after the last dose. Figure 4A shows the weekly body weights of BALB / c female mice. Data are presented as mean ± SEM. Figures 4B-4E show bronchoalveolar lavage fluid (BALF) (Figures 4B-4C) and whole lungs (Figures 4D-4E) collected postmortem and analyzed for IL-12 and IL-2 concentrations by ELISA. Whole lung homogenate cytokine concentrations were normalized to total protein. Data are presented as mean ± SEM, and symbols represent independent animals. n = 2–3 per group. For Figures 4B–4E, statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc analysis. *p = <0.05; ** = p <0.01. Figures 4F–4I show in vivo evaluation in healthy mice after intratracheal administration of HSV-IL12 / HSV-IL2 (approximately 10 total PFU) or intravenous administration of IL-12 and IL-2 recombinant proteins (125 ng and 600 ng, respectively) at mouse doses equivalent to those that have demonstrated clinical efficacy and toxicity in humans. Untreated animals served as negative controls. Figures 4F–4G show serum samples taken 5 min and 24 h after administration and assayed by ELISA to compare peak systemic cytokine exposure for treatment with recombinant proteins and vectors, respectively. Values ​​indicate the magnitude difference between peak exposures. Figures 4H-4I show whole lungs harvested at the indicated time points to measure IL-12 and IL-2 concentrations. Cytokine concentrations were normalized to total protein. For Figures 4F-4I, data are presented as mean ± SEM, symbols indicate independent animals (n = 2-3 per group), and statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc analysis. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 4I] See legend to Figure 4A. [Figure 5A]Figures 5A-5S show the efficacy of HSV-IL12 / HSV-IL2 in an in vivo mouse model of melanoma. Figures 5A-5B show that combined HSV-IL12 / HSV-IL2 administered via intratumoral injection enhanced survival of B16F10 melanoma-bearing mice compared with control or single-vector treatment. Figure 5A shows the experimental design. Criteria for euthanasia were either a tumor area of ​​150 mm or greater or a weight loss of 20% or greater from pre-experimental body weight. Vectors were administered at approximately 10 total PFU. SC: Subcutaneous. Figure 5B shows survival curves for mice administered vehicle control, or HSV-IL12 alone, HSV-IL2 alone, HSV-GMCSF alone, and combined HSV-IL12 / HSV-IL2. Survival data are presented as individual animals and analyzed using the log-rank test corrected for multiple comparisons to compare each treatment group to the vehicle control. ns = non-significant. **=p<0.01. Figures 5C-5E show that weekly intratumoral injections of HSV-IL12 / HSV-IL2 and additional maintenance doses improved survival of B16F10 melanoma-bearing mice. Figure 5C shows the experimental design. Criteria for euthanasia were either a tumor area of ​​150 mm or greater or a weight loss of 20% or greater than pre-experimental body weight. Vectors were administered at approximately 10 total PFU. SC: subcutaneous. Figure 5D shows tumor measurements for each treatment group. Data are presented as mean ± SEM for n=10 animals per group and analyzed using mixed-effects analysis. Figure 5E shows survival data presented as individual animals and analyzed using the log-rank test. ***=p<0.001. Figures 5F-5I show that HSV-IL12 / HSV-IL2 treatment of primary B16F10 melanoma resulted in control of rechallenge tumors. Figure 5F shows the experimental design. The criteria for euthanasia were either a tumor area of ​​150 mm or greater or a weight loss of 20% or greater than pre-experimental body weight. The vector was administered at approximately 10 total PFU. SC: Subcutaneous. Figures 5G-5H show tumor measurements for each treatment group in the initial phase (primary tumor; Figure 5G) and reload phase (reload tumor; Figure 5H). Data are presented as the mean ± SEM for n = 4-5 animals per group.For the rechallenge phase, five age-matched naïve C57BL / 6 animals were inoculated with tumors to serve as positive controls for tumor growth. Figure 5I shows survival data displayed as individual animals. Statistical significance was determined using a mixed-effects model (Figures 5G-5H) or a log-rank test (Figure 5I). * = p < 0.05; ** = p < 0.01. Figures 5J-5S show that HSV-IL12 / HSV-IL2 treatment of primary B16F10 melanoma resulted in an abscopal effect on secondary B16F10 tumors. Figure 5J shows an overview of tumor inoculation and experimental design. Euthanasia criteria were either a tumor area of ​​150 mm or greater or a weight loss of 20% or greater from pre-experimental weight. Vectors were administered at approximately 108 total PFU. Figures 5K-5L show tumor measurements of the primary / treated tumors (Figure 5K) and secondary tumors (Figure 5L) in the group inoculated with secondary tumors on day 0. Data are presented as mean ± SEM for n = 5 animals per group. Figure 5M shows survival data for the group inoculated with secondary tumors on day 0. Data are presented for individual animals. Figures 5N-5O show tumor measurements of the primary / treated tumors (Figure 5N) and secondary tumors (Figure 5O) in the group inoculated with secondary tumors on day 4. Data are presented as mean ± SEM for n = 5 animals per group. Figure 5P shows survival data for the group inoculated with secondary tumors on day 4. Data are presented for individual animals. Figures 5Q-5R show tumor measurements of the primary / treated tumors (Figure 5Q) and secondary tumors (Figure 5R) in the group inoculated with secondary tumors on day 10. Data are presented as mean ± SEM for 5 animals per group. Figure 5S shows survival data for the group inoculated with secondary tumors on day 10. Data are presented as individual animals. Statistical significance was determined using mixed-effects analysis (tumor area) or the log-rank test (survival). *=p<0.05; **=p<0.01; ****=p<0.0001. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 5G] See legend to Figure 5A. [Figure 5H] See legend to Figure 5A. [Figure 5I] See legend to Figure 5A. [Figure 5J] See legend to Figure 5A. [Figure 5K] See legend to Figure 5A. [Figure 5L] See legend to Figure 5A. [Figure 5M] See legend to Figure 5A. [Figure 5N] See legend to Figure 5A. [Figure 5O] See legend to Figure 5A. [Figure 5P] See legend to Figure 5A. [Figure 5Q] See legend to Figure 5A. [Figure 5R] See legend to Figure 5A. [Figure 5S] See legend to Figure 5A. [Figure 6A]Figures 6A-6H show the efficacy of HSV-IL12 / HSV-IL2 in an in vivo mouse model of osteosarcoma (e.g., osteosarcoma lung metastasis). Figures 6A-6D show that combined HSV-IL12 / HSV-IL2 administered intratracheally enhanced tumor regression and survival in the K7M2 osteosarcoma lung metastasis model compared to control or single-vector treatment. Figure 6A shows the experimental design. Animals were euthanized if they lost more than 20% of their pre-experimental body weight. All vectors were administered at approximately 107 total PFU. IV: intravenous. Figures 6B-6C show data on body weight (Figure 6B) and survival (Figure 6C). Body weight data are presented as the mean ± SEM for n = 5 animals per group. Survival data are presented for individual animals, and statistical significance was determined using the log-rank test corrected for multiple comparisons. Figure 6D shows a representative H&E-stained lung section from an animal that survived to day 100. Figures 6E–6H show that intratracheal administration of HSV-IL12 / HSV-IL2 minimizes initial K7M2 lung tumor growth and delays tumor recurrence without additional therapeutic intervention. Figure 6E shows an overview of the experimental design. Animals were euthanized if body weight loss reached 20% or more of their preexperimental weight. All vectors were administered at approximately 107 total PFU. For the rechallenge phase, tumors were inoculated into five untreated age-matched BALB / c animals to serve as positive controls for tumor growth. IV: intravenous. Figures 6F–6G show animal weight measurements during the first phase (Figure 6F) and the rechallenge phase (Figure 6G). Data are presented as the mean ± SEM for n = 4–5 animals per group. Figure 6H shows survival data presented as individual animals. Statistical significance was determined using the log-rank test. ** = p < 0.01; *** = p < 0.001. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is provided as a description of example embodiments.

[0022] I. General Techniques The techniques and procedures described or referred to herein are generally well understood by those skilled in the art and are commonly employed using conventional methodologies, e.g., those described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988); Oligonucleotide Synthesis (M.J.Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (RIFreshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Gene Transfer Vectors for Mammalian Cells (JMMiller and PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); and a widely used methodology described in Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0023] II. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0024] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and so forth.

[0025] As used herein, the term "and / or" can include any and all combinations of one or more of the associated listed items. For example, the term "a and / or b" can refer to "a alone," "b alone," "a or b," or "a and b"; the term "a, b, and / or c" can refer to "a alone," "b alone," "c alone," "a or b," "a or c," "b or c," "a, b, or c," "a and b," "a and c," "b and c," or "a, b, and c"; and so forth.

[0026] As used herein, the term "about" refers to a normal error range for the respective value, which is readily known to one of ordinary skill in the art. As used herein, reference to a value or parameter with "about" includes (describes) aspects related to the value or parameter itself.

[0027] It is understood that aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0028] As used herein, the terms "polynucleotide," "nucleic acid sequence," "nucleic acid," and variations thereof are intended to refer collectively to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, provided that the polymers contain nucleic acid bases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, such as substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications.

[0029] As used herein, a nucleic acid is "operatively linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosomal binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" or "operably linked" means that the DNA or RNA sequences being linked are contiguous.

[0030] As used herein, "open reading frame" or "ORF" refers to a continuous stretch of nucleic acid, either DNA or RNA, that encodes a protein or polypeptide. Typically, the nucleic acid includes a translation initiation signal or start codon, e.g., ATG or AUG, and a stop codon.

[0031] As used herein, "untranslated region" or "UTR" refers to nucleic acid at the 5' and / or 3' end of an open reading frame that is not translated. When one or more UTRs are included in a polynucleotide, they can affect post-transcriptional regulation, mRNA stability, translation of the polynucleotide, etc.

[0032] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and may refer to a polymer of two or more amino acids.

[0033] As used herein, "subject," "host," or "individual" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sport, or pet animals, such as dogs, horses, cats, cows, and animals used in research, such as mice, rats, hamsters, rabbits, and non-human primates. In some embodiments, the mammal is a human.

[0034] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject receiving the composition or formulation. A "pharmaceutically acceptable" excipient (e.g., vehicle, additive) is one that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0035] As used herein, an "effective amount" is at least the minimum amount required to affect a measurable improvement or prevention of one or more symptoms of a particular disorder. An "effective amount" may vary depending on factors such as the patient's condition, age, sex, and weight. An effective amount is also an amount in which the therapeutically beneficial effects outweigh the toxic or detrimental effects of treatment. For prophylactic use, beneficial or desired results include results such as elimination or reduction of the risk, reduction in severity, or delay in onset of the disease, its complications, and intermediate pathological phenotypes manifesting during disease development. For therapeutic use, beneficial or desired results include clinical results such as reduction in one or more symptoms resulting from the disease, improvement in the quality of life of the person affected by the disease, reduction in the dose of other drug treatments used to treat the symptoms of the disease, delay in disease progression, and / or prolonged survival. An effective amount may be administered in one or more administrations. For purposes of this disclosure, an effective amount of a recombinant polynucleotide, gene delivery vehicle, pharmaceutical composition, and / or medicament comprising the recombinant polynucleotide is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective amount of a recombinant polynucleotide, gene delivery vehicle comprising the recombinant polynucleotide, pharmaceutical composition, and / or medicament may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if the desired result can be or is achieved in combination with one or more other agents.

[0036] As used herein, "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during clinical pathology. The desired effects of treatment include a reduction in the rate of progression of the disease / disorder / defect, an improvement or alleviation of the state of the disease / disorder / defect, and remission, or an improved prognosis.

[0037] As used herein, the term "delaying the progression" of a disease / disorder / defect refers to deferring, hindering, slowing, preventing, stabilizing, and / or postponing the onset of the disease / disorder / defect. This delay can be of varying length or time, depending on the history of the disease / disorder / defect and / or the individual receiving treatment. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.

[0038] Throughout this disclosure, various aspects are presented in range format. Description in range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the disclosure. Therefore, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, as well as other ranges described or intermediate values ​​of that described range, are encompassed by the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are encompassed by the disclosure, except for any specifically excluded limits in the described range. When a described range includes one or both of the limits, ranges excluding one or both of the included limits are also encompassed by the disclosure. This is true regardless of the width of the range.

[0039] III. Polynucleotides Polynucleotides encoding IL-2 and / or IL-12 polypeptides In some embodiments, the present disclosure relates to a first polynucleotide (e.g., a first recombinant polynucleotide, a first isolated polynucleotide) encoding an IL-2 polypeptide, and a second polynucleotide (e.g., a second recombinant polynucleotide, a second isolated polynucleotide) encoding an IL-12 polypeptide. Any suitable IL-2 and / or IL-12 polypeptide described herein or known in the art, including, for example, human IL-2 and / or human IL-12, can be encoded by one or more polynucleotides of the present disclosure.

[0040] In some embodiments, the polynucleotides of the present disclosure comprise the wild-type coding sequence of any IL-2 gene and / or IL-12 gene (including any isoforms or splice variants thereof) described herein or known in the art, including, for example, the IL2 gene (e.g., NCBI Gene ID: 3558; SEQ ID NO: 5), the IL12A gene (e.g., NCBI Gene ID: 3592; SEQ ID NO: 6), the IL12B gene (e.g., NCBI Gene ID: 3593; SEQ ID NO: 7), etc. In some embodiments, the polynucleotides of the present disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the human IL-2 and / or IL-12 genes (and / or their coding sequences) described herein or known in the art. In some embodiments, the polynucleotides of the present disclosure comprise a codon-optimized variant of the wild-type coding sequence of any IL-2 and / or IL-12 gene described herein or known in the art. In some embodiments, the use of a codon-optimized variant of a gene's coding sequence increases the stability and / or yield of heterologous expression (RNA and / or protein) of the encoded polypeptide in a target cell compared to the stability and / or yield of heterologous expression of the corresponding non-codon-optimized wild-type sequence. Any suitable method known in the art for performing codon optimization of a sequence of interest for expression in one or more target cells (e.g., one or more human cells) may be used, including, for example, the method described by Fath et al. (PLoS One. 2011 Mar 3;6(3):e17596).

[0041] In some embodiments, the first polynucleotide of the present disclosure encodes an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a human IL-2 polypeptide (see, e.g., UniProt Accession No. P60568). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type IL2 gene (see, e.g., NCBI Gene ID: 3558, SEQ ID NO: 5) or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 8). In some embodiments, the polynucleotide encoding an IL-2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide encoding an IL-2 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0042] In some embodiments, the first polynucleotide encoding an IL-2 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 1. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, or fewer than 153 consecutive amino acids of SEQ ID NO: 1.

[0043] In some embodiments, the second polynucleotide of the present disclosure encodes an IL-12 subunit alpha polypeptide. In some embodiments, the IL-12 subunit alpha polypeptide is a human IL-12 subunit alpha polypeptide (see, e.g., UniProt Accession No. P29459). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type IL12A gene (see, e.g., NCBI Gene ID: 3592, SEQ ID NO: 6) or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 9). In some embodiments, the polynucleotide encoding the IL-12 subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:2. In some embodiments, the polynucleotide encoding the IL-12 subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0044] In some embodiments, the second polynucleotide encoding the IL-12 subunit alpha polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 2. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, or fewer than 219 consecutive amino acids of SEQ ID NO: 2.

[0045] In some embodiments, the second polynucleotide of the present disclosure encodes an IL-12 subunit β polypeptide. In some embodiments, the IL-12 subunit β polypeptide is a human IL-12 subunit β polypeptide (see, e.g., UniProt Accession No. P29460). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type IL12B gene (see, e.g., NCBI Gene ID: 3593, SEQ ID NO: 7) or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 10). In some embodiments, the recombinant polynucleotide encoding the IL-12 subunit beta polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding the IL-12 subunit beta polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

[0046] In some embodiments, the second polynucleotide encoding the IL-12 subunit β polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 3. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, or fewer than 328 consecutive amino acids of SEQ ID NO: 3.

[0047] In some embodiments, the polynucleotide encoding an IL-2 polypeptide, the polynucleotide encoding an IL-12 subunit α polypeptide, and / or the polynucleotide encoding an IL-12 subunit β polypeptide is a single contiguous polynucleotide (e.g., a single contiguous DNA, a single contiguous RNA (e.g., a synthetic mRNA)). In some embodiments, the polynucleotide encoding an IL-2 polypeptide, the polynucleotide encoding an IL-12 subunit α polypeptide, and / or the polynucleotide encoding an IL-12 subunit β polypeptide is a single contiguous polynucleotide contained within a single DNA molecule. In some embodiments, the polynucleotide encoding an IL-2 polypeptide, the polynucleotide encoding an IL-12 subunit α polypeptide, and / or the polynucleotide encoding an IL-12 subunit β polypeptide is a single contiguous polynucleotide contained within a single RNA molecule (e.g., a synthetic RNA molecule). In some embodiments, the polynucleotide encoding an IL-2 polypeptide, the polynucleotide encoding an IL-12 subunit α polypeptide, and / or the polynucleotide encoding an IL-12 subunit β polypeptide is a single contiguous polynucleotide contained within a single mRNA (e.g., a single synthetic mRNA or mmRNA). In some embodiments, the single contiguous polynucleotide encodes an IL-2 polypeptide on a first open reading frame (ORF) and an IL-12 polypeptide on a second open reading frame (ORF). In some embodiments, the two ORFs are separated by an intracellular ribosome entry site (IRES).

[0048] In some embodiments, the single contiguous polynucleotide encodes an IL-2 polypeptide on a first open reading frame (ORF), an IL-12 subunit alpha polypeptide on a second open reading frame (ORF), and an IL-12 subunit beta polypeptide on a third open reading frame (ORF). In some embodiments, the first, second, and third ORFs are each separated by an IRES.

[0049] Examples of suitable IRESs may include, but are not limited to, IRESs derived from viruses (e.g., IRESs derived from poliovirus, rhinovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis C virus, hog cholera virus, Rous sarcoma virus, human immunodeficiency virus, cricket paralysis virus, Kaposi's sarcoma-associated herpesvirus, etc.) and IRESs derived from cellular mRNAs (e.g., IRESs derived from growth factor mRNAs such as fibroblast growth factor 2, platelet-derived growth factor B, and vascular endothelial growth factor, IRESs derived from transcription factor mRNAs such as antennapedia, ultrapithoraxm, and NF-κB inhibitor, IRESs derived from oncogene mRNAs such as c-myc, pim-1, and protein kinase p58PITSLRE, etc.).

[0050] In some embodiments, a polynucleotide encoding any of the chimeric polypeptides described herein (e.g., including an IL-12 subunit beta polypeptide and an IL-12 subunit alpha polypeptide) is encoded on a single ORF.

[0051] In some embodiments, the polynucleotides of the present disclosure encode one or more of an interleukin-2 (IL-2) peptide, an interleukin-12 subunit alpha (IL-12α) peptide, an interleukin-12 subunit beta (IL-12β) peptide, and / or any chimeric polypeptides thereof, in any combination. In some embodiments, the chimeric polypeptide comprises a linker polypeptide.

[0052] In some embodiments, the linker polypeptide is a cleavable linker polypeptide. Any cleavable linker polypeptide known in the art can be used in the chimeric polypeptides of the present disclosure, including, for example, a T2A linker, a P2A linker, an E2A linker, an F2A linker, and the like. In some embodiments, the linker polypeptide is a T2A linker polypeptide. An exemplary nucleic acid sequence encoding a T2A linker polypeptide is provided as SEQ ID NO: 12. An exemplary amino acid sequence of a T2A linker polypeptide is provided as SEQ ID NO: 16. In some embodiments, the linker polypeptide is a P2A linker polypeptide. An exemplary nucleic acid sequence encoding a P2A linker polypeptide is provided as SEQ ID NO: 13. An exemplary amino acid sequence of a P2A linker polypeptide is provided as SEQ ID NO: 17. In some embodiments, the linker polypeptide is an E2A linker polypeptide. An exemplary nucleic acid sequence encoding an E2A linker polypeptide is provided as SEQ ID NO: 14. An exemplary amino acid sequence of an E2A linker polypeptide is provided as SEQ ID NO: 18. In some embodiments, the linker polypeptide is an F2A linker polypeptide. An exemplary nucleic acid sequence encoding an F2A linker polypeptide is provided as SEQ ID NO:15. An exemplary amino acid sequence of an F2A linker polypeptide is provided as SEQ ID NO:19. In some embodiments, the linker polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:16-19. In some embodiments, the linker polypeptide comprises a sequence selected from SEQ ID NOs:16-19.

[0053] In some embodiments, the linker polypeptide is a non-cleavable linker polypeptide. For example, Any non-cleavable linker polypeptide known in the art can be used in the chimeric polypeptides of the present disclosure, including, for example, TIFF2026508358000002.tif47166. In some embodiments, the linker polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs:20-31. In some embodiments, the linker polypeptide comprises a sequence selected from SEQ ID NOs:20-31.

[0054] In some embodiments, the second polynucleotide of the disclosure encodes an IL-12 subunit alpha polypeptide and an IL-12 subunit beta polypeptide. In some embodiments, the IL-12 subunit alpha polypeptide is a human IL-12 subunit alpha polypeptide (see, e.g., UniProt Accession No. P29459). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type IL12A gene (see, e.g., NCBI Gene ID: 3592, SEQ ID NO: 6) or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 9). In some embodiments, the polynucleotide encoding the IL-12 subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide encoding the IL-12 subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IL-12 subunit beta polypeptide is a human IL-12 subunit beta polypeptide (see, e.g., UniProt Accession No. P29460). In some embodiments, the polynucleotide comprises the coding sequence of the wild-type IL12B gene (see, e.g., NCBI Gene ID: 3593, SEQ ID NO: 7) or a codon-optimized variant thereof (see, e.g., SEQ ID NO: 10).In some embodiments, the recombinant polynucleotide encoding the IL-12 subunit beta polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encoding the IL-12 subunit beta polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the IL-as subunit alpha polypeptide and the subunit beta polypeptide are separated by a linker polypeptide.

[0055] In some embodiments, the second polynucleotide of the present disclosure encodes a chimeric polypeptide comprising an IL-12 subunit beta polypeptide and an IL-12 subunit alpha polypeptide. In some embodiments, the chimeric polypeptide further comprises a linker polypeptide linking the IL-12 subunit beta polypeptide and the IL-12 subunit alpha polypeptide. In some embodiments, the chimeric polypeptide comprises, from N-terminus to C-terminus, an IL-12 subunit alpha polypeptide - a linker polypeptide - an IL-12 subunit beta polypeptide. In some embodiments, the chimeric polypeptide comprises, from N-terminus to C-terminus, an IL-12 subunit beta polypeptide - a linker polypeptide - an IL-12 subunit alpha polypeptide. An exemplary nucleic acid sequence encoding a chimeric polypeptide comprising a human IL12 subunit beta polypeptide, a linker polypeptide, and a human IL12 subunit alpha polypeptide is provided as SEQ ID NO:11. An exemplary amino acid sequence of a chimeric polypeptide comprising a human IL12 subunit beta polypeptide, a linker polypeptide, and a human IL12 subunit alpha polypeptide is provided as SEQ ID NO:4. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 4. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0056] In some embodiments, the polynucleotides of the present disclosure do not encode an interleukin-4 (IL-4) polypeptide. In some embodiments, the polynucleotides of the present disclosure do not encode an interleukin-10 (IL-10) polypeptide. In some embodiments, the polynucleotides of the present disclosure do not encode a granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide. In some embodiments, the polynucleotides of the present disclosure do not encode an interleukin-4 (IL-4) polypeptide, an interleukin-10 (IL-10) polypeptide, and / or a granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide. In some embodiments, the first polynucleotide encoding an IL-2 polypeptide and / or the second polynucleotide encoding an IL-12 polypeptide encodes one or more additional polypeptides. In some embodiments, the first polynucleotide encoding an IL-2 polypeptide and / or the second polynucleotide encoding an IL-12 polypeptide does not encode an additional polypeptide. In some embodiments, the first polynucleotide encoding an IL-2 polypeptide and / or the second polynucleotide encoding an IL-12 polypeptide encodes one or more additional cytokines and / or chemokines. In some embodiments, the first polynucleotide encoding an IL-2 polypeptide and / or the second polynucleotide encoding an IL-12 polypeptide does not encode an additional cytokine and / or chemokine.

[0057] A polynucleotide (e.g., a recombinant polynucleotide) of the present disclosure that encodes a polypeptide (e.g., an IL-2 polypeptide and / or an IL-12 polypeptide) may further encode additional coding and non-coding sequences. Examples of additional coding and non-coding sequences include, but are not limited to, sequences encoding additional polypeptide tags (e.g., encoded in frame with the polypeptide to produce a fusion protein), introns (e.g., natural, modified, or heterologous introns), 5' and / or 3' UTRs (e.g., natural, modified, or heterologous 5' and / or 3' UTRs), etc. Examples of suitable polypeptide tags include, but are not limited to, any combination of purification tags, such as his tags, flag tags, maltose-binding protein tags, and glutathione-S-transferase tags; detection tags, such as tags that can be detected photometrically (e.g., green fluorescent protein, red fluorescent protein, etc.); tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.); secretory sequences; signal sequences; leader sequences; and / or stabilizing sequences; tags containing protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites, etc.); and the like. In some embodiments, the 5' and / or 3' UTRs increase the stability, localization, and / or translation efficiency of the polynucleotide. In some embodiments, the 5' and / or 3' UTRs improve the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTRs comprise elements (e.g., one or more miRNA binding sites, etc.) that can block or reduce off-target expression (e.g., by inhibiting expression in particular cell types (e.g., neuronal cells), at particular points in the cell cycle, at particular developmental stages, etc.). In some embodiments, the 5' and / or 3' UTRs comprise elements (e.g., one or more miRNA binding sites, etc.) that can enhance expression of the encoded polypeptide in particular cell types.

[0058] In some embodiments, a polynucleotide (e.g., a recombinant polynucleotide) of the present disclosure encoding a polypeptide (e.g., an IL-2 polypeptide and / or an IL-12 polypeptide) is operably linked to one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) regulatory sequences. The term "regulatory sequence" may include enhancers, insulators, promoters, and other expression control elements (e.g., polyadenylation signals). Any suitable enhancer known in the art may be used, including, for example, enhancer sequences derived from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), enhancer sequences derived from eukaryotic viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.), and any combination thereof. Any suitable insulator known in the art may be used, including, for example, the HSV chromatin boundary (CTRL / CTCF binding / insulator) elements CTRL1 and / or CTRL2, the chicken hypersensitive site 4 insulator (cHS4), the human HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE), the scaffold / matrix attachment region (S / MAR) derived from the human interferon beta gene (IFNB1), and any combination thereof.Any suitable promoter known in the art (e.g., suitable for transcription in mammalian host cells) can be used, so long as it is compatible with the host cell, including, for example, promoters obtained from the genomes of viruses (e.g., polyomavirus, poultry virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), promoters derived from heterologous mammalian genes (e.g., actin promoters (e.g., β-actin promoter), ubiquitin promoters (e.g., ubiquitin C (UbC) promoter), phosphoglycerate kinase (PGK) promoter, immunoglobulin promoters, heat shock promoters, etc.), promoters derived from homologous mammalian genes, synthetic promoters (e.g., CAG promoter), and any combination thereof. Regulatory sequences can include those that direct constitutive expression of the nucleic acid, as well as tissue-specific regulatory sequences and / or inducible or repressible sequences.

[0059] In some embodiments, the polynucleotide (e.g., recombinant polynucleotide) of the present disclosure is operably linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of a constitutive promoter, a tissue-specific promoter, a temporal promoter, a spatial promoter, an inducible promoter, and a repressible promoter. In some embodiments, the one or more heterologous promoters are one or more of a human cytomegalovirus (HCMV) immediate early promoter, a human elongation factor 1 (EF1) promoter, a human β-actin promoter, a human UbC promoter, a human PGK promoter, a synthetic CAG promoter, and any combination thereof.

[0060] In some embodiments, a polynucleotide (e.g., a recombinant polynucleotide) of the present disclosure encoding a polypeptide (e.g., an IL-2 polypeptide and / or an IL-12 polypeptide) expresses the polypeptide when the polynucleotide is delivered to one or more target cells of a subject (e.g., one or more cancer cells, one or more healthy (non-cancerous) cells, one or more cells of the respiratory tract, airway, lung, etc.). In some embodiments, expression of a polypeptide (e.g., an IL-2 polypeptide and / or an IL-12 polypeptide) enhances, increases, augments, and / or supplements the level, function, and / or activity of the polypeptide in one or more target cells of the subject (e.g., compared to before expression of the polypeptide in the cell, compared to the expression level of the endogenous polypeptide, etc.). In some embodiments, expression of the polypeptides (e.g., IL-2 polypeptide and / or IL-12 polypeptide) provides prophylactic, symptomatic, or therapeutic alleviation of one or more signs or symptoms of cancer (e.g., solid tumor, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, etc.) in a subject (e.g., compared to before expression of the polypeptides).

[0061] In some embodiments, a polynucleotide (e.g., a recombinant polynucleotide) of the present disclosure does not include a coding sequence (e.g., a transgene encoding) for a collagen alpha 1 (VII) chain polypeptide (COL7). In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence (e.g., a transgene encoding) for a lysyl hydroxylase 3 polypeptide (LH3). In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence (e.g., a transgene encoding) for a keratin type I cytoskeleton 17 polypeptide (KRT17). In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence (e.g., a transgene encoding) for a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide, e.g., a human TGM1 polypeptide and / or a human TGM5 polypeptide). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a transgene encoding) for a cosmetic protein (e.g., a collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional skin extracellular matrix proteins, etc.). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a transgene encoding) for an antibody (e.g., a full-length antibody, an antibody fragment, etc.). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a transgene encoding) for a Kazal-type serine protease inhibitor (SPINK) polypeptide (e.g., a human SPINK polypeptide, e.g., a SPINK5 polypeptide). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a transgene encoding) for a filaggrin or filaggrin 2 polypeptide (e.g., a human filaggrin or filaggrin 2 polypeptide). In some embodiments, the polynucleotides of the disclosure comprise a coding sequence for a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide (e.g., a human CFTR polypeptide) (e.g.,In some embodiments, the polynucleotide of the present disclosure does not comprise an ichthyosis-associated polypeptide (e.g., an ATP-binding cassette subfamily A member 12 polypeptide, an 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 polypeptide, an aldehyde dehydrogenase family 3 member A2 polypeptide, an arachidonate 12-lipoxygenase type 12R polypeptide, a hydroperoxide isomerase ALOXE3 polypeptide, an AP-1 complex subunit sigma-1A polypeptide, an arylsulfatase E polypeptide, a caspase 14 polypeptide, a corneodesmosin polypeptide, a ceramide synthase 3 polypeptide, a carbohydrate sulfotransferase 8 polypeptide, a claudin 1 polypeptide, a cystatin A polypeptide, a cytochrome P450 4F22 polypeptide, a 3-beta-hydroxysteroid-delta(8),delta(7)-isomerase polypeptide, an elongation of very long chain fatty acids (ELO) polypeptide, a phosphodiesterase (PDE ... (protein) amino acid (protein 4) polypeptide, filaggrin polypeptide, filaggrin 2 polypeptide, gap junction beta 2 polypeptide, gap junction beta 3 polypeptide, gap junction beta 4 polypeptide, gap junction beta 6 polypeptide, 3-ketodihydrosphingosine reductase polypeptide, keratin, type II cytoskeletal 1 polypeptide, keratin, type II cytoskeletal 2 epidermal polypeptide, keratin, type I cytoskeletal 9 polypeptide, keratin, type I cytoskeletal 10 polypeptide, lipase member N polypeptide, loricrin polypeptide, membrane-bound transcription factor site 2 protease polypeptide, magnesium transporter NIPA4 polypeptide, sterol-4-alpha-carboxylic acid 3-dehydrogenase, decarboxylation polypeptide, peroxisomal targeting signal 2 receptor polypeptide, D-3-phosphoglycerate dehydrogenase polypeptide, phytanoyl-CoA dioxygenase, peroxisomal polypeptide,In some embodiments, the polynucleotides of the present disclosure do not include the coding sequence (e.g., a transgene encoding) of a patatin-like phospholipase domain-containing protein 1 polypeptide, a proteasome maturation protein polypeptide, a phosphoserine aminotransferase polypeptide, a short-chain dehydrogenase / reductase family 9C member 7 polypeptide, a serpin B8 polypeptide, a long-chain fatty acid transport protein 4 polypeptide, a synaptosomal-associated protein 29 polypeptide, a suppressor of tumorigenicity 14 protein polypeptide, a sterylsulfatase polypeptide, a vacuolar protein sorting-associated protein 33B polypeptide, and a CAAX prenyl protease 1 homolog polypeptide. In some embodiments, the polynucleotides of the present disclosure do not include the coding sequence (e.g., a transgene encoding) of a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or any chimeric polypeptide thereof. In some embodiments, the polynucleotides of the disclosure are selected from the group consisting of collagen alpha 1(VII) chain polypeptide, lysyl hydroxylase 3 polypeptide, keratin type I cytoskeleton 17 polypeptide, transglutaminase (TGM) polypeptide, filaggrin polypeptide, cosmetic protein, antibody, SPINK polypeptide, CFTR polypeptide, ichthyosis-associated polypeptide, alpha-1-antitrypsin polypeptide, sodium-dependent phosphate transport protein 2B polypeptide, dynein heavy chain 5 axonemal polypeptide, dynein heavy chain 11 axonemal polypeptide, coiled-coil domain-containing protein 39 polypeptide, dynein intermediate chain 1 axonemal polypeptide, coiled-coil domain-containing protein 40 polypeptide, coiled-coil domain-containing protein 103 polypeptide, sperm associated antigen 1 polypeptide, zinc finger MYND domain-containing protein 10 polypeptide, armadillo repeat-containing protein 4 polypeptide, coiled-coil domain-containing protein 151 polypeptide, dynein intermediate chain 2 axonemal polypeptide, radial spokehead 1 homolog polypeptide,Coiled-coil domain-containing protein 114 polypeptide, radial spokehead protein 4 homolog A polypeptide, dynein assembly factor 1 axonemal polypeptide, dynein assembly factor 2 axonemal polypeptide, leucine-rich repeat-containing protein 6 polypeptide, pulmonary surfactant-associated protein B polypeptide, pulmonary surfactant-associated protein C polypeptide, homeobox protein Nkx-2.1 polypeptide, ATP-binding cassette subfamily A member 3 polypeptide, cytokine receptor common subunit beta polypeptide, granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide, bone morphogenetic protein receptor type 2 polypeptide, sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, serine / threonine protein kinase receptor R3 polypeptide, endoglin polypeptide, Mothers against decapentaplegic homolog The present invention does not include a coding sequence (e.g., a transgene encoding) for a telomere elongation helicase 1 polypeptide, a poly(A)-specific ribonuclease PARN polypeptide, a TERF1-interacting nuclear factor 2 polypeptide, a H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, a mucin 5B polypeptide, a desmoplakin polypeptide, a CST complex subunit STN1 polypeptide, a dipeptidyl peptidase 9 polypeptide, and / or any chimeric polypeptide thereof.

[0062] Synthetic RNA polynucleotides In some embodiments, the present disclosure relates to a synthetic RNA containing one or more polynucleotides described herein. In some embodiments, the synthetic RNA is a synthetic mRNA containing one or more polynucleotides described herein. In some embodiments, the synthetic RNA comprises a 5' cap structure. Examples of 5' cap structures may include, but are not limited to, cap0, cap1, cap2, and cap3 structures, and any combination and / or derivative thereof. In some embodiments, the synthetic RNA comprises a 3' poly(A) tail. In some embodiments, the synthetic RNA comprises one or more 5' and / or 3' UTRs adjacent to one or more coding sequences contained within the synthetic RNA. In some embodiments, the 5' and / or 3' UTRs increase the stability, localization, and / or translation efficiency of the synthetic RNA. In some embodiments, the 5' and / or 3' UTRs are modified to increase the stability, localization, and / or translation efficiency of the synthetic RNA. In some embodiments, the 5' and / or 3' UTRs improve the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTR is modified to improve the level and / or duration of protein expression. In some embodiments, the 5' and / or 3' UTR contains elements (e.g., miRNA binding sites, etc.) that can limit off-target expression (e.g., by inhibiting expression in specific cell types (e.g., neuronal cells), specific times in the cell cycle, specific developmental stages, etc.). In some embodiments, the 5' UTR contains a Kozak sequence. In some embodiments, the Kozak sequence is identical to or substantially similar to the Kozak consensus sequence. Methods for producing synthetic RNAs containing one or more polynucleotides of interest are well known to those skilled in the art.

[0063] In some embodiments, the synthetic RNA of the present disclosure comprises one or more modified ribonucleotides. Examples of modified ribonucleotides include 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-aminouridine, 5-hydroxyuridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio -5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methyl- 5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio N4-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidineN4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5- Aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine Cytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxy 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio- N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine,2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methyl Cytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-aza These may include, but are not limited to, adenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0064] In some embodiments, the polynucleotide encoding the IL-2 polypeptide and the polynucleotide encoding the IL-12 polypeptide are contained within two separate synthetic RNA molecules (e.g., two separate synthetic mRNAs). In some embodiments, the polynucleotide encoding the IL-2 polypeptide, the polynucleotide encoding the IL-12 subunit α polypeptide, and / or the polynucleotide encoding the IL-12 subunit β polypeptide are contained within three separate synthetic RNA molecules (e.g., three separate synthetic mRNAs).

[0065] IV. Recombinant Nucleic Acids Certain aspects of the present disclosure relate to recombinant nucleic acids comprising one or more of the polynucleotides described herein (e.g., a first recombinant polynucleotide encoding an IL-2 polypeptide and / or a second recombinant polynucleotide encoding an IL-12 polypeptide). In some embodiments, the recombinant nucleic acid is linear DNA, circular DNA, closed-ended DNA (ceDNA), RNA (e.g., synthetic RNA), mRNA, and / or mmRNA. In some embodiments, the recombinant nucleic acid is a recombinant viral genome.

[0066] In some embodiments, the recombinant nucleic acid of the present disclosure is linear DNA. In some embodiments, the linear DNA is linear expression template (LET). As will be understood by those skilled in the art, LET can include a promoter region, one or more recombinant polynucleotides described herein, and / or a transcription terminator. As will be understood by those skilled in the art, LET can be generated in vitro, for example, from plasmid DNA or genomic DNA via polymerase chain reaction (PCR).

[0067] In some embodiments, the recombinant nucleic acids of the present disclosure are close-ended DNA (ceDNA; e.g., as disclosed in U.S. Patent Application Publication No. 2021 / 0059953 and / or U.S. Patent Application Publication No. 2020 / 0283794, the contents of which are incorporated by reference in their entireties.

[0068] In some embodiments, the recombinant nucleic acid of the present disclosure is circular DNA.In some embodiments, the circular DNA is a plasmid (for example, as disclosed in U.S. Patent Application Publication No. 2013 / 0195800 and U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859, the contents of which are incorporated herein by reference in their entirety), cosmid or bacterial artificial chromosome (BAC).

[0069] In some embodiments, the circular DNA is a plasmid. The plasmid may be in the form of a circular double-stranded DNA. In some embodiments, the plasmid may contain an autonomously replicating sequence, a genome integration sequence, and / or a phage or nucleotide sequence. In some embodiments, the plasmid may be linear, circular, or supercoiled, and may be single-stranded or double-stranded DNA or RNA. The plasmids described herein may be derived from any source, in which multiple nucleotide sequences are joined or recombined into a unique construct that can introduce a promoter fragment and a selected gene product DNA sequence into a cell along with appropriate 3' untranslated sequences.

[0070] In some embodiments, the circular DNA is a cosmid. As will be understood by those skilled in the art, a cosmid is a type of plasmid containing a lambda phage cos sequence. A cosmid may contain 37 to 52 kb of DNA. In some embodiments, a cosmid is replicable if it has an origin of replication (e.g., SV40 in mammalian cells). In some embodiments, a cosmid can be packaged into a phage capsid. In some embodiments, a cosmid is a plasmid containing a bacterial oriV, an antibiotic selection marker, and / or a cloning site. In some embodiments, a cosmid may have one or more (e.g., two) cos sites derived from bacteriophage lambda.

[0071] In some embodiments, the circular DNA is a bacterial artificial chromosome (BAC). As will be understood by those skilled in the art, a BAC is a DNA construct based on a functional fertility plasmid (e.g., an F plasmid). In some embodiments, the BAC can accommodate an insert size of 150-350 kbp. In some embodiments, the BAC can include oriS and / or repE·F for plasmid replication and / or copy number control; parA and / or parB, which can partition F plasmid DNA to daughter cells during division and ensure stable maintenance of the BAC; a selectable marker; and / or T7 and / or Sp6 for transcription of the inserted gene.

[0072] In some embodiments, the recombinant nucleic acids of the present disclosure are RNA, mRNA, and / or mmRNA (e.g., as disclosed in U.S. Patent Nos. 10,898,574, 10,702,600, and 10,933,127, the contents of which are incorporated by reference in their entireties).

[0073] In some embodiments, the RNA, mRNA, and / or mmRNA may not only enhance the efficiency of protein production, the intracellular retention of nucleic acids, and the viability of contacted cells, but also possess reduced immunogenicity.

[0074] In some embodiments, the RNA, mRNA, and / or mmRNA is greater than about 30 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1 , 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 nucleotides or more, up to 100,000 nucleotides).

[0075] In some embodiments, the RNA, mRNA, and / or mmRNA may contain one or more modified nucleosides (e.g., as described above), which may have useful properties, including a lack of substantial induction of the innate immune system of cells into which the RNA, mRNA, and / or mmRNA is introduced.

[0076] In some embodiments, modified nucleosides may not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may be present at various positions in the nucleic acid. Those skilled in the art will understand that nucleotide analogs or other modifications may be located at any position in the nucleic acid without substantially reducing the function of the nucleic acid. Modifications may be 5' or 3' terminal modifications. The nucleic acid may contain at least one, up to 100% modified nucleotides, or any intermediate percentage, e.g., at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.

[0077] In some embodiments, modified nucleosides include pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl- These may include 1-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl -pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0078] In some embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N ... May include adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0079] In some embodiments, modified nucleosides can include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0080] Several different 5' cap structures can be used to generate the 5' cap of the RNA, mRNA, and / or mmRNA described herein. Many chemical cap analogs have been used to co-transcriptionally cap synthetic mRNA molecules. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine bond, with one guanine containing an N7 methyl group and a 3'-O-methyl group. While chemical cap analogs allow for co-capping of RNA molecules, up to 20% of the transcripts remain uncapped, and synthetic cap analogs are not identical to the endogenous 5' cap structure of authentic cellular mRNA. This can lead to reduced translational capacity and reduced cellular stability.

[0081] In some embodiments, the RNA, mRNA, and / or mmRNA comprises a poly-A tail. In some embodiments, the poly-A tail is designed relative to the entire length of the RNA, mRNA, and / or mmRNA to be modified. This design may be based on the length of the coding region of the RNA to be modified, the length of a particular feature or region of the RNA (e.g., mRNA) to be modified, or the length of the final product expressed from the RNA to be modified. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the RNA or feature to be modified. The poly-A tail may be designed as a fraction of the modified RNA to which it belongs. In this context, the poly-A tail may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, or the total length of the construct minus the poly-A tail.

[0082] In some embodiments, the recombinant nucleic acid of the present disclosure is one or more recombinant viral genomes and / or one or more non-viral nucleic acids. In some embodiments, the one or more recombinant viral genomes and / or one or more non-viral nucleic acids comprise DNA or RNA vectors. Generally, a vector suitable for maintaining, propagating, and / or expressing a polynucleotide to produce one or more polypeptides in a subject can be used. Examples of suitable vectors can include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, poxvirus vectors, bacteriophage vectors, alphavirus vectors, picornavirus vectors, iridovirus vectors, Newcastle disease virus vectors, baculovirus vectors, geminivirus vectors, caulimovirus vectors, Sindbis virus vectors, measles vectors, herpesvirus vectors, lentivirus vectors, retrovirus vectors, etc.). In some embodiments, the one or more recombinant viral genomes and / or one or more non-viral nucleic acids are replicable in a host cell. In some embodiments, one or more recombinant viral genomes and / or one or more non-viral nucleic acids are replication-incompetent in host cells. In some embodiments, one or more recombinant viral genomes and / or one or more non-viral nucleic acids can be integrated into host DNA. In some embodiments, one or more recombinant viral genomes and / or one or more non-viral nucleic acids cannot be integrated into host DNA (e.g., are episomal).

[0083] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant adenovirus genomes. The recombinant adenovirus genome can be a recombinant genome derived from any member of the Adenoviridae family of viruses known in the art, including, for example, recombinant Atadenovirus genomes, recombinant Aviadenovirus genomes, recombinant Ichtadenovirus genomes, recombinant Mastadenovirus genomes, recombinant Siadenovirus genomes, recombinant Testadenovirus genomes, and any combination or derivative thereof. In some embodiments, the recombinant adenovirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. As used herein, an "inactivating mutation" may refer to any mutation that results in a gene or regulon product (RNA or protein) that is reduced in amount and / or function, undetectable, or eliminated (e.g., compared to the corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements in the transcriptional regulatory sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. Any suitable method known in the art for measuring the amount of a gene or regulon product may be used, including, for example, qPCR, Northern blot, RNA sequencing, Western blot, ELISA, etc. In some embodiments, one or more inactivating mutations are present in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) adenoviral genes.

[0084] In some embodiments, the recombinant atadenovirus genome can be a recombinant bovine atadenovirus D genome, a recombinant bovine atadenovirus E genome, a recombinant deer atadenovirus A genome, a recombinant duck atadenovirus A genome, a recombinant lizard atadenovirus A genome, a recombinant lizard atadenovirus B genome, a recombinant ovine atadenovirus D genome, a recombinant possum atadenovirus A genome, a recombinant parrot atadenovirus A genome, a recombinant snake atadenovirus A genome, and any combination or derivative thereof.

[0085] In some embodiments, the recombinant Aviadenovirus genome can be a recombinant duck Aviadenovirus B genome, a recombinant peregrine falcon Aviadenovirus A genome, a recombinant poultry Aviadenovirus A genome, a recombinant poultry Aviadenovirus B genome, a recombinant poultry Aviadenovirus C genome, a recombinant poultry Aviadenovirus D genome, a recombinant poultry Aviadenovirus E genome, a recombinant goose Aviadenovirus A genome, a recombinant pigeon Aviadenovirus A genome, a recombinant pigeon Aviadenovirus B genome, a recombinant parrot Aviadenovirus B genome, a recombinant parrot Aviadenovirus C genome, a recombinant turkey Aviadenovirus B genome, a recombinant turkey Aviadenovirus C genome, a recombinant turkey Aviadenovirus D genome, and any combination or derivative thereof.

[0086] In some embodiments, the recombinant Ictadenovirus genome can be a recombinant Sturgeon's Ictadenovirus A.

[0087] In some embodiments, the recombinant mastadenovirus genome is a recombinant bat mastadenovirus A genome, a recombinant bat mastadenovirus B genome, a recombinant bat mastadenovirus C genome, a recombinant bat mastadenovirus D genome, a recombinant bat mastadenovirus E genome, a recombinant bat mastadenovirus F genome, a recombinant bat mastadenovirus G genome, a recombinant bat mastadenovirus H genome, a recombinant bat mastadenovirus I genome, a recombinant bat mastadenovirus J genome, Genome, recombinant bovine mastadenovirus A genome, recombinant bovine mastadenovirus B genome, recombinant bovine mastadenovirus C genome, recombinant canine mastadenovirus A genome, recombinant deer mastadenovirus B genome, recombinant dolphin mastadenovirus A genome, recombinant dolphin mastadenovirus B genome, recombinant equine mastadenovirus A genome, recombinant equine mastadenovirus B genome, recombinant guinea pig mastadenovirus A genome, recombinant human mastadenovirus A genome, recombinant human mastadenovirus B genome , recombinant human mastadenovirus C genome, recombinant human mastadenovirus D genome, recombinant human mastadenovirus E genome, recombinant human mastadenovirus F genome, recombinant human mastadenovirus G genome, recombinant mouse mastadenovirus A genome, recombinant mouse mastadenovirus B genome, recombinant mouse mastadenovirus C genome, recombinant ovine mastadenovirus A genome, recombinant ovine mastadenovirus B genome, recombinant ovine mastadenovirus C genome, recombinant platyrrhine mastadenovirus A genome , recombinant polar bear mammary gland adenovirus A genome, recombinant porcine mammary gland adenovirus A genome, recombinant porcine mammary gland adenovirus B genome, recombinant porcine mammary gland adenovirus C genome, recombinant sea lion mammary gland adenovirus A genome, recombinant simian mammary gland adenovirus A genome, recombinant simian mammary gland adenovirus B genome, recombinant simian mammary gland adenovirus C genome, recombinant simian mammary gland adenovirus D genome, recombinant simian mammary gland adenovirus E genome, recombinant simian mammary gland adenovirus F genome, recombinant simian mammary gland adenovirus G genome,The recombinant simian adenovirus H genome, recombinant simian adenovirus I genome, recombinant skunk bear adenovirus A genome, recombinant lizard adenovirus A genome, recombinant tupaima adenovirus A genome, and any combination or derivative thereof.

[0088] In some embodiments, the recombinant mastadenovirus genome can be a recombinant human mastadenovirus A genome, a recombinant human mastadenovirus B genome, a recombinant human mastadenovirus C genome, a recombinant human mastadenovirus D genome, a recombinant human mastadenovirus E genome, a recombinant human mastadenovirus F genome, or a recombinant human mastadenovirus G genome, and any combination or derivative thereof.

[0089] In some embodiments, the recombinant human mastadenovirus A genome can be recombinant human mastadenovirus A serotype 12, 18, 31, and any combination or derivative thereof.

[0090] In some embodiments, the recombinant human mastadenovirus B genome can be recombinant human mastadenovirus B serotype 3, 7, 11, 14, 16, 21, 34, 35, 50, 55, and any combination or derivative thereof.

[0091] In some embodiments, the recombinant human mastadenovirus C genome can be recombinant human mastadenovirus C serotype 1, 2, 5, 6, 57, and any combination or derivative thereof.

[0092] In some embodiments, the recombinant human mastadenovirus D genome can be recombinant human mastadenovirus D serotype 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 51, 53, 54, 56, 58, 59, 60, 62, 63, 64, 65, 67, 69, 70, 71, 72, 73, 74, 75, and any combination or derivative thereof.

[0093] In some embodiments, the recombinant human mastadenovirus E genome can be recombinant human mastadenovirus E serotype 4.

[0094] In some embodiments, the recombinant human mastadenovirus F genome can be recombinant human mastadenovirus F serotype 40, 41, and any combination or derivative thereof.

[0095] In some embodiments, the recombinant human mastadenovirus G genome can be recombinant human mastadenovirus G serotype 52.

[0096] In some embodiments, the recombinant siadenovirus genome can be a recombinant frog siadenovirus A genome, a recombinant great tit siadenovirus A genome, a recombinant penguin siadenovirus A genome, a recombinant parrot siadenovirus D genome, a recombinant parrot siadenovirus E genome, a recombinant raptor siadenovirus A genome, a recombinant skua siadenovirus A genome, a recombinant turkey siadenovirus A genome, and any combination or derivative thereof.

[0097] In some embodiments, the recombinant adenoviral genome can be a recombinant test adenoviral genome.

[0098] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant retrovirus genomes. Recombinant retrovirus genomes can be recombinant genomes derived from any member of the Retroviridae family of viruses known in the art, including, for example, recombinant Alpharetrovirus genomes, recombinant Betaretrovirus genomes, recombinant Gammaretrovirus genomes, recombinant Deltaretrovirus genomes, recombinant Epsilonretrovirus genomes, recombinant Lentivirus genomes, recombinant Bovispumavirus genomes, recombinant Equispumavirus genomes, recombinant Felispumavirus genomes, recombinant Prosimiispumavirus genomes, recombinant Simiispumavirus genomes, and any combinations or derivatives thereof. In some embodiments, the recombinant retroviral genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) retroviral genes.

[0099] In some embodiments, the recombinant alpharetroviral genome can be a recombinant avian tumor Mill Hill virus 2 genome, a recombinant avian leukosis virus genome, a recombinant avian myeloblastosis virus genome, a recombinant avian myelocytomatosis virus 29 genome, a recombinant avian sarcoma virus CT10 genome, a recombinant Fujinami sarcoma virus genome, a recombinant Rous sarcoma virus genome, a recombinant UR2 sarcoma virus genome, a recombinant Y73 sarcoma virus genome, and any combination or derivative thereof.

[0100] In some embodiments, the recombinant betaretroviral genome can be a recombinant Jaagsiekte sheep retrovirus genome, a recombinant langur virus genome, a recombinant Mason-Pfizer monkey virus genome, a recombinant mouse mammary tumor virus genome, a recombinant squirrel monkey retrovirus genome, and any combination or derivative thereof.

[0101] In some embodiments, the recombinant gammaretroviral genome is a recombinant chick syncytial virus genome, a recombinant feline leukemia virus genome, a recombinant Finkel-Biskis-Jinkins murine sarcoma virus genome, a recombinant Gardner-Arnstein feline sarcoma virus genome, a recombinant gibbon ape leukemia virus genome, a recombinant guinea pig type C oncovirus genome, a recombinant Hardy-Zuckerman feline sarcoma virus genome, a recombinant Harvey murine sarcoma virus genome, a recombinant guinea pig type C oncovirus ... Genome, recombinant Kirsten murine sarcoma virus genome, recombinant koala retrovirus genome, recombinant Moloney murine sarcoma virus genome, recombinant murine leukemia virus genome, recombinant porcine type C oncovirus genome, recombinant reticuloendotheliosis virus genome, recombinant Snyder-Theilen feline sarcoma virus genome, recombinant Trager duck spleen necrosis virus genome, recombinant Viper retrovirus genome, recombinant Woolly simian sarcoma virus genome, and any combination or derivative thereof.

[0102] In some embodiments, the recombinant deltaretrovirus genome can be a recombinant bovine leukemia virus genome, a recombinant primate T-lymphotropic virus 1 genome, a recombinant primate T-lymphotropic virus 2 genome, a recombinant primate T-lymphotropic virus 3 genome, and any combination or derivative thereof.

[0103] In some embodiments, the recombinant epsilon retroviral genome can be a recombinant walleye dermal sarcoma virus genome, a recombinant walleye epidermal hyperplasia virus 1 genome, a recombinant walleye epidermal hyperplasia virus 2 genome, and any combination or derivative thereof.

[0104] In some embodiments, the recombinant lentiviral genome can be a recombinant bovine immunodeficiency virus genome, a recombinant caprine arthritis-encephalitis virus genome, a recombinant equine infectious anemia virus genome, a recombinant feline immunodeficiency virus genome, a recombinant human immunodeficiency virus 1 genome, a recombinant human immunodeficiency virus 2 genome, a recombinant Jembrana disease virus genome, a recombinant puma lentivirus genome, a recombinant simian immunodeficiency virus genome, a recombinant Visna-maedi virus genome, and any combination or derivative thereof.

[0105] In some embodiments, the recombinant retroviral genome can be a recombinant bovine pumavirus genome.

[0106] In some embodiments, the recombinant retroviral genome may be a recombinant equine puma virus genome.

[0107] In some embodiments, the recombinant retroviral genome can be a recombinant feline spumavirus genome.

[0108] In some embodiments, the recombinant retroviral genome can be a recombinant prosimian spumavirus genome.

[0109] In some embodiments, the recombinant simian spumavirus genome can be a recombinant simian foamy virus genome, a recombinant human foamy virus genome, and any combination or derivative thereof.

[0110] In some embodiments, one or more recombinant nucleic acids described herein are recombinant adeno-associated virus genomes. The recombinant adeno-associated virus genome can be a recombinant genome derived from any member of the Dependoparvoviridae family of viruses known in the art, including, for example, a recombinant adeno-associated dependoparvovirus A genome, a recombinant adeno-associated dependoparvovirus B genome, and any combination or derivative thereof. In some embodiments, the recombinant adeno-associated virus genome contains one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) adeno-associated virus genes.

[0111] In some embodiments, the recombinant adeno-associated virus (AAV) genome can be a recombinant AAV serotype 1 genome, a recombinant AAV serotype 2 genome, a recombinant AAV serotype 3 genome, a recombinant AAV serotype 4 genome, a recombinant AAV serotype 5 genome, a recombinant AAV serotype 6 genome, a recombinant AAV serotype 7 genome, a recombinant AAV serotype 8 genome, a recombinant AAV serotype 9 genome, a recombinant AAV serotype 10 genome, a recombinant AAV serotype 11 genome, and any combination or derivative thereof.

[0112] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant herpes simplex virus (HSV) amplicons. Herpes virus amplicons, including their structural features and methods of production, are generally known to those skilled in the art (see, for example, de Silva S. and Bowers W, "Herpes Virus Amplicon Vectors", Viruses 2009, 1, 594-629). In some embodiments, the herpes simplex virus amplicon is an HSV-1 amplicon. In some embodiments, the herpes simplex virus amplicon is an HSV-1 hybrid amplicon. Examples of HSV-1 hybrid amplicons can include, but are not limited to, an HSV / AAV hybrid amplicon, an HSV / EBV hybrid amplicon, an HSV / EBV / RV hybrid amplicon, and / or an HSV / Sleeping Beauty hybrid amplicon. In some embodiments, the amplicon is an HSV / AAV hybrid amplicon. In some embodiments, the amplicon is an HSV / Sleeping Beauty hybrid amplicon.

[0113] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant herpesvirus genomes. The recombinant herpesvirus genome can be a recombinant genome derived from any member of the Herpesviridae family of DNA viruses known in the art, including, for example, a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof. In some embodiments, the recombinant herpesvirus genome contains one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) herpesvirus genes.

[0114] In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus (HSV) genome. In some embodiments, the recombinant herpes virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant HSV-1 genome is, for example, strain 17, Ty25, R62, S25, Ku86, S23, R11, Ty148, Ku47, H166 syn, 1319-2005, F-13, M-12, 90237, F-17, KOS, 3083-2008, F12g, L2, CD38, H193, M-15, India 2011, 0116209, F-11I, 66-207, 2762, 369-2007, 3355, MacIntyre, McKrae, 7862, 7-h se, HF10, 1394,2005, 270-2007, OD4, SC16, M-19, 4J1037, 5J1060, J1060, KOS79, 132- The recombinant HSV-1 genome may be derived from any HSV-1 strain known in the art, including 1988, 160-1982, H166, 2158-2007, RE, 78326, F18g, F11, 172-2010, H129, F, E4, CJ994, F14g, E03, E22, E10, E06, E11, E25, E23, E35, E15, E07, E12, E14, E08, E19, E13, ATCC 2011, etc. (See, e.g., Bowen et al. J Virol. 2019 Apr 3;93(8)). In some embodiments, the recombinant HSV-1 genome is derived from a KOS strain. In some embodiments, the recombinant HSV-1 genome is not derived from a McKrae strain.

[0115] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant poxvirus genomes. The recombinant poxvirus genome can be a recombinant genome derived from any member of the Poxvirus family of viruses known in the art, including, for example, a recombinant smallpox virus genome, a recombinant vaccinia virus genome, a recombinant cowpox virus genome, a recombinant empox virus genome, a recombinant orf virus genome, a recombinant pseudocowpox virus genome, a recombinant bovine papular stomatitis virus genome, a recombinant tanapox virus genome, a recombinant yabasa tumor virus genome, a recombinant molluscum contagiosum virus genome, and any combination or derivative thereof. In some embodiments, the recombinant poxvirus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) poxvirus genes.

[0116] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant bacteriophage genomes, such as recombinant 186 phage genome, recombinant Escherichia virus Lambda genome, recombinant Pseudomonas virus Phi6 genome, recombinant Bacillus virus Φ29 genome, recombinant Escherichia virus ΦX174 genome, recombinant bacteriophage ΦCb5 genome, recombinant Escherichia virus G4 genome, recombinant Escherichia virus M13 genome, recombinant Emesvirus zinderi genome, recombinant N4 phage genome, recombinant Escherichia virus P1 genome, recombinant Escherichia virus P2 genome, recombinant Enterobacteriaceae phage genome, recombinant Escherichia virus Ψ6 ... The recombinant bacteriophage genome may be a recombinant bacteriophage P4 genome, a recombinant R17 phage genome, a recombinant Enterobacteriaceae phage T2 genome, a recombinant Escherichia virus T4 genome, a recombinant Escherichia virus T7 genome, a recombinant bacteriophage T12 genome, and any combination or derivative thereof. In some embodiments, the recombinant bacteriophage genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) bacteriophage genes.

[0117] In some embodiments, one or more of the recombinant nucleic acids described herein is a recombinant alphavirus genome. Recombinant alphavirus genomes include, for example, a recombinant Aura virus genome, a recombinant Barmah Forest virus genome, a recombinant Bebaru virus genome, a recombinant Caaingua virus genome, a recombinant Cabassou virus genome, a recombinant Chikungunya virus genome, a recombinant Eastern Equine Encephalitis virus genome, a recombinant Eilat virus genome, a recombinant Everglades virus genome, a recombinant Fort Morgan virus genome, a recombinant Getah virus genome, a recombinant Highlands J virus genome, a recombinant Madariaga virus genome, a recombinant Mayaro virus genome, a recombinant Middelburg virus genome, a recombinant Mosso das Pedras virus genome, a recombinant E. coli ... The recombinant genome may be derived from any member of the Alphaviridae family of viruses known in the art, including a recombinant Pedras virus genome, a recombinant Mucambo virus genome, a recombinant Ndumu virus genome, a recombinant O'nyong'nyong virus genome, a recombinant Pixuna virus genome, a recombinant Rio Negro virus genome, a recombinant Ross River virus genome, a recombinant salmon pancreatic disease virus genome, a recombinant Semliki Forest virus genome, a recombinant Sindbis virus genome, a recombinant southern elephant seal virus genome, a recombinant Tonate virus genome, a recombinant Trocara virus genome, a recombinant Una virus genome, a recombinant Venezuelan equine encephalitis virus genome, a recombinant Western equine encephalitis virus genome, a recombinant Whataroa virus genome, and any combination or derivative thereof.In some embodiments, the recombinant alphavirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) alphavirus genes.

[0118] In some embodiments, one or more of the recombinant nucleic acids described herein is a recombinant picornavirus genome. Recombinant picornavirus genomes include, for example, recombinant Aalivirus genomes, recombinant Ailurivirus genomes, recombinant Ampivirus genomes, recombinant Anativirus genomes, recombinant Aphthovirus genomes, recombinant Aquamavirus genomes, recombinant Avihepatovirus genomes, recombinant Avisivirus genomes, recombinant Boosepivirus genomes, recombinant Bopivirus genomes, recombinant Caecilivirus genomes, recombinant Cardiovirus genomes, recombinant Cosavirus genomes, recombinant Crahelivirus genomes, recombinant Crohivirus genomes, recombinant Danipivirus genomes, recombinant Dicipiv irus genome, recombinant Diresapivirus genome, recombinant Enterovirus genome, recombinant Erbovirus genome, recombinant Felipivirus genome, recombinant Fipivirus genome, recombinant Gallivirus genome, recombinant Gruhelivirus genome, recombinant Grusopivirus genome, recombinant Harkavirus genome, recombinant Hemipivirus genome, recombinant Hepatovirus genome, recombinant Hunnivirus genome, recombinant Kobuvirus genome, recombinant Kunsagivirus genome, recombinant Limnipivirus genome, recombinant Livupivirus genome, recombinant Ludopivirus genome,Recombinant Malagasivirus genome, Recombinant Marsupivirus genome, Recombinant Megrivirus genome, Recombinant Mischivirus genome, Recombinant Mosavirus genome, Recombinant Mupivirus genome, Recombinant Myrropivirus genome, Recombinant Orivirus genome, Recombinant Oscivirus genome, Recombinant Parabovirus genome, Recombinant Parechovirus genome, Recombinant Pasivirus genome, Recombinant Passerivirus genome, Recombinant Pemapivirus genome, Recombinant Poecivirus genome, Recombinant Potamipivirus genome, Recombinant Pygoscepivirus genome, Recombinant Rabovirus genome, Recombinant Rafivirus (R afivirus genome, recombinant Rajidapivirus genome, recombinant Rohelivirus genome, recombinant Rosavirus genome, recombinant Sakobuvirus genome, recombinant Salivirus genome, recombinant Sapelovirus genome, recombinant Senecavirus genome, recombinant Shanbavirus genome, recombinant Sicinivirus genome, recombinant Symapivirus genome, recombinant Teschovirus genome, recombinant Torchivirus genome, recombinant Tottorivirus genome, recombinant Tremovirus genome, recombinant Tropivirus genome, and any combination or derivative thereof. In some embodiments,The recombinant picornavirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) picornavirus genes.

[0119] In some embodiments, the recombinant enterovirus genome can be a recombinant coxsackievirus genome, a recombinant poliovirus genome, a recombinant echovirus genome, a recombinant bovine enterovirus genome, and any combination or derivative thereof.

[0120] In some embodiments, the recombinant Seneca virus genome can be a recombinant Seneca Valley virus genome.

[0121] In some embodiments, the recombinant cardiovirus genome can be a recombinant Theiler's murine encephalomyelitis genome, a recombinant encephalomyocarditis virus genome, a recombinant mengovirus genome, and any combination or derivative thereof.

[0122] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant iridovirus genomes. The recombinant iridovirus genome can be a recombinant genome derived from any member of the Iridoviridae family of viruses known in the art, including, for example, a recombinant invertebrate iridescent virus 6 (IIV-6) genome, a recombinant invertebrate iridescent virus 31 (IIV-31) genome, and any combination or derivative thereof. In some embodiments, the recombinant iridovirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) iridovirus genes.

[0123] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant Newcastle disease virus genomes. In some embodiments, the recombinant Newcastle disease virus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) Newcastle virus genes.

[0124] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant baculovirus genomes. The recombinant baculovirus genome can be a recombinant genome derived from any member of the Baculoviridae family of viruses known in the art, including, for example, a recombinant alphabaculovirus genome, a recombinant betabaculovirus genome, a recombinant deltabaculovirus genome, a recombinant gammabaculovirus genome, and any combination or derivative thereof. In some embodiments, the recombinant Baculoviridae genome contains one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) baculovirus genes.

[0125] In some embodiments, the recombinant alphabaculovirus genome is a recombinant Adoxophyes honmai nucleopolyhedrovirus genome, a recombinant Agrotis ipsilon multiple nucleopolyhedrovirus genome, a recombinant Agrotis segetum nucleopolyhedrovirus A genome, a recombinant Agrotis seg- etum nucleopolyhedrovirus B genome, a recombinant Antheraea pernyi nucleopolyhedrovirus genome, a recombinant Anticarsia gemmatalis multiple nucleopolyhedrovirus genome, a recombinant Autographa californica multiple nucleopolyhedrovirus genome, a recombinant Bombyx mori nucleopolyhedrovirus genome, a recombinant Buzura suppressaria suppressaria nuclear polyhedrosis virus genome, recombinant Catopsilia pomona nuclear polyhedrosis virus genome, recombinant Choristoneura fumiferana DEF multiple nucleopolyhedrosis virus genome, recombinant Choristoneura fumiferana multiple nucleopolyhedrosis virus genome, recombinant Choristoneura murinana nuclear polyhedrosis virus genome, recombinant Choristoneura rosaceana nuclear polyhedrosis virus genome, recombinant Chrysodeixis chalcites nuclear polyhedrosis virus genome, recombinant Chrysodeixis includens nuclear polyhedrosis virus genome, recombinant Clanis virineata bilineata nuclear polyhedrosis virus genome, recombinant Condylorrhiza vestigialis nuclear polyhedrosis virus genome, recombinant Cryptophlebia peltastica nuclear polyhedrosis virus genome, recombinant Cyclophragma undansundans nuclear polyhedrosis virus genome, recombinant Ectropis obliqua nuclear polyhedrosis virus genome, recombinant Epiphyas postvittana nuclear polyhedrosis virus genome, recombinant Euproctis pseudoconspersa nuclear polyhedrosis virus genome, recombinant Helicoverpa armigera nuclear polyhedrosis virus genome, recombinant Hemileuca species nuclear polyhedrosis virus genome, recombinant Hyphantria cunea nuclear polyhedrosis virus genome, recombinant Hyposidra talaca nuclear polyhedrosis virus genome, recombinant Lambdina fiscellaria nuclear polyhedrosis virus genome, recombinant Leucania separata separata nuclear polyhedrosis virus genome, recombinant Lonomia obliqua nuclear polyhedrosis virus genome, recombinant Lymantria dispar nuclear polyhedrosis virus genome, recombinant Lymantria xylina nuclear polyhedrosis virus genome, recombinant Mamestra brassicae nuclear polyhedrosis virus genome, recombinant Mamestra configurata nuclear polyhedrosis virus A genome, recombinant Mamestra configurata nuclear polyhedrosis virus B genome, recombinant Maruca vitrata nuclear polyhedrosis virus genome, recombinant Mythimna unipuncta unipuncta nuclear polyhedrosis virus A genome, recombinant Misimna unipuncta nuclear polyhedrosis virus B genome, recombinant Operophtera brumata nuclear polyhedrosis virus genome, recombinant Orgyia leucostigma nuclear polyhedrosis virus genome, recombinant Orgyia pseudotsugata multiple nuclear polyhedrosis virus genome, recombinant Oxyplax ochraceaochracea nuclear polyhedrosis virus genome, recombinant Peridroma saucia nuclear polyhedrosis virus genome, recombinant Perigonia lusca nuclear polyhedrosis virus genome, recombinant Spodoptera eridania nuclear polyhedrosis virus genome, recombinant Spodoptera exempta nuclear polyhedrosis virus genome, recombinant Spodoptera exigua nuclear polyhedrosis virus A genome, recombinant Spodoptera exigua nuclear polyhedrosis virus B genome, recombinant Spodoptera frugiperda nuclear polyhedrosis virus genome, recombinant Spodoptera littoralis nuclear polyhedrosis virus genome, recombinant Spodoptera The recombinant nucleopolyhedrovirus may be a recombinant Lactobacillus litura nucleopolyhedrovirus genome, a recombinant Sucra jujuba nucleopolyhedrovirus genome, a recombinant Thysanoplusia orichalcea nucleopolyhedrovirus genome, a recombinant Trichoplusia ni nucleopolyhedrovirus genome, a recombinant Urbanus proteus nucleopolyhedrovirus genome, a recombinant Wisea signata nucleopolyhedrovirus genome, and any combination or derivative thereof.

[0126] In some embodiments, the recombinant beta baculovirus genome is a recombinant Adoxophyes orana granulovirus genome, a recombinant Agrotis segmentum granulovirus genome, a recombinant Artogeia rapae granulovirus genome, a recombinant Choristoneura fumiferana granulovirus genome, a recombinant Clostera anachoreta granulovirus genome, a recombinant Clostera anastomosis granulovirus A genome, a recombinant Clostera anastomosis granulovirus B genome, a recombinant Cnaphalocrocis medinalis granulovirus genome, a recombinant Cryptophlebia leucotreta granulovirus genome, a recombinant Cydia pomonella granulovirus genome, a recombinant Diatraea saccharalis granulovirus genome, ... Adoxophyes orana granulovirus genome, a recombinant Adoxophyes orana granulovirus genome, a recombinant Adoxophyes orana granulovirus genome, a recombinant Adoxophyes orana granulovirus genome, a recombinant Adoxophyes orana granulovirus genome, a recombinant Adoxophyes orana granulovirus genome, a recombinant saccharalis granulovirus genome, recombinant Epinotia aporema granulovirus genome, recombinant Erinnyis ello granulovirus genome, recombinant Harrisina brillians granulovirus genome, recombinant Helicoverpa armigera granulovirus genome, recombinant Lacanobia oleracea granulovirus genome, recombinant Mocis latipes granulovirus genome, recombinant Misimuna unipuncta granulovirus A genome, recombinant Misimuna unipuncta granulovirus B genome, recombinant Phthorimaea operculella granulovirus genome, recombinant Plodia interpunctella granulovirus genome, recombinant Plutella xylostellaxylostella granulovirus genome, recombinant Spodoptera frugiperda granulovirus genome, recombinant Spodoptera litura granulovirus genome, recombinant Trichoplusia ni granulovirus genome, recombinant Xestia c-nigrum granulovirus genome, and any combination or derivative thereof.

[0127] In some embodiments, the recombinant delta baculovirus genome can be a recombinant Culex nigripalpus nuclear polyhedrosis virus genome.

[0128] In some embodiments, the recombinant gamma baculovirus genome can be a recombinant Neodiprion lecontei nuclear polyhedrosis virus genome, a recombinant Neodiprion sertifer nuclear polyhedrosis virus genome, and any combination or derivative thereof.

[0129] In some embodiments, one or more of the recombinant nucleic acids described herein are recombinant geminivirus genomes. The recombinant geminivirus genome can be a recombinant genome derived from any member of the Geminiviridae family of viruses known in the art, including, for example, a recombinant Becurtovirus genome, a recombinant Begomovirus genome, a recombinant Capulavirus genome, a recombinant Citlodavirus genome, a recombinant Curtovirus genome, a recombinant Eragrovirus genome, a recombinant Grablovirus genome, a recombinant Maldovirus genome, a recombinant Mastrevirus genome, a recombinant Mulcrilevirus genome, a recombinant Opunvirus genome, a recombinant Topilevirus genome, a recombinant Topocuvirus genome, a recombinant Turncurtovirus genome, and any combination or derivative thereof. In some embodiments, the recombinant geminivirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) geminivirus genes.

[0130] In some embodiments, one or more of the recombinant nucleic acids described herein is a recombinant caulimovirus genome. The recombinant caulimovirus genome can be a recombinant genome derived from any member of the Caulimoviridae family of viruses known in the art, including, for example, a recombinant Angelica bushy stunt virus genome, a recombinant Atractylodes mild mottle virus genome, a recombinant Carnation etched ring virus genome, a recombinant Cauliflower mosaic virus genome, a recombinant Dahlia mosaic virus genome, a recombinant Figwort mosaic virus genome, a recombinant Horseradish latent virus genome, a recombinant Lamium malformed leaf virus genome, a recombinant Mirabilis mosaic virus genome, a recombinant Soybean Putnam virus genome, a recombinant Strawberry vein banding virus genome, a recombinant Thistle mottle virus genome, and any combination or derivative thereof. In some embodiments, the recombinant caulimovirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) caulimovirus genes.

[0131] In some embodiments, one or more of the recombinant nucleic acids described herein is a recombinant anellovirus genome. Recombinant anellovirus genomes include, for example, a recombinant Aleptorquevirus genome, a recombinant Alphatorquevirus genome, a recombinant Betatorquevirus genome, a recombinant Chitorquevirus genome, a recombinant Dalettorquevirus genome, a recombinant Deltatorquevirus genome, a recombinant Epsilontorquevirus genome, a recombinant Etatorquevirus genome, a recombinant Gammatorquevirus genome, a recombinant Gimeltorquevirus genome, a recombinant Gyrovirus genome, a recombinant Hetorquevirus genome, a recombinant Iotatorquevirus genome, or a recombinant Kappatorquevirus genome. (Kappatorquevirus) genome, recombinant Lambdatorquevirus genome, recombinant Mutorquevirus genome, recombinant Nutorquevirus genome, recombinant Omegatorquevirus genome, recombinant Omicrontorquevirus genome, recombinant Pitorquevirus genome, recombinant Psitorquevirus genome, recombinant Rhotorquevirus genome, recombinant Sigmatorquevirus genome, recombinant Tettorquevirus genome, recombinant Thetatorquevirus genome, recombinant Upsilontorquevirus genome, recombinant Wawtorquevirus genome,The recombinant genome may be derived from any member of the Anelloviridae family of viruses known in the art, including a recombinant Xitorquevirus genome, a recombinant Zayintorquevirus genome, a recombinant Zetatorquevirus genome, and any combination or derivative thereof. In some embodiments, the recombinant anellovirus genome comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.) anellovirus genes.

[0132] V. Gene Delivery Vehicles Viral Vectors or Viral Gene Delivery Vehicles Certain aspects of the present disclosure relate to viral vectors or viral gene delivery vehicles comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the virus is capable of infecting one or more target cells of a subject (e.g., a human). In some embodiments, the virus is suitable for delivering polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human). In some embodiments, the one or more target cells are human cells. In some embodiments, the one or more target cells are one or more cancer cells. In some embodiments, the one or more target cells are one or more non-cancerous (e.g., healthy) cells. In some embodiments, the one or more target cells are one or more airway epithelial cells. In some embodiments, the one or more target cells are one or more cells of the respiratory tract (e.g., airway epithelial cells (e.g., goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, oncocytes, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (e.g., type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); bronchial salivary gland cells (e.g., serous cells, mucous cells, and / or duct cells), etc.). In some embodiments, the one or more target cells are one or more cells of the lung.

[0133] Any suitable virus known in the art can be used, including, for example, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, papillomavirus, herpesvirus (e.g., herpes simplex virus), vaccinia virus (e.g., poxvirus), bacteriophage, alphavirus, picornavirus, iridovirus, Newcastle virus, baculovirus, geminivirus, caulimovirus, anellovirus, and / or any hybrid or derivative thereof. In some embodiments, the virus is attenuated. In some embodiments, the virus is replication-competent. In some embodiments, the virus is replication-defective. In some embodiments, the virus is not oncolytic. In some embodiments, the virus has been modified to alter its tissue tropism compared to the tissue tropism of the corresponding unmodified wild-type virus. In some embodiments, the virus has reduced cytotoxicity (e.g., in target cells) compared to the corresponding wild-type virus. Methods for producing viruses containing polynucleotides and / or recombinant nucleic acids are well known to those skilled in the art.

[0134] In some embodiments, viral gene delivery vehicle is adenovirus.The adenovirus can be derived from any member of the adenoviridae family of viruses known in the art, including, for example, recombinant atadenovirus, recombinant aviadenovirus, recombinant ictadenovirus, recombinant mastadenovirus, recombinant siadenovirus, recombinant testadenovirus, and any combination or derivative thereof.In some embodiments, the adenovirus is replication-competent.In some embodiments, the adenovirus is replication-defective or replication-defective.In some embodiments, the adenovirus is not oncolytic.

[0135] In some embodiments, the viral gene delivery vehicle is a retrovirus.The retrovirus can be derived from any member of the retrovirus family known in the art, including, for example, recombinant alpharetrovirus, recombinant betaretrovirus, recombinant gammaretrovirus, recombinant deltaretrovirus, recombinant epsilonretrovirus, recombinant lentivirus, recombinant bovine spumavirus, recombinant equine spumavirus, recombinant feline spumavirus, recombinant prosimian spumavirus, recombinant simian spumavirus, and any combination or derivative thereof.In some embodiments, the retrovirus is replication-competent.In some embodiments, the retrovirus is replication-defective or replication-defective.In some embodiments, the retrovirus is not oncolytic.

[0136] In some embodiments, viral gene delivery vehicle is adeno-associated virus (AAV).AAV can be derived from any member of the Dependoparvoviridae family of viruses known in the art, including, for example, recombinant adeno-associated dependoparvovirus A, recombinant adeno-associated dependoparvovirus B, and any combination or derivative thereof.In some embodiments, AAV is replication-competent.In some embodiments, AAV is replication-defective or replication-defective.In some embodiments, AAV is not oncolytic.

[0137] In some embodiments, the AAV may be derived from any serotype known in the art, including, for example, serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, AAV serotype 9, serotype 10, serotype 11, and any combination or derivative thereof.

[0138] In some embodiments, the viral gene delivery vehicle is a member of the Herpesviridae family of DNA viruses, including, for example, herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, and any combination or derivative thereof. In some embodiments, the Herpesviridae virus is replication-competent. In some embodiments, the Herpesviridae virus is replication-defective or replication-incompetent. In some embodiments, the Herpesviridae virus is not oncolytic.

[0139] In some embodiments, the herpes virus is a herpes simplex virus. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any combination or derivative thereof. In some embodiments, the herpes simplex virus is an HSV-1 virus.

[0140] In some embodiments, the viral gene delivery vehicle is a poxvirus.The poxvirus can be derived from any member of the Poxvirus family of viruses known in the art, including, for example, recombinant smallpox virus, recombinant vaccinia virus, recombinant cowpox virus, recombinant empox virus, recombinant orf virus, recombinant pseudocowpox virus, recombinant bovine papular stomatitis virus, recombinant tanapox virus, recombinant yabasa tumor virus, recombinant molluscum contagiosum virus, and any combination or derivative thereof.In some embodiments, the poxvirus is replication-competent.In some embodiments, the poxvirus is replication-defective or replication-incompetent.In some embodiments, the poxvirus is not oncolytic.

[0141] In some embodiments, the viral gene delivery vehicle is a bacteriophage. The bacteriophage can be any bacteriophage known in the art, including, for example, recombinant 186 phage, recombinant Escherichia virus lambda, recombinant Pseudomonas virus phi6, recombinant Bacillus virus Φ29, recombinant Escherichia virus ΦX174, recombinant bacteriophage ΦCb5, recombinant Escherichia virus G4, recombinant Escherichia virus M13, recombinant Emes virus Zinderi, recombinant N4 phage, recombinant Escherichia virus P1, recombinant Escherichia virus P2, recombinant Enterobacteriaceae phage P4, recombinant R17 phage, recombinant Enterobacteriaceae phage T2, recombinant Escherichia virus T4, recombinant Escherichia virus T7, recombinant bacteriophage T12, and any combination or derivative thereof. In some embodiments, the bacteriophage is replication-competent. In some embodiments, the bacteriophage is replication-defective or replication-incompetent. In some embodiments, the bacteriophage is not oncolytic.

[0142] In some embodiments, the viral gene delivery vehicle is an alphavirus, such as, for example, a recombinant Aura virus, a recombinant Barmah Forest virus, a recombinant Bebaru virus, a recombinant Ka-Ingua virus, a recombinant Kabaso virus, a recombinant Chikungunya virus, a recombinant Eastern Equine Encephalitis virus, a recombinant Eilat virus, a recombinant Everglades virus, a recombinant Fort Morgan virus, a recombinant Getah virus, a recombinant Highland J virus, a recombinant Madariaga virus, a recombinant Mayaro virus, a recombinant Middelburg virus, a recombinant Moso das Pedras virus, a recombinant Mucambo virus, a recombinant Ndum virus, a recombinant The alphavirus may be derived from any member of the Alphavirus family of viruses known in the art, including recombinant O'Nyong Nyong virus, recombinant Pixuna virus, recombinant Rio Negro virus, recombinant Ross River virus, recombinant salmon pancreatic disease virus, recombinant Semliki Forest virus, recombinant Sindbis virus, recombinant southern elephant seal virus, recombinant Tonate virus, recombinant Trocara virus, recombinant Una virus, recombinant Venezuelan equine encephalitis virus, recombinant Western equine encephalitis virus, recombinant Wataroa virus, and any combination or derivative thereof. In some embodiments, the alphavirus is replication-competent. In some embodiments, the alphavirus is replication-defective or replication-incompetent. In some embodiments, the alphavirus is not oncolytic.

[0143] In some embodiments, the viral gene delivery vehicle is a picornavirus, such as a recombinant Aarivirus, recombinant Ailurvirus, recombinant Ampivirus, recombinant Anativirus, recombinant Aphthovirus, recombinant Akuamavirus, recombinant Avihepatovirus, recombinant Abyssivirus, recombinant Bucepivirus, recombinant Bopivirus, recombinant Caecilivirus, recombinant Cardiovirus, recombinant Cosavirus, recombinant Krahelivirus, recombinant Kurohivirus, recombinant Danipivirus, recombinant Dicipivirus, recombinant Diresapivirus, recombinant Enterovirus, recombinant Erbovirus, recombinant Felippivirus, recombinant Phippivirus, recombinant Galivirus, recombinant Gruhelivirus, recombinant Grussopivirus, recombinant Harkavirus, recombinant Hemipivirus, recombinant Hepatovirus, recombinant Hunnivirus, recombinant Kobuvirus, recombinant Kunsaguivirus, recombinant Limnipivirus, recombinant Ribpivirus, recombinant Ludpivirus, recombinant Malagasyvirus, recombinant Mals The picornavirus may be derived from any member of the Picornaviridae family of viruses known in the art, including Pivirus, recombinant Meghrivirus, recombinant Miskivirus, recombinant Mosavirus, recombinant Mupivirus, recombinant Miropivirus, recombinant Ocivirus, recombinant Parabovirus, recombinant Parechovirus, recombinant Pacivirus, recombinant Parsleyvirus, recombinant Pemapivirus, recombinant Poesivirus, recombinant Potamipivirus, recombinant Pygoseppivirus, recombinant Rabovirus, recombinant Rafivirus, recombinant Rajidapivirus, recombinant Rohelivirus, recombinant Rosavirus, recombinant Sacobuvirus, recombinant Salivirus, recombinant Sapelovirus, recombinant Senecavirus, recombinant Shambavirus, recombinant Sisinivirus, recombinant Shimapivirus, recombinant Teschovirus, recombinant Torkivirus, recombinant Tottorivirus, recombinant Tremovirus, recombinant Tropivirus, and any combination or derivative thereof. In some embodiments, the picornavirus is replication-competent. In some embodiments, the picornavirus is replication-defective or replication-incompetent. In some embodiments, the picornavirus is not oncolytic.

[0144] In some embodiments, the viral gene delivery vehicle is an iridovirus.The iridovirus can be derived from any member of the Iridoviridae family of viruses known in the art, including, for example, recombinant invertebrate iridescent virus 6 (IIV-6), recombinant invertebrate iridescent virus 31 (IIV-31), and any combination or derivative thereof.In some embodiments, the iridovirus is replication-competent.In some embodiments, the iridovirus is replication-defective or replication-incompetent.In some embodiments, the iridovirus is not oncolytic.

[0145] In some embodiments, the viral gene delivery vehicle is a Newcastle disease virus. In some embodiments, the Newcastle disease virus is replication-competent. In some embodiments, the Newcastle disease virus is replication-defective or replication-incompetent. In some embodiments, the Newcastle disease virus is not oncolytic.

[0146] In some embodiments, the viral gene delivery vehicle is a baculovirus.The baculovirus can be derived from any member of the baculovirus family of viruses known in the art, including, for example, recombinant alpha baculovirus, recombinant beta baculovirus, recombinant delta baculovirus, recombinant gamma baculovirus, and any combination or derivative thereof.In some embodiments, the baculovirus is replication-competent.In some embodiments, the baculovirus is replication-defective or replication-incompetent.In some embodiments, the baculovirus is not oncolytic.

[0147] In some embodiments, the viral gene delivery vehicle is a gemininvirus. The gemininvirus can be derived from any member of the Geminiviridae family of viruses known in the art, including, for example, recombinant vekurtviruses, recombinant begomoviruses, recombinant capraviruses, recombinant citrodaviruses, recombinant kurtoviruses, recombinant elagroviruses, recombinant glabroviruses, recombinant maldoviruses, recombinant mastreviruses, recombinant marculireviruses, recombinant opunviruses, recombinant topileviruses, recombinant topokuviruses, recombinant turnkurtviruses, and any combination or derivative thereof. In some embodiments, the gemininvirus is replication-competent. In some embodiments, the gemininvirus is replication-defective or replication-incompetent. In some embodiments, the gemininvirus is not oncolytic.

[0148] In some embodiments, the viral gene delivery vehicle is a caulimovirus. The caulimovirus can be derived from any member of the Caulimoviridae family of viruses known in the art, including, for example, recombinant angelica bushy stunt virus, recombinant Okra mild mottle virus, recombinant carnation etched dring virus, recombinant cauliflower mosaic virus, recombinant dahlia mosaic virus, recombinant figwort mosaic virus, recombinant horseradish latent virus, recombinant dead nettle leaf malformation virus, recombinant Mirabilis mosaic virus, recombinant soybean Putnam virus, recombinant strawberry vein banding virus, recombinant thistle mottle virus, and any combination or derivative thereof. In some embodiments, the caulimovirus is replication-competent. In some embodiments, the caulimovirus is replication-defective or replication-incompetent. In some embodiments, the caulimovirus is not oncolytic.

[0149] In some embodiments, the viral gene delivery vehicle is an anellovirus. The anellovirus can be derived from any member of the anelloviridae family of viruses known in the art, including, for example, recombinant aleptorq virus, recombinant alphatorq virus, recombinant betatorq virus, recombinant chitorq virus, recombinant daletorq virus, recombinant deltatorq virus, recombinant epsilontorq virus, recombinant etatorq virus, recombinant gammatorq virus, recombinant zimertorq virus, recombinant gyrovirus, recombinant heetorq virus, recombinant iotatorq virus, recombinant kappatorq virus, recombinant lambdatorq virus, recombinant mutorq virus, recombinant nutorq virus, recombinant omegatorq virus, recombinant omicrontorq virus, recombinant pitorq virus, recombinant psitorq virus, recombinant rhotorq virus, recombinant sigmatorq virus, recombinant tetorq virus, recombinant thetatorq virus, recombinant upsilontorq virus, recombinant wautorq virus, recombinant xitorq virus, recombinant zeintorq virus, recombinant zetatorq virus, and any combination or derivative thereof. In some embodiments, the anellovirus is replication-competent. In some embodiments, the anellovirus is replication-defective or replication-incompetent. In some embodiments, the anellovirus is not oncolytic.

[0150] In some embodiments, provided herein are recombinant viruses, which may or may not be pseudotyped, that produce IL-2 and IL-12 polypeptides for the treatment of cancer, including solid tumors (e.g., advanced solid tumors) and hematological malignancies. In some embodiments, the therapeutic polypeptides produced by the recombinant viruses described herein mediate or enhance an anti-tumor effect, for example, by effector cell-mediated tumor cell lysis.

[0151] Non-viral Vectors or Non-viral Gene Delivery Vehicles Certain aspects of the present disclosure relate to non-viral vectors or non-viral gene delivery vehicles comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the non-viral vectors or non-viral gene delivery vehicles are capable of delivering the polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human). In some embodiments, the non-viral vectors or non-viral gene delivery vehicles are suitable for delivering the polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human). In some embodiments, the one or more target cells are human cells. In some embodiments, the one or more target cells are one or more cancer cells. In some embodiments, the one or more target cells are one or more non-cancerous (e.g., healthy) cells. In some embodiments, the one or more target cells are one or more airway epithelial cells. In some embodiments, the one or more target cells are one or more cells of the respiratory tract (e.g., airway epithelial cells (e.g., goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, oncocytes, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (e.g., type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); bronchial salivary gland cells (e.g., serous cells, mucous cells, and / or duct cells), etc.). In some embodiments, the one or more target cells are one or more cells of the lung.

[0152] Any non-viral vector or non-viral gene delivery vehicle known in the art can be used, including, for example, chemical compounds, bacteria, mammalian cells, or physical delivery systems, and / or any hybrids or derivatives thereof. Methods for producing non-viral vectors or non-viral gene delivery vehicles containing polynucleotides and / or recombinant nucleic acids are well known to those skilled in the art.

[0153] In some embodiments, the non-viral vector or gene delivery vehicle is a chemical compound, hi some embodiments, the chemical compound may be a polymeric compound, a lipid compound, an inorganic compound, and any combination or derivative thereof.

[0154] In some embodiments, the polymeric compound can be a natural polymer, a synthetic polymer, a biopolymer, a biodegradable polymer, a cationic polymer, a protein polymer, a polysaccharide polymer, and any combination or derivative thereof.

[0155] In some embodiments, the polymeric compound is selected from the group consisting of poly-L-lactide (PLA), chitosan, pectin, polyethyleneimine (PEI), poly-L-lysine (PLL), poly 2-N-dimethylaminoethyl methacrylate (PDMAEMA), polyaminoamines, polyaminoesters, polyamidoamines, polyvinylimidazole (PVI), polybeta-aminoesters (PBAE), gelatin, albumin, chitosan, beta-cyclodextrin, dextran, hyaluronic acid, polyesters, alpha-pyrrolidinohexyl phenoxyethanol, and the like. The polymer may be poly(PHP), poly(2-acrylamidoglycolic acid) (PAGA), poly(delta-valerolactone) (PVL), polyphenyl ether (PPE), poly(beta-amino-ester) (PBAE), polyamine-co-ester (PACE), polylactic-co-glycolic acid (PLGA), polycarbonate, polyurethane, micelles, cationic emulsions, exosomes, Reb blood cell membranes, DNA-protein complexes, plasmid DNA, naked DNA, pBR322, pUC, and any combination or derivative thereof.

[0156] In some embodiments, the lipid compound may be a nanoparticle (e.g., as disclosed in U.S. Patent Application Publication No. 2010 / 0015232, the entire contents of which are incorporated herein by reference), a lipid nanoparticle (LNP; e.g., as disclosed in U.S. Patent Application Publication Nos. 10,898,574, 10,702,600, and 10,933,127, the entire contents of which are incorporated herein by reference), a cell-targeted lipid nanoparticle (ctLNP; e.g., as disclosed in U.S. Patent Application Publication Nos. 2021 / 0059953, 2020 / 0283794, and / or 2021 / 0108228, the entire contents of which are incorporated herein by reference), a liposome, a cationic liposome, a solid lipid nanoparticle, a lipid emulsion, a lipidoid, a cytofectin, a lipid emulsion, a surfactant, a gemini surfactant, and any combination or derivative thereof.

[0157] In some embodiments, the inorganic compound may be a nanocarrier, a DNA nanoparticle, a gold nanoparticle, a carbon nanotube, graphene, a quantum dot, an upconversion nanoparticle, a silica nanoparticle, iron oxide, ferritin, and any combination or derivative thereof.

[0158] In some embodiments, the non-viral vector or non-viral gene delivery vehicle is a physical delivery system.In some embodiments, the physical delivery system can be electroporation, gene gun, jet gun, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, DNA bombardment injection, sonoporation, photoporation, laser pulse, magnetofection, magnetoporation, magnetic particle, hydroporation, and any combination or derivative thereof.

[0159] VI. Pharmaceutical Compositions and Formulations Certain aspects of the present disclosure relate to pharmaceutical compositions or formulations comprising any of a polynucleotide, a recombinant nucleic acid, and / or a gene delivery vehicle comprising a polynucleotide and / or a recombinant nucleic acid, and a pharmaceutically acceptable excipient or carrier.

[0160] Pharmaceutical compositions and formulations can be prepared by mixing an active ingredient (e.g., a polynucleotide, a recombinant nucleic acid comprising a polynucleotide, or a gene delivery vehicle comprising a polynucleotide and / or recombinant nucleic acid) having a desired degree of purity with one or more pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients are generally nontoxic to recipients at the dosages and concentrations employed and may include buffers (e.g., phosphate, citric acid, acetic acid, and other organic acids); antioxidants (e.g., ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); low molecular weight (less than about 10 residues) polypeptides; proteins (e.g., serum albumin, etc.). chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); counterions that form salts (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). A thorough discussion of pharmaceutically acceptable carriers is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0161] In some embodiments, the pharmaceutical composition or formulation comprises one or more lipid (e.g., cationic lipid) carriers. In some embodiments, the pharmaceutical composition or formulation comprises one or more nanoparticle carriers. Using methods well known in the art, a variety of molecules (e.g., proteins, peptides, recombinant nucleic acids, etc.) can be efficiently encapsulated in nanoparticles. In some embodiments, a molecule "encapsulated" in a nanoparticle can refer to a molecule contained within the nanoparticle, or a molecule attached to and / or associated with the surface of the nanoparticle, or any combination thereof. Nanoparticles for use in the compositions or formulations described herein can be any type of biocompatible nanoparticle known in the art, including, for example, nanoparticles comprising poly(lactic acid), poly(glycolic acid), PLGA, PLA, PGA, and any combination thereof (see, e.g., Vauthier et al. Adv Drug Del Rev. (2003) 55:519-48; US 2007 / 0148074; US 2007 / 0092575; US 2006 / 0246139; US 5,753,234; US 7,081,483; and US 2008 / 0260851, all of which are incorporated herein by reference in their entireties).

[0162] In some embodiments, the pharmaceutically acceptable carrier or excipient may be suitable for any administration route known in the art, including, for example, intratumoral, intravenous, intramuscular, subcutaneous, cutaneous, oral, intranasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, implant, inhalation, intrathecal, intraventricular, intracranial, and / or intranasal administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is suitable for intranasal and / or inhalation administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is suitable for inhalation administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is suitable for intratumoral administration.

[0163] In some embodiments, the pharmaceutical composition or formulation is suitable for any administration route known in the art, including, for example, intratumoral, intravenous, intramuscular, subcutaneous, cutaneous, oral, intranasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, implant, inhalation, intrathecal, intraventricular, intracranial, or intranasal administration. In some embodiments, the pharmaceutical composition or formulation is suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is suitable for intranasal and / or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is suitable for inhalation administration. In some embodiments, the pharmaceutical composition or formulation is suitable for intratumoral administration.

[0164] In some embodiments, the pharmaceutical composition or formulation further comprises one or more additional ingredients. Examples of additional ingredients may include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); humectants (e.g., sodium lauryl sulfate, etc.); salt solutions; alcohol; polyethylene glycol; gelatin; lactose; amylase; magnesium stearate; talc; silicic acid; viscous paraffin; hydroxymethylcellulose; polyvinylpyrrolidone; sweeteners; flavorings; fragrances; colorants; moisturizers; sunscreens; antibacterial agents; agents that can stabilize or prevent the degradation of polynucleotides, etc. In some embodiments, the pharmaceutical composition or formulation comprises a methylcellulose gel (e.g., hydroxypropylmethylcellulose, carboxymethylcellulose, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer. In some embodiments, the pharmaceutical composition or formulation comprises glycerol (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer and glycerol.

[0165] Pharmaceutical compositions and formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0166] In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used to deliver one or more polynucleotides encoding IL-2 and IL-12 polypeptides to one or more cells of a subject (e.g., one or more cells of the subject's respiratory tract). In some embodiments, the subject is afflicted with cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of carcinoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of lymphoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of blastoma.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of sarcoma.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of neuroendocrine tumors.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of mesothelioma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of schwannoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of meningioma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of adenocarcinoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of melanoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of leukemia. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of lymphoid malignancies.

[0167] In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used to deliver one or more polynucleotides encoding IL-2 and IL-12 polypeptides to one or more cells of a subject (e.g., one or more cells of the subject's respiratory tract). In some embodiments, the subject is afflicted with cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of solid tumors. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of hematological cancers. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of bladder cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of brain cancer.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of breast cancer.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of colon cancer.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of gastric cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of glioma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of head cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of leukemia. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of liver cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of lung cancer.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of lymphoma.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of myeloma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of cervical cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of ovarian cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of melanoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of pancreatic cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of kidney cancer.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of salivary gland cancer.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, atypical nevi, and / or Merkel cell carcinoma). In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of gastric cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of thymic epithelial cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of thyroid cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of osteosarcoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of leptomeningeal carcinoma.

[0168] In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used to deliver one or more polynucleotides encoding IL-2 polypeptides and IL-12 polypeptides to one or more cells of a subject (e.g., one or more cells of the subject's respiratory tract). In some embodiments, the subject is afflicted with lung cancer. Lung cancer is often classified into the broad categories of small cell lung cancer (SCLC), also known as oat cell carcinoma, and non-small cell lung cancer (NSCLC). NSCLC is further classified into three major types: squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of small cell lung cancer. In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of lung adenocarcinoma.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of lung squamous cell carcinoma.In some embodiments, any of the polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein can be used in the treatment of non-small cell lung cancer.

[0169] VII. Method Certain aspects of the present disclosure relate to methods of delivering one or more polynucleotides to one or more cells of a subject (e.g., one or more cancer cells, one or more non-cancerous (e.g., healthy) cells, one or more cells of the respiratory tract, e.g., airway epithelial cells (goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, oncocytes, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); bronchial salivary gland cells (serous cells, mucous cells, and / or duct cells); etc.), comprising administering to the subject any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the polynucleotide, the recombinant nucleic acid comprising the polynucleotide, the gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or the pharmaceutical composition or formulation is administered to a subject topically, transdermally, subcutaneously, epidermally, intradermally, orally, sublingually, bucally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, epichoroidally, intracranially, intrathecally, intraventricularly, intraarticularly, periarticularly, intratumorally, locally, or via inhalation. In some embodiments, the polynucleotide, the recombinant nucleic acid comprising the polynucleotide, the gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or the pharmaceutical composition or formulation is administered to a subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the polynucleotide, a recombinant nucleic acid comprising the polynucleotide, a gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or a pharmaceutical composition or formulation is administered to a subject intranasally or via inhalation.In some embodiments, the polynucleotide, the recombinant nucleic acid comprising the polynucleotide, the gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or the pharmaceutical composition or formulation is administered to a subject via inhalation. In some embodiments, the polynucleotide, the recombinant nucleic acid comprising the polynucleotide, the gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or the pharmaceutical composition or formulation is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the polynucleotide, the recombinant nucleic acid comprising the polynucleotide, the gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or the pharmaceutical composition or formulation is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer. In some embodiments, the polynucleotide, the recombinant nucleic acid comprising the polynucleotide, the gene delivery vehicle comprising the polynucleotide and / or the recombinant nucleic acid, and / or the pharmaceutical composition or formulation is administered intratumorally.

[0170] In some embodiments, the subject is human.In some embodiments, the subject is suffering from cancer.In some embodiments, the cancer is acute myeloid leukemia (LAML or AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), brainstem glioma, brain low-grade glioma (LGG), brain tumor, breast cancer (BRCA), bronchial tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, Cervical adenocarcinoma (CESC), childhood cancer, bile duct cancer (CHOL), chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer (adenocarcinoma) (COAD), colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer (ESCA), olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, Gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioblastoma multiforme, glioma (GBM), hairy cell leukemia, head and neck cancer (HNSD), cardiac cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leptomeningeal cancer, lip cancer, liver cancer, lymphoid neoplasms, diffuse large B-cell lymphoma (DLBCL), malignant fibrous Histiocytoma, bone cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma (MESO), metastatic squamous cell neck cancer of occult primary, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma (PCPG), intermediate pineal parenchymal tumor,Pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma (PRAD), rectal cancer, renal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (SARC), Sézary syndrome, cutaneous melanoma (SKCM), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric (s The cancer is selected from gastric (tonach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, testicular germ cell tumor (TGCT), throat cancer, thymic cancer, thymoma (THYM), thyroid cancer (THCA), transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma (UVM), vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and / or Wilms' tumor. In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-associated cancer (e.g., HPV-associated cancer of the back of the throat, cervix, anus, vulva, penis, and / or vagina). In some embodiments, the cancer is not a skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, atypical nevus, and / or Merkel cell carcinoma). In some embodiments, the cancer is not a melanoma. In some embodiments, the subject is afflicted with one or more of carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies. In some embodiments, the subject is afflicted with one or more of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, skin cancer, stomach cancer, thymic epithelial carcinoma, and thyroid cancer. In some embodiments, the subject has one or more of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the subject has osteosarcoma.

[0171] Another aspect of the present disclosure relates to a method for providing preventive, symptomatic, or therapeutic relief of one or more signs or symptoms of cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles, medicaments, and / or pharmaceutical compositions or formulations comprising the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the subject is human. In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is acute myeloid leukemia (LAML or AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), brainstem glioma, brain low-grade glioma (LGG), brain tumor, breast cancer (BRCA), bronchial tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor ... Nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma (CESC), childhood cancer, bile duct cancer (CHOL), chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer (adenocarcinoma) (COAD), colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer (ESCA), olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor , extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioblastoma multiforme, glioma GBM), hairy cell leukemia, head and neck cancer (HNSD), cardiac cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leptomeningeal cancer, lip cancer, liver cancer, lymphoid neoplasms, diffuse large intestine Cancerous large B-cell lymphoma (DLBCL), malignant fibrous histiocytoma, bone cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma (MESO), metastatic squamous cell neck cancer of unknown primary origin, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma,Non-melanoma skin cancer, non-small cell lung cancer, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma (PCPG), intermediate pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma (PRAD), rectal cancer, kidney cancer, renal cell (kidney) cancer, renal cell carcinoma, respiratory cancer, retinoblastoma In some embodiments, the cancer is selected from the group consisting of rhabdomyosarcoma, salivary gland cancer, sarcoma (SARC), Sézary syndrome, cutaneous melanoma (SKCM), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, testicular germ cell tumor (TGCT), throat cancer, thymic cancer, thymoma (THYM), thyroid cancer (THCA), transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma (UVM), vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and / or Wilms' tumor. In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-associated cancer (e.g., HPV-associated cancer of the back of the throat, cervix, anus, vulva, penis, and / or vagina). In some embodiments, the cancer is not a skin cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, atypical nevus, and / or Merkel cell carcinoma). In some embodiments, the cancer is not melanoma. In some embodiments, the subject is suffering from one or more of carcinoma, lymphoma, blastoma, sarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, leukemia, and lymphoid malignancies. In some embodiments, the subject has a cancer of the following type: a solid tumor, a blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer,and thyroid cancer. In some embodiments, the subject is afflicted with one or more of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma. In some embodiments, the subject is afflicted with osteosarcoma.

[0172] VIII.Host cells Certain aspects of the present disclosure relate to one or more host cells comprising any of the polynucleotides and / or recombinant nucleic acids described herein. For example, prokaryotic cells, e.g., eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, e.g., Escherichia (e.g., E. coli), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), Salmonella spp., Salmonella typhimurium, ... typhimurium), Serratia (e.g., S. marcescans), and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; fungal cells (e.g., S. cerevisiae); insect cells (e.g., S2 cells, etc.); and mammalian cells, such as SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC Any suitable host cell (prokaryotic or eukaryotic) known in the art may be used, including mouse ovarian cancer cells (Human Breast Cancer Cells (HBMC), ATCC CCL 1606), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (Hep G2), Chinese hamster ovary (CHO) cells, e.g., DHFR-CHO cells, and myeloma cell lines, e.g., NS0 and Sp2 / 0. In some embodiments, the host cell is a human cell (e.g., a T cell, a NK cell, etc.) or a non-human primate cell.In some embodiments, the host cell is a cell from a cell line. Examples of suitable host cells or cell lines may include, but are not limited to, 293 cells, HeLa cells, SH-Sy5y cells, Hep G2 cells, CACO-2 cells, A549 cells, L929 cells, 3T3 cells, K562 cells, CHO-K1 cells, MDCK cells, HUVEC cells, Vero cells, N20 cells, COS-7 cells, PSN1 cells, VcaP cells, CHO cells, etc.

[0173] IX. Manufactured Articles or Kits Certain aspects of the present disclosure relate to articles of manufacture or kits that include any of the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the articles of manufacture or kits include a package insert that includes instructions for administering the polynucleotides, recombinant nucleic acids comprising the polynucleotides, gene delivery vehicles comprising the polynucleotides and / or recombinant nucleic acids, medicaments, and / or pharmaceutical compositions or formulations.

[0174] Suitable containers for polynucleotides, recombinant nucleic acids comprising polynucleotides, gene delivery vehicles comprising polynucleotides and / or recombinant nucleic acids, medicaments, and / or pharmaceutical compositions or formulations can include, for example, bottles, vials, bags, tubes, and syringes. Containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container comprises a label on or associated with the container providing instructions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, package inserts, etc.

[0175] X. Enumerated Aspects Embodiment 1: A composition comprising a first recombinant polynucleotide encoding an interleukin (IL)-2 polypeptide and a second recombinant polynucleotide encoding an IL-12 polypeptide.

[0176] Embodiment 2: A pharmaceutical composition comprising the first recombinant polynucleotide and the second recombinant polynucleotide of embodiment 1.

[0177] Embodiment 3: The pharmaceutical composition of embodiment 2, comprising a recombinant nucleic acid comprising said first polynucleotide and a recombinant nucleic acid comprising said second polynucleotide.

[0178] Embodiment 4: The pharmaceutical composition of either embodiment 2 or 3, wherein the recombinant nucleic acid comprising said first polynucleotide and the recombinant nucleic acid comprising said second polynucleotide are the same recombinant nucleic acid.

[0179] Embodiment 5: The pharmaceutical composition of any of embodiments 2 to 4, wherein the recombinant nucleic acid comprising the first polynucleotide and the recombinant nucleic acid comprising the second polynucleotide are different recombinant nucleic acids.

[0180] Embodiment 6: The pharmaceutical composition of any of embodiments 2-5, comprising one or more gene delivery vehicles comprising said first polynucleotide and said second polynucleotide.

[0181] Embodiment 7: The pharmaceutical composition of any of embodiments 2 to 6, wherein said first polynucleotide and said second polynucleotide are contiguous.

[0182] Embodiment 8: The pharmaceutical composition of any of embodiments 2 to 7, wherein said first polynucleotide and said second polynucleotide are not contiguous.

[0183] Embodiment 9: The pharmaceutical composition of any of embodiments 2 to 8, wherein said first polynucleotide and / or said second polynucleotide comprises deoxyribonucleic acid (DNA).

[0184] Embodiment 10: The pharmaceutical composition of any of embodiments 2 to 9, wherein said first polynucleotide or said second polynucleotide comprises deoxyribonucleic acid (DNA).

[0185] Embodiment 11: The pharmaceutical composition of any of embodiments 2 to 10, wherein said first polynucleotide and said second polynucleotide comprise deoxyribonucleic acid (DNA).

[0186] Embodiment 12: The pharmaceutical composition of any of embodiments 2 to 11, wherein said first polynucleotide comprises deoxyribonucleic acid (DNA).

[0187] Embodiment 13: The pharmaceutical composition of any of embodiments 2 to 12, wherein said second polynucleotide comprises deoxyribonucleic acid (DNA).

[0188] Embodiment 14: The pharmaceutical composition of any of embodiments 2 to 13, wherein the DNA is linear DNA or circular DNA.

[0189] Embodiment 15: The pharmaceutical composition of any of embodiments 2 to 14, wherein the DNA is linear DNA.

[0190] Embodiment 16: The pharmaceutical composition of any of embodiments 2 to 15, wherein the DNA is circular DNA.

[0191] Embodiment 17: The pharmaceutical composition of any of embodiments 2 to 16, wherein said first polynucleotide and / or said second polynucleotide comprises ribonucleic acid (RNA), messenger RNA (mRNA), or modified mRNA (mmRNA).

[0192] Embodiment 18: The pharmaceutical composition of any of embodiments 2 to 17, wherein said first polynucleotide comprises RNA.

[0193] Embodiment 19: The pharmaceutical composition of any of embodiments 2 to 18, wherein said first polynucleotide comprises mRNA.

[0194] Embodiment 20: The pharmaceutical composition of any of embodiments 2 to 19, wherein said first polynucleotide comprises mmRNA.

[0195] Embodiment 21: The pharmaceutical composition of any of embodiments 2 to 20, wherein said second polynucleotide comprises RNA.

[0196] Embodiment 22: The pharmaceutical composition of any of embodiments 2 to 21, wherein said second polynucleotide comprises mRNA.

[0197] Embodiment 23: The pharmaceutical composition of any of embodiments 2 to 22, wherein said second polynucleotide comprises an mmRNA.

[0198] Embodiment 24: The pharmaceutical composition of any of embodiments 2 to 23, wherein said first polynucleotide and said second polynucleotide comprise RNA.

[0199] Embodiment 25: The pharmaceutical composition of any of embodiments 2 to 24, wherein said first polynucleotide and said second polynucleotide comprise mRNA.

[0200] Embodiment 26: The pharmaceutical composition of any of embodiments 2 to 25, wherein said first polynucleotide and said second polynucleotide comprise mmRNA.

[0201] Embodiment 27: The pharmaceutical composition of any of embodiments 2 to 26, wherein said mRNA and / or said mmRNA further comprises a 5' untranslated region (UTR), a 3' UTR, a polyadenylation (poly(A)) tail, and / or a 5' cap analog.

[0202] Embodiment 28: The pharmaceutical composition of any of embodiments 2 to 27, wherein said mRNA further comprises a 5' untranslated region (UTR), a 3' UTR, a polyadenylation (poly(A)) tail, and a 5' cap analog.

[0203] Embodiment 29: The pharmaceutical composition of any of embodiments 2 to 28, wherein said mmRNA further comprises a 5' untranslated region (UTR), a 3' UTR, a polyadenylation (poly(A)) tail, and a 5' cap analog.

[0204] Embodiment 30 The pharmaceutical composition of any of embodiments 2 to 29, wherein said 5' cap analog is 7mG(5')ppp(5')NlmpNp.

[0205] Embodiment 31: The pharmaceutical composition of any of embodiments 2 to 30, wherein said mRNA and / or said mmRNA further comprises 1-methylpseudouridine modifications and / or 1-ethylpseudouridine modifications.

[0206] Embodiment 32: The pharmaceutical composition of any of embodiments 2 to 31, wherein said mRNA further comprises a 1-methylpseudouridine modification.

[0207] Embodiment 33: The pharmaceutical composition of any of embodiments 2 to 32, wherein said mRNA further comprises a 1-ethylpseudouridine modification.

[0208] Embodiment 34: The pharmaceutical composition of any of embodiments 1 to 33, wherein said mmRNA further comprises a 1-methylpseudouridine modification.

[0209] Embodiment 35: The pharmaceutical composition of any of embodiments 2 to 34, wherein said mmRNA further comprises a 1-ethylpseudouridine modification.

[0210] Embodiment 36: The pharmaceutical composition of any of embodiments 2 to 35, wherein said mRNA further comprises 1-methylpseudouridine modifications and 1-ethylpseudouridine modifications.

[0211] Embodiment 37: The pharmaceutical composition of any of embodiments 2 to 36, wherein said mmRNA further comprises 1-methylpseudouridine modifications and 1-ethylpseudouridine modifications.

[0212] Embodiment 38: The pharmaceutical composition of any of embodiments 2 to 37, wherein said first polynucleotide and / or said second polynucleotide is codon-optimized.

[0213] Embodiment 39: The pharmaceutical composition of any of embodiments 2 to 38, wherein said first polynucleotide is codon optimized.

[0214] Embodiment 40: The pharmaceutical composition of any of embodiments 2 to 29, wherein said second polynucleotide is codon optimized.

[0215] Embodiment 41: The pharmaceutical composition of any of embodiments 2 to 40, wherein said first polynucleotide and said second polynucleotide are codon-optimized.

[0216] Embodiment 42: The pharmaceutical composition of any of embodiments 2 to 41, wherein said first polynucleotide and / or said second polynucleotide is codon-optimized for human codon usage.

[0217] Embodiment 43 The pharmaceutical composition of any of embodiments 2 to 42, wherein said first polynucleotide is codon-optimized for human codon usage.

[0218] Embodiment 44 The pharmaceutical composition of any of embodiments 2 to 43, wherein said second polynucleotide is codon-optimized for human codon usage.

[0219] Embodiment 45 The pharmaceutical composition of any of embodiments 2 to 44, wherein said first polynucleotide and said second polynucleotide are codon-optimized for human codon usage.

[0220] Embodiment 46: The pharmaceutical composition of any of embodiments 2 to 45, wherein said IL-2 polypeptide is a human IL-2 polypeptide.

[0221] Embodiment 47: The pharmaceutical composition of any of embodiments 2 to 46, wherein said IL-2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1.

[0222] Embodiment 48: The pharmaceutical composition of any of embodiments 2 to 47, wherein said IL-12 polypeptide is a human IL-12 polypeptide.

[0223] Embodiment 49: The pharmaceutical composition of any of embodiments 2 to 48, wherein said IL-12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs:2-4.

[0224] Embodiment 50: The pharmaceutical composition of any of embodiments 2 to 49, wherein said IL-12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0225] Embodiment 51: The pharmaceutical composition of any of embodiments 2 to 50, wherein said IL-12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3.

[0226] Embodiment 52: The pharmaceutical composition of any of embodiments 2 to 51, wherein said IL-12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4.

[0227] Embodiment 53: The pharmaceutical composition of any of embodiments 2 to 52, wherein said one or more gene delivery vehicles are one or more of viral gene delivery vehicles and / or non-viral gene delivery vehicles.

[0228] Embodiment 54: The pharmaceutical composition of any of embodiments 2 to 53, wherein said one or more gene delivery vehicles are one or more viral gene delivery vehicles.

[0229] Embodiment 55: The pharmaceutical composition of any of embodiments 2 to 54, wherein said one or more gene delivery vehicles are one or more of non-viral gene delivery vehicles.

[0230] Embodiment 56: The pharmaceutical composition of any of embodiments 2 to 55, wherein said one or more gene delivery vehicles are one or more of viral and non-viral gene delivery vehicles.

[0231] Embodiment 57: The pharmaceutical composition of any of embodiments 2 to 56, wherein said viral gene delivery vehicle comprises a recombinant viral genome.

[0232] Embodiment 58: The pharmaceutical composition of any of embodiments 2 to 57, wherein said recombinant viral genome comprises said first polynucleotide and / or said second polynucleotide.

[0233] Embodiment 59: The pharmaceutical composition of any of embodiments 2 to 58, wherein said recombinant viral genome comprises said first polynucleotide.

[0234] Embodiment 60: The pharmaceutical composition of any of embodiments 2 to 59, wherein said recombinant viral genome comprises said second polynucleotide.

[0235] Embodiment 61: The pharmaceutical composition of any of embodiments 2 to 60, wherein said recombinant viral genome comprises said first polynucleotide and said second polynucleotide.

[0236] Embodiment 62: The pharmaceutical composition of any of embodiments 2 to 61, wherein said recombinant viral genome is replication-competent or replication-defective.

[0237] Embodiment 63: The pharmaceutical composition of any of embodiments 2 to 62, wherein the recombinant viral genome is replication competent.

[0238] Embodiment 64: The pharmaceutical composition of any of embodiments 2 to 63, wherein said recombinant viral genome is replication-defective.

[0239] Embodiment 65: The pharmaceutical composition of any of embodiments 2 to 64, wherein said recombinant viral genome is a recombinant oncolytic viral genome, or wherein said recombinant viral genome is not a recombinant oncolytic viral genome.

[0240] Embodiment 66: The pharmaceutical composition of any of embodiments 2 to 65, wherein said recombinant viral genome is a recombinant oncolytic viral genome.

[0241] Embodiment 67: The pharmaceutical composition of any of embodiments 2 to 66, wherein said recombinant viral genome is not a recombinant oncolytic viral genome.

[0242] Embodiment 68: The pharmaceutical composition of any of embodiments 2 to 67, wherein said recombinant viral genome is selected from the group consisting of a recombinant adenovirus genome, a recombinant retrovirus genome, a recombinant adeno-associated virus genome, a recombinant herpesvirus genome, a recombinant poxvirus genome, a recombinant bacteriophage genome, a recombinant alphavirus genome, a recombinant picornavirus genome, a recombinant iridovirus genome, a recombinant Newcastle disease virus genome, a recombinant baculovirus genome, a recombinant geminivirus genome, a recombinant caulimovirus genome, and a combination thereof.

[0243] Embodiment 69: The pharmaceutical composition of any of embodiments 2 to 68, wherein said recombinant viral genome is a recombinant adenoviral genome.

[0244] Embodiment 70: The pharmaceutical composition of any of embodiments 2 to 69, wherein said recombinant viral genome is a recombinant retroviral genome.

[0245] Embodiment 71: The pharmaceutical composition of any of embodiments 2 to 70, wherein said recombinant viral genome is a recombinant adeno-associated viral genome.

[0246] Embodiment 72: The pharmaceutical composition of any of embodiments 2 to 71, wherein said recombinant viral genome is a recombinant herpesvirus genome.

[0247] Embodiment 73: The pharmaceutical composition of any of embodiments 2 to 72, wherein said recombinant viral genome is a recombinant poxvirus genome.

[0248] Embodiment 74: The pharmaceutical composition of any of embodiments 2 to 73, wherein said recombinant viral genome is a recombinant bacteriophage genome.

[0249] Embodiment 75: The pharmaceutical composition of any of embodiments 2 to 74, wherein said recombinant viral genome is a recombinant alphavirus genome.

[0250] Embodiment 76: The pharmaceutical composition of any of embodiments 2 to 75, wherein said recombinant viral genome is a recombinant picornavirus genome.

[0251] Embodiment 77: The pharmaceutical composition of any of embodiments 2 to 76, wherein said recombinant viral genome is a recombinant iridovirus genome.

[0252] Embodiment 78: The pharmaceutical composition of any of embodiments 2 to 77, wherein said recombinant viral genome is a recombinant Newcastle Disease Virus genome.

[0253] Embodiment 79: The pharmaceutical composition of any of embodiments 2 to 78, wherein said recombinant viral genome is a recombinant baculovirus genome.

[0254] Embodiment 80: The pharmaceutical composition of any of embodiments 2 to 79, wherein said recombinant viral genome is a recombinant geminivirus genome.

[0255] Embodiment 81: The pharmaceutical composition of any of embodiments 2 to 80, wherein said recombinant viral genome is a recombinant caulimovirus genome.

[0256] Embodiment 82: The pharmaceutical composition of any of embodiments 2 to 81, wherein said recombinant adenovirus genome is selected from the group consisting of a recombinant atadenovirus genome, a recombinant aviadenovirus genome, a recombinant iktadenovirus genome, a recombinant mastadenovirus genome, a recombinant siadenovirus genome, a recombinant testadenovirus genome, and any derivative thereof.

[0257] Embodiment 83: The pharmaceutical composition of any of embodiments 2 to 82, wherein said recombinant retroviral genome is selected from the group consisting of a recombinant alpharetroviral genome, a recombinant betaretroviral genome, a recombinant gammaretroviral genome, a recombinant deltaretroviral genome, a recombinant epsilonretroviral genome, a recombinant lentiviral genome, a recombinant bovine spumavirus genome, a recombinant equine spumavirus genome, a recombinant feline spumavirus genome, a recombinant prosimian spumavirus genome, a recombinant simian spumavirus genome, and any derivative thereof.

[0258] Embodiment 84: The pharmaceutical composition of any of embodiments 2 to 83, wherein said recombinant adeno-associated virus genome is selected from the group consisting of a recombinant adeno-associated dependoparvovirus A genome, a recombinant adeno-associated dependoparvovirus B genome, and any derivative thereof.

[0259] Embodiment 85: The pharmaceutical composition of any of embodiments 2 to 84, wherein said recombinant adeno-associated virus (AAV) genome is a recombinant AAV serotype 1 genome, a recombinant AAV serotype 2 genome, a recombinant AAV serotype 3 genome, a recombinant AAV serotype 4 genome, a recombinant AAV serotype 5 genome, a recombinant AAV serotype 6 genome, a recombinant AAV serotype 7 genome, a recombinant AAV serotype 8 genome, a recombinant AAV serotype 9 genome, a recombinant AAV serotype 10 genome, a recombinant AAV serotype 11 genome, or a combination thereof.

[0260] Embodiment 86: The pharmaceutical composition of any of embodiments 2 to 85, wherein said recombinant herpesvirus genome is selected from the group consisting of a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any derivative thereof.

[0261] Embodiment 87: The pharmaceutical composition of any of embodiments 2 to 86, wherein said recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof.

[0262] Embodiment 88: The pharmaceutical composition of any of embodiments 2 to 87, wherein said recombinant poxvirus genome is selected from the group consisting of a recombinant smallpox virus genome, a recombinant vaccinia virus genome, a recombinant cowpox virus genome, a recombinant empox virus genome, a recombinant orf virus genome, a recombinant pseudocowpox virus genome, a recombinant bovine papular stomatitis virus genome, a recombinant tanapox virus genome, a recombinant yabasa tumor virus genome, a recombinant molluscum contagiosum virus genome, and any derivative thereof.

[0263] Aspect 89: The pharmaceutical composition of any of aspects 2 to 88, wherein said recombinant bacteriophage genome is selected from the group consisting of a recombinant 186 phage genome, a recombinant Escherichia virus Lambda genome, a recombinant Pseudomonas virus Phi6 genome, a recombinant Bacillus virus Φ29 genome, a recombinant Escherichia virus ΦX174 genome, a recombinant bacteriophage ΦCb5 genome, a recombinant Escherichia virus G4 genome, a recombinant Escherichia virus M13 genome, a recombinant Emes virus Zinderi genome, a recombinant N4 phage genome, a recombinant Escherichia virus P1 genome, a recombinant Escherichia virus P2 genome, a recombinant Enterobacteriaceae phage P4 genome, a recombinant R17 phage genome, a recombinant Enterobacteriaceae phage T2 genome, a recombinant Escherichia virus T4 genome, a recombinant Escherichia virus T7 genome, a recombinant bacteriophage T12 genome, and any derivative thereof.

[0264] Aspect 90: The recombinant alphavirus genome is a recombinant Aura virus genome, a recombinant Barmah Forest virus genome, a recombinant Bebaru virus genome, a recombinant Ka-Ingua virus genome, a recombinant Kabaso virus genome, a recombinant Chikungunya virus genome, a recombinant Eastern Equine Encephalitis virus genome, a recombinant Eilat virus genome, a recombinant Everglades virus genome, a recombinant Fort Morgan virus genome, a recombinant Getah virus genome, a recombinant Highland J virus genome, a recombinant Madariaga virus genome, a recombinant Mayaro virus genome, a recombinant Middelburg virus genome, a recombinant Moso das Pedras virus genome, a recombinant Mucambouil virus genome, a recombinant Afghanistan ... 90. The pharmaceutical composition of any of aspects 2-89, wherein the recombinant human encephalitis virus is selected from the group consisting of a recombinant Streptococcus aureus virus genome, ...

[0265] Aspect 91: The recombinant picornavirus genome is a recombinant Aarivirus genome, a recombinant Ailur virus genome, a recombinant Ampivirus genome, a recombinant Anachivirus genome, a recombinant Aphthovirus genome, a recombinant Akuamavirus genome, a recombinant Avihepatovirus genome, a recombinant Abyssivirus genome, a recombinant Bucepivirus genome, a recombinant Bopivirus genome, a recombinant Caecilivirus genome, a recombinant Cardiovirus genome, a recombinant Cosavirus genome, a recombinant Krahelivirus genome, a recombinant Kurohivirus genome, a recombinant Danipivirus genome, a recombinant Dicipivirus genome, a recombinant Diresapivirus, a recombinant Enterovirus genome, a recombinant Erbovirus genome, a recombinant Felippivirus genome, a recombinant Phippivirus genome, a recombinant Gallivirus genome, a recombinant Guruhelivirus genome, a recombinant Gurusoppivirus genome, a recombinant Harkavirus genome, a recombinant Hemipivirus genome, a recombinant Hepatovirus genome, a recombinant Hunnivirus genome, a recombinant Kobuvirus genome, a recombinant Kunsaguivirus genome, a recombinant Limnipivirus genome, a recombinant Ribpivirus genome ... Nom, recombinant Rudpivirus genome, recombinant Malagasyvirus genome, recombinant Marspivirus genome, recombinant Meghrivirus genome, recombinant Miskivirus genome, recombinant Mosavirus genome, recombinant Mupivirus genome, recombinant Miropivirus genome, recombinant Orivirus genome, recombinant Osivirus genome, recombinant Parabovirus genome, recombinant Parechovirus genome, recombinant Pacivirus genome, recombinant Parsleyvirus genome, recombinant Pemapivirus genome, recombinant Poesivirus genome, recombinant Potamipivirus genome, recombinant Pygoseppivirus genome, recombinant Labovirus genome, recombinant Rafivirus genome, recombinant Rajidapivirus genome, recombinant Rohelivirus genome, recombinant Rosavirus genome, recombinant Sacobuvirus genome, recombinant Salivirus genome, recombinant Sapelovirus genome, recombinant Senecavirus genome, recombinant Shambavirus genome, recombinant Sisinivirus genome, recombinant Shimapivirus genome, recombinant Teschovirus genome, recombinant Torchivirus genome, recombinant Tottorivirus genome, recombinant Tremovirus genome, recombinant Tropivirus genomeThe pharmaceutical composition of any of embodiments 2 to 90, wherein the composition is selected from the group consisting of:

[0266] Embodiment 92: The pharmaceutical composition of any of embodiments 2 to 91, wherein said recombinant iridovirus genome is a recombinant Invertebrate iridescent virus 6 (IIV-6) genome or a recombinant Invertebrate iridescent virus 31 (IIV-31) genome.

[0267] Embodiment 93: The pharmaceutical composition of any of embodiments 2 to 92, wherein said recombinant baculovirus genome is selected from the group consisting of a recombinant alphabaculovirus genome, a recombinant betabaculovirus genome, a recombinant deltabaculovirus genome, a recombinant gammabaculovirus genome, and any derivative thereof.

[0268] Embodiment 94: The pharmaceutical composition of any of embodiments 2 to 93, wherein said recombinant geminivirus genome is selected from the group consisting of a recombinant bekurtvirus genome, a recombinant begomovirus genome, a recombinant capravirus genome, a recombinant citrodavirus genome, a recombinant kurtovirus genome, a recombinant elagrovirus genome, a recombinant glabrovirus genome, a recombinant maldovirus genome, a recombinant mastrevirus genome, a recombinant marculirevirus genome, a recombinant opunvirus genome, a recombinant topilevirus genome, a recombinant topokuvirus genome, a recombinant turnkurtvirus genome, and any derivative thereof.

[0269] Aspect 95: The pharmaceutical composition of any of Aspects 2 to 94, wherein said recombinant caulimovirus genome is selected from the group consisting of a recombinant Angelica bushy stunt virus genome, a recombinant Okra mild mottle virus genome, a recombinant Carnation etched ring virus genome, a recombinant Cauliflower mosaic virus genome, a recombinant Dahlia mosaic virus genome, a recombinant Scrophulariaceae mosaic virus genome, a recombinant Horseradish latent virus genome, a recombinant Dead Nettle Leaf Distortion Virus genome, a recombinant Mirabilis mosaic virus genome, a recombinant Soybean Putnam virus genome, a recombinant Strawberry Vein Banding Virus genome, a recombinant Thistle Mottle genome, and any derivative thereof.

[0270] Embodiment 96: The pharmaceutical composition of any of embodiments 2 to 95, wherein said gene delivery vehicle is a non-viral gene delivery vehicle.

[0271] Embodiment 97: The pharmaceutical composition of any of embodiments 2 to 96, wherein said non-viral gene delivery vehicle is a chemical compound, a bacterium, a mammalian cell, or a physical delivery system.

[0272] Embodiment 98: The pharmaceutical composition of any of embodiments 2 to 97, wherein said non-viral gene delivery vehicle is a chemical compound.

[0273] Embodiment 99: The pharmaceutical composition of any of embodiments 2 to 98, wherein said non-viral gene delivery vehicle is a bacterium.

[0274] Embodiment 100: The pharmaceutical composition of any of embodiments 2 to 99, wherein said non-viral gene delivery vehicle is a mammalian cell.

[0275] Embodiment 101: The pharmaceutical composition of any of embodiments 2 to 100, wherein said non-viral gene delivery vehicle is a physical delivery system.

[0276] Embodiment 102: The pharmaceutical composition of any of embodiments 2 to 101, wherein said chemical compound is selected from the group consisting of a polymeric compound, a lipid compound, an inorganic compound, and combinations thereof.

[0277] Embodiment 103: The pharmaceutical composition of any of embodiments 2 to 102, wherein said chemical compound is a polymeric compound.

[0278] Embodiment 104: The pharmaceutical composition of any of embodiments 2 to 103, wherein said chemical compound is a lipid compound.

[0279] Embodiment 105: The pharmaceutical composition of any of embodiments 2 to 104, wherein said chemical compound is an inorganic compound.

[0280] Embodiment 106: The pharmaceutical composition of any of embodiments 2 to 105, wherein said polymeric compound is a natural polymer, a synthetic polymer, a biopolymer, a biodegradable polymer, a cationic polymer, a protein polymer, a polysaccharide polymer, or any combination thereof.

[0281] Aspect 107: The polymer compound is selected from the group consisting of poly-L-lactide (PLA), chitosan, pectin, polyethyleneimine (PEI), poly-L-lysine (PLL), poly 2-N-dimethylaminoethyl methacrylate (PDMAEMA), polyaminoamine, polyaminoester, polyamidoamine, polyvinylimidazole (PVI), polybeta-aminoester (PBAE), gelatin, albumin, chitosan, beta-cyclodextrin, dextran, hyaluronic acid, polyester, alpha-pyrrolidinohexiophenone (PHP), poly 2-aminopropanol, polyisopropyl ... 107. The pharmaceutical composition of any of embodiments 2-106, wherein the polymer is selected from the group consisting of acrylamidoglycolic acid (PAGA), polydelta-valerolactone (PVL), polyphenyl ether (PPE), polybeta-amino-ester (PBAE), polyamine-co-ester (PACE), polylactic-co-glycolic acid (PLGA), polycarbonate, polyurethane, micelles, cationic emulsions, exosomes, Reb blood cell membranes, DNA-protein complexes, plasmid DNA, naked DNA, pBR322, pUC, and any derivatives thereof.

[0282] Embodiment 108: The pharmaceutical composition of any of embodiments 2 to 107, wherein said lipid compound is selected from the group consisting of nanoparticles, lipid nanoparticles, liposomes, cationic liposomes, solid lipid nanoparticles, lipid emulsions, lipidoids, cytofectins, lipid emulsions, surfactants, gemini surfactants, and any derivatives thereof.

[0283] Embodiment 109: The pharmaceutical composition of any of embodiments 2 to 108, wherein the inorganic compound is selected from the group consisting of nanocarriers, DNA nanoparticles, gold nanoparticles, carbon nanotubes, graphene, quantum dots, upconversion nanoparticles, silica nanoparticles, iron oxide, ferritin, and any derivatives thereof.

[0284] Embodiment 110: The pharmaceutical composition of any of embodiments 2 to 109, wherein said physical delivery system is selected from the group consisting of electroporation, a gene gun, a jet gun, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, ballistic DNA injection, sonoporation, photoporation, laser pulses, magnetofection, magnetoporation, magnetic particles, hydroporation, and any combination thereof.

[0285] Embodiment 111: Use of a pharmaceutical composition according to any of embodiments 2 to 110 in the manufacture of a medicament for treating cancer.

[0286] Aspect 112: The cancer is selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, brain stem glioma, brain low-grade glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma, pediatric cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, uterine Endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, heart cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leptomeningeal cancer, lip cancer, liver cancer, lymphoid neoplasms, diffuse large B-cell lymphoma, malignant fibrous histiocytoma, bone Cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous cell neck cancer of occult primary, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, sinus cancer, Parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma, rectal cancer, kidney cancer, renal cell carcinoma, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, cutaneous melanoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer,The use of embodiment 111, wherein the cancer is selected from the group consisting of testicular germ cell tumor, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0287] Embodiment 113: A method of expressing, enhancing, increasing, augmenting and / or supplementing the level of an IL-2 polypeptide and / or an IL-12 polypeptide in one or more cells of a subject, said method comprising administering to said subject an effective amount of the pharmaceutical composition of any of embodiments 2 to 112.

[0288] Embodiment 114: The method of embodiment 113, wherein said one or more cells are one or more cells of the respiratory tract, airway epithelium, and / or lung.

[0289] Embodiment 115: The method of any of embodiments 113 or 114, wherein said one or more cells are one or more cells of the respiratory tract.

[0290] Embodiment 116: The method of any of embodiments 113 to 115, wherein said one or more cells are one or more of an airway epithelium.

[0291] Embodiment 117: The method of any of embodiments 113 to 116, wherein said one or more cells are one or more of the lung.

[0292] Embodiment 118: A method of providing prophylactic, symptomatic, or therapeutic alleviation of one or more signs or symptoms of cancer in a subject in need thereof, said method comprising administering to said subject an effective amount of the pharmaceutical composition of any of embodiments 2 to 112.

[0293] Embodiment 119: A method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of the pharmaceutical composition of any of embodiments 2 to 112.

[0294] Aspect 120: The cancer is selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, brain stem glioma, brain low-grade glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma, childhood cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, uterine Endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, heart cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leptomeningeal cancer, lip cancer, liver cancer, lymphoid neoplasms, diffuse large B-cell lymphoma, malignant fibrous histiocytoma, bone Cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous cell neck cancer of occult primary, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, sinus cancer, Parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma, rectal cancer, kidney cancer, renal cell carcinoma, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, cutaneous melanoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer,120. The method of any of embodiments 118 or 119, wherein the cancer is selected from the group consisting of testicular germ cell tumor, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0295] Embodiment 121: The method of any of embodiments 118 to 120, wherein the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer.

[0296] Embodiment 122: The method of any one of embodiments 118 to 121, wherein the cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma.

[0297] Embodiment 123: The method of any one of embodiments 118 to 122, wherein the cancer is non-small cell lung cancer.

[0298] Embodiment 124: The method of any one of embodiments 113 to 123, wherein the subject is a human.

[0299] Embodiment 125: The method of any of embodiments 113-124, wherein the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epidermally, intradermally, orally, sublingually, bucally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, epichoroidally, intracranially, intrathecally, intraventricularly, intraarticularly, periarticularly, intratumorally, locally, or via inhalation.

[0300] Embodiment 126: The method of any of embodiments 113 to 125, wherein said pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation.

[0301] Embodiment 127: The method of any of embodiments 113 to 126, wherein the pharmaceutical composition is administered intratumorally to the subject.

[0302] Embodiment 128: The method of any of embodiments 113 to 127, wherein the pharmaceutical composition is administered intratracheally to the subject.

[0303] The specification is considered to be sufficient to enable one skilled in the art to practice the disclosure. Various modifications of the disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. [Example]

[0304] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that in light of them, various modifications or changes will be suggested to those skilled in the art, which are to be included within the spirit and scope of the present application and the appended claims.

[0305] Example 1: Construction of modified herpes simplex viruses encoding human IL-12 and IL-2 The following example describes the design of recombinant herpes simplex virus type 1 (HSV-1) that encodes human IL-12 and IL-2 and successfully expresses full-length human IL-12 and IL-2 proteins.

[0306] A recombinant HSV-1 was engineered incorporating expression cassettes for human IL-12 and IL-2 containing heterologous promoters and poly(A) sequences. Viral plaques suspected to contain the human IL-12 and IL-2 cassettes were selected and screened by infection of complementing cell lines to test for human IL-12 and IL-2 protein expression via Western blot analysis (data not shown). A highly expressing clone, designated HSV-IL12 / IL2, was then selected for further in vitro analysis.

[0307] Human embryonic kidney (HEK) 293FT cells were mock-infected with a vehicle control or infected with HSV-IL12 / IL2 at a multiplicity of infection (MOI) of 1 in serum-free cell culture medium. At 24 or 48 hours postinfection, cell pellets were harvested, lysed in RIPA buffer containing protease inhibitors, and protein content was quantified via BCA assay. 30–40 μg of each sample was loaded onto a 4–20% acrylamide gel, and expression of HSV-encoded human proteins was determined via Western blot analysis (data not shown). Recombinant human IL-12 or IL-2 was loaded onto the gel as a positive control. Neither human IL-12 nor IL-2 was detectable in uninfected control cells, but robust expression of human IL-12 (data not shown) and IL-2 (data not shown) was observed following infection with HSV-IL12 / IL2.

[0308] Because IL-12 and IL-2 are naturally secreted proteins, HEK293FT cell culture supernatants were also collected and tested for the presence of human proteins by ELISA. Consistent with the Western blot data, human IL-12 (>3,000 ng / mL; Fig. 1A) and IL-2 (>400 ng / mL; Fig. 1B) were detected in the supernatants of HEK293FT cells infected with HSV-IL12 / IL2 at an MOI of 1, suggesting that the full-length human proteins were properly processed and secreted after expression from the recombinant vectors.

[0309] In summary, the data presented in this example demonstrated that recombinant HSV-1 vectors efficiently infected multiple cell types and were capable of expressing the human IL-12 and IL-2 transgenes encoded within the vectors. Furthermore, the data demonstrated that the exogenous human proteins were subsequently secreted (appropriately) from the infected cells.

[0310] Example 2: In vitro murine IL-12 and IL-2 bioactivity assay The following example describes the design of recombinant herpes simplex virus type 1 (HSV-1) that encodes murine IL-12 or IL-2 and successfully expresses full-length murine IL-12 or IL-2 proteins.

[0311] The purpose of this experiment was, in part, to determine whether murine IL-12 and IL-2 proteins produced from recombinant HSV-1 vectors have similar bioactivity to commercially available recombinant IL-12 and IL-2 proteins. HEK293FT cells were infected with HSV-IL12 or HSV-IL2 at an MOI of 1 for 24 hours in serum-free cell culture medium. Cell culture supernatants were collected, and IL-12 and IL-2 concentrations were determined by ELISA. As shown in Figures 2A-2B, serial dilutions of supernatants from HEK293FT infections with HSV-IL12 / HSV-IL2 were added to murine BALB / c splenocytes (Figure 2A) or HEK-Blue™-IL2 cells (Figure 2B) to determine the bioactivity of vector-derived IL-12 or IL-2, respectively. For splenocyte stimulation, commercially available mouse αCD3 / αCD28-coated beads were washed and added to the cultures to induce stimulation and interferon (IFN)γ secretion. For comparison, commercially available recombinant IL-12 protein (Figure 2A) or IL-2 protein (Figure 2B) was added to some cultures at similar concentrations. Twenty-four hours after stimulation, culture supernatants of mouse splenocytes or HEK-Blue™-IL2 cells were collected and assayed by IFNγ ELISA (Figure 2A) or for secreted fetal alkaline phosphatase (SEAP) activity (Figure 2B).

[0312] As shown in Figure 2A, addition of αCD3 / αCD28-coated beads resulted in the release of IFNγ from mouse splenocytes. In comparison, addition of HSV-IL12-derived viral supernatant to αCD3 / αCD28-stimulated splenocytes induced higher levels of IFNγ secretion compared with αCD3 / αCD28 stimulation alone. Furthermore, HSV-IL12-dependent IFNγ release was comparable to that induced by recombinant IL-12. As shown in Figure 2B, HSV-IL2-dependent SEAP activity in HEK-Blue™ IL-2 reporter cells was comparable to that of recombinant IL-2.

[0313] Taken together, these results suggest that the IL-12 and IL-2 proteins produced from recombinant herpes simplex viruses have similar biological activities to commercially available recombinant IL-12 and IL-2 proteins, respectively.

[0314] Example 3: Intradermal administration and in vivo evaluation of HSV-IL12 / HSV-IL2 in healthy mice Having confirmed the transgene bioactivity of IL-12 and IL-2 in the above experiments, the next step was to test HSV-IL12 / HSV-IL2 in vivo. In this experiment, a single dose (8 × 10 8 IL-12 and / or IL-2 expression was assessed after intradermal administration of 1000 PFU (1000 ng / mL) of HSV-IL12 / HSV-IL2 to C57BL / 6 animals. IL-12 and IL-2 expression was determined by nucleic acid and protein analysis in skin tissue at the indicated time points after vector administration. All procedures performed complied with applicable animal welfare laws and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0315] Nucleic acid analysis of skin tissue after sacrifice showed that vector genomes and transcripts of IL-12 (Figure 3A) and IL-2 (Figure 3C) were detectable for at least 168 hours after administration. Furthermore, skin biopsies were homogenized for protein analysis of IL-12 (Figure 3B) and IL-2 (Figure 3D) by ELISA. Peak cytokine protein levels were observed 8 hours after injection, and these protein levels remained detectable up to 7 days after intradermal (ID) administration of HSV-IL12 / HSV-IL2.

[0316] Another experiment was performed to compare local effector concentrations in the skin and systemic cytokine exposure after ID administration of HSV-IL12 / HSV-IL2 with intravenously administered recombinant IL-12 and IL-2 proteins (Figures 3E-3H). C57BL / 6 mice were treated with ID injection (approximately 10 8Mice were treated with HSV-IL12 / HSV-IL2 via IV administration (through a total PFU) or intravenously with recombinant IL-12 and IL-2 proteins (125 ng and 600 ng, respectively) at mouse doses equivalent to those of human IL-12 and IL-2, whose clinical efficacy and toxicity have been demonstrated in humans. Untreated animals served as negative controls. Serum samples and skin biopsies were collected 5 minutes or 24 hours after treatment for analysis by ELISA. As shown in Figures 3E–3H, compared with intravenous (IV) recombinant protein treatment, minimal systemic cytokine exposure was observed after ID administration of HSV-IL12 / HSV-IL2 (Figures 3E–3F), in addition to robust expression of vector-derived IL-12 and IL-2 observed in skin tissue (Figures 3G–3H). Taken together, these data demonstrate that vector-mediated intradermal delivery of IL-12 and IL-2 minimizes systemic cytokine exposure while enhancing local effector concentrations compared with intravenously administered recombinant proteins at clinically relevant doses.

[0317] Example 4: Intratracheal administration and in vivo evaluation of HSV-IL12 / HSV-IL2 in healthy mice The purpose of this experiment was, in part, to evaluate transgene expression in the lung following intratracheal (IT) administration of HSV-IL12 / HSV-IL2.

[0318] On days 0 and 7 of the experiment, one of two doses (high or medium dose, respectively) was administered. 8 or about 10 7A single dose of HSV-IL12 / HSV-IL2 (total PFU) was administered intratracheally to BALB / c animals. On day 8, 24 h after the final administration, animals were sacrificed. To assess toxicity, body weight was measured weekly, and IL-12 and IL-2 protein expression in lung tissue and bronchoalveolar lavage fluid (BALF) was determined by ELISA. Results from this experiment show no significant differences in body weight between groups (Figure 4A). Regarding effector expression, IL-12 and IL-2 protein levels in BALF and lung tissue were significantly higher in animals treated with HSV-IL12 / HSV-IL2 compared to vehicle controls (Figures 4B-4D). Collectively, these data suggest that intratracheally administered HSV-IL12 / HSV-IL2 is well tolerated and results in detectable, dose-dependent IL-12 and IL-2 expression in the lungs.

[0319] Next, experiments were performed to compare local effector concentrations in the lung and systemic cytokine exposure after IT administration of HSV-IL12 / HSV-IL2 with intravenously administered recombinant IL-12 and IL-2 proteins. BALB / c mice were injected with HSV-IL12 / HSV-IL2 (approximately 10 7Mice were treated intratracheally with 100 mg of recombinant IL-12 and IL-2 (total PFU) or intravenously with mouse doses of recombinant IL-12 and IL-2 proteins (125 ng and 600 ng, respectively) equivalent to human IL-12 and IL-2, whose clinical efficacy and toxicity have been demonstrated in humans. Untreated animals served as negative controls. Serum samples and lungs were collected 5 min or 24 h after treatment for analysis by ELISA (Figures 4F-4I). As shown in Figures 4F-4I, minimal systemic cytokine exposure was observed after administration of HSV-IL12 / HSV-IL2 compared to IV recombinant protein treatment (Figures 4F-4G), in addition to more robust IL-12 and IL-2 expression in lung homogenates (Figures 4H-4I). Taken together, these data demonstrate that vector-mediated IT delivery of IL-12 and IL-2 minimizes systemic cytokine exposure while increasing IL-12 and IL-2 concentrations in the lung compared with recombinant proteins administered intravenously at clinically relevant doses.

[0320] Example 5: Efficacy of HSV-IL12 / HSV-IL2 in an in vivo mouse model of melanoma The purpose of this experiment was, in part, to evaluate the efficacy of intradermal administration of HSV-IL12 / HSV-IL2 treatment in inhibiting B16F10 melanoma tumor growth in the skin.

[0321] The B16F10 model of melanoma following subcutaneous (SC) administration is well established. As shown in Figure 5A, animals received the indicated vectors via intratumoral injection 7, 14, and 21 days after SC administration of B16F10 cells (approximately 10 8PFU). As shown in Figure 5B, the greatest survival benefit was observed in animals treated with the combination HSV-IL12 / HSV-IL2 compared with the groups treated with a single vector or vehicle control. Furthermore, as shown in Figures 5C–5E, this survival benefit (Figure 5E) was enhanced when mice received a maintenance dose of HSV-IL12 / HSV-IL2 on day 42 after SC administration of B16F10 cells (Figure 5C). Notably, tumor area (Figure 5D) was significantly reduced in the HSV-IL12 / HSV-IL2-treated group compared with the vehicle control. These data indicate that intratumoral injection of HSV-IL12 / HSV-IL2 improves the survival of B16F10 melanoma-bearing mice.

[0322] Next, experiments were performed to determine whether HSV-IL12 / HSV-IL2 treatment of primary B16F10 melanoma resulted in the control of tumor recurrence (Figures 5F-5I). As shown in Figure 5F, animals were administered the indicated drugs via intratumoral injection on days 7, 14, and 21 after SC administration of B16F10 cells (approximately 10 8 PFU) (initial phase). After this initial phase, surviving mice received an additional SC injection of B16F10 cells 55 days after the initial B16F10 tumor challenge to mimic tumor recurrence (rechallenge phase). As shown in Figures 5G–5I, intratumoral administration of HSV-IL12 / HSV-IL2 resulted in significant tumor area reduction compared with vehicle control or untreated tumors in both the initial phase (Figure 5G) and the rechallenge phase (Figure 5H), respectively. Notably, an HSV-IL12 / HSV-IL2-dependent survival benefit was observed in both the initial and rechallenge phases (Figure 5I). Therefore, this experiment suggests that HSV-IL12 / HSV-IL2 treatment of primary B16F10 melanoma results in the control of rechallenge melanoma tumors, suggesting that vector-derived IL-12 and IL-2 treatment induces a sustained antitumor memory response.

[0323] Next, experiments were performed to determine whether HSV-IL12 / HSV-IL2 treatment of primary B16F10 melanoma resulted in an abscopal effect on secondary B16F10 tumors (Figures 5J-5S). As shown in Figure 5J, 7, 14, and 21 days after SC administration of B16F10 cells (primary tumor), animals were administered the indicated vectors via intratumoral injection (approximately 10 8 Furthermore, secondary tumor inoculation (1.0 × 10 5 B16F10 cells) were transfected on either day 0, day 4, or day 10 after the initiation of the experiment (Figure 5J). Animals treated with HSV-IL12 / HSV-IL2 showed reduced primary tumor growth, regardless of secondary tumor administration (Figures 5K, 5N, and 5Q). Notably, an abscopal effect of HSV-IL12 / HSV-IL2 treatment was observed in groups receiving secondary tumors on either day 4 (Figure 5O) or day 10 (Figure 5R) after the initiation of the experiment; no effect was demonstrated in day 0 secondary tumor recipients (Figure 5L). Overall, a survival benefit was observed in mice receiving HSV-IL12 / HSV-IL2 treatment compared with vehicle controls in all groups (Figures 5M, 5P, and 5S). Collectively, these data suggest that HSV-IL12 / HSV-IL2 treatment of primary melanoma tumors results in an abscopal effect on secondary melanoma tumors.

[0324] Example 6: Efficacy of HSV-IL12 / HSV-IL2 in an in vivo mouse model of osteosarcoma The purpose of this experiment was, in part, to evaluate the efficacy of intratracheal administration of HSV-IL12 / HSV-IL2 therapy in inhibiting metastatic osteosarcoma lung tumor growth.

[0325] The K7M2 BALB / c model of osteosarcoma lung metastasis after intravenous (IV) administration is well established. As shown in Figure 6A, 14, 21, and 28 days after IV administration of K7M2 cells, animals were administered the indicated vectors via IT administration (approximately 10 7(Total PFU). Animals receiving the cytokine-expressing vectors had higher body weights compared with vehicle and vector control recipients (Figure 6B). Regarding survival, a survival benefit was observed with single-agent treatment with HSV-IL12 or HSV-IL2. Unexpectedly, combined treatment with HSV-encoded IL-12 and IL-2 resulted in a synergistic effect, with this group of animals having a 100% survival rate over 100 days after tumor injection (Figure 6C). Evidence of tumor regression in the lungs was also observed histologically (Figure 6D). Collectively, these data demonstrate that intratracheally administered combination HSV-IL12 / HSV-IL2 enhances tumor regression and survival compared with control or single-vector treatment in the K7M2 osteosarcoma lung metastasis model.

[0326] Further experiments were then performed to address whether IT administration of HSV-IL12 / HSV-IL2 could minimize initial lung tumor growth and delay tumor recurrence without additional therapeutic intervention. As shown in Figure 6E, animals were administered the indicated vectors (approximately 10 μg / mL) 14, 21, and 28 days after IV administration of K7M2 cells. 7 (total PFU) (Phase 1). On day 75, surviving animals received a second inoculation of K7M2 cells without additional therapeutic intervention (rechallenge phase). As shown in Figures 6F–6G, animal weights were higher in animals treated with HSV-IL12 / HSV-IL2 compared with controls in both Phase 1 and Phase 2. Notably, the survival benefit observed in the HSV-IL12 / HSV-IL2 group was maintained in the rechallenge phase (Figure 6H). Collectively, these data indicate that IT administration of HSV-IL12 / HSV-IL2 not only minimizes initial K7M2 lung tumor growth but also delays tumor recurrence without additional therapeutic intervention.

[0327] Example 7: Recombinant nucleic acids as linear DNA encoding IL-12 and IL-2 A first recombinant nucleic acid is designed as a linear DNA comprising a recombinant polynucleotide encoding human IL-12. A second recombinant nucleic acid is designed as a linear DNA comprising a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests are not available at the time of filing.

[0328] The recombinant nucleic acids are designed as linear DNAs containing a recombinant polynucleotide encoding human IL-12 and a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for their effectiveness as cancer therapeutics. These results are not available at the time of filing.

[0329] Example 8: Recombinant nucleic acids as circular DNA encoding IL-12 and IL-2 The first recombinant nucleic acid is designed as a circular DNA containing a recombinant polynucleotide encoding human IL-12. The second recombinant nucleic acid is designed as a circular DNA containing a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests are not available at the time of filing.

[0330] The recombinant nucleic acids are designed as circular DNAs containing a recombinant polynucleotide encoding human IL-12 and a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for their effectiveness as cancer therapeutics. These results are not available at the time of filing.

[0331] Example 9: Recombinant nucleic acids as closed-end DNA encoding IL-12 and IL-2 A first recombinant nucleic acid is designed as a closed-end DNA containing a recombinant polynucleotide encoding human IL-12. A second recombinant nucleic acid is designed as a closed-end DNA containing a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests are not available at the time of filing.

[0332] The recombinant nucleic acids are designed as closed-end DNAs containing a recombinant polynucleotide encoding human IL-12 and a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for their effectiveness as cancer therapeutics. These results are not available at the time of filing.

[0333] Example 10: Recombinant nucleic acids as RNA encoding IL-12 and IL-2 The first recombinant nucleic acid is designed as an RNA comprising a recombinant polynucleotide encoding human IL-12. The second recombinant nucleic acid is designed as an RNA comprising a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests are not available at the time of filing.

[0334] The recombinant nucleic acids are designed as RNAs containing a recombinant polynucleotide encoding human IL-12 and a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for their effectiveness as cancer therapeutics. These results are not available at the time of filing.

[0335] Example 11: Recombinant nucleic acids as mRNA encoding IL-12 and IL-2 The first recombinant nucleic acid is designed as an mRNA containing a recombinant polynucleotide encoding human IL-12. The second recombinant nucleic acid is designed as an mRNA containing a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests are not available at the time of filing.

[0336] The recombinant nucleic acids are designed as mRNAs containing a recombinant polynucleotide encoding human IL-12 and a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for their effectiveness as cancer therapeutics. These results are not available at the time of filing.

[0337] Example 12: Recombinant nucleic acids as mmRNA encoding IL-12 and IL-2 The first recombinant nucleic acid is designed as an mmRNA containing a recombinant polynucleotide encoding human IL-12. The second recombinant nucleic acid is designed as an mmRNA containing a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests are not available at the time of filing.

[0338] The recombinant nucleic acids are designed as mmRNAs containing a recombinant polynucleotide encoding human IL-12 and a recombinant polynucleotide encoding human IL-2. The recombinant nucleic acids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for their effectiveness as cancer therapeutics. These results are not available at the time of filing.

[0339] Example 13: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant adenoviral genome The recombinant nucleic acids described in any one or more of Examples 7-12 will be engineered into a recombinant adenoviral genome, optionally in an adenoviral gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0340] Example 14: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant retroviral genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered into recombinant retroviral genomes, optionally in retroviral gene delivery vehicles, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0341] Example 15: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant adeno-associated virus genome The recombinant nucleic acids described in any one or more of Examples 7-12 will be engineered into a recombinant adeno-associated viral genome, optionally in an adeno-associated viral gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0342] Example 16: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant poxvirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 will be engineered into a recombinant poxvirus genome, optionally in a poxvirus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0343] Example 17: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant bacteriophage genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered in recombinant bacteriophage genomes, optionally in bacteriophage gene delivery vehicles, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0344] Example 18: Recombinant nucleic acids described in any one or more of Examples 7-12 in a recombinant alphavirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 will be engineered into recombinant alphavirus genomes, optionally in alphavirus gene delivery vehicles, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0345] Example 19: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant picornavirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 will be engineered in a recombinant picornavirus genome, optionally in a picornavirus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0346] Example 20: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant iridovirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered in a recombinant iridovirus genome, and optionally in an iridovirus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0347] Example 21: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant Newcastle disease virus genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered into a recombinant Newcastle Disease Virus genome, and optionally in a Newcastle Disease Virus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0348] Example 22: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant baculovirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered in a recombinant baculovirus genome, optionally in a baculovirus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0349] Example 23: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant geminivirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered into recombinant geminiviral genomes, optionally in geminiviral gene delivery vehicles, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0350] Example 24: Recombinant nucleic acids described in any one or more of Examples 7-12 in a recombinant caulimovirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 will be engineered in a recombinant caulimovirus genome, and optionally in a caulimovirus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0351] Example 25: Recombinant nucleic acids described in any one or more of Examples 7 to 12 in a recombinant anellovirus genome The recombinant nucleic acids described in any one or more of Examples 7-12 are engineered into a recombinant anellovirus genome, and optionally in an anellovirus gene delivery vehicle, and tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0352] Example 26: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more chemical compounds, bacteria, mammalian cells, and / or physical delivery systems A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more chemical compounds, bacteria, mammalian cells, and / or physical delivery systems as non-viral gene delivery vehicles. The one or more chemical compounds, bacteria, mammalian cells, and / or physical delivery systems are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. Results of these tests are not available at the time of filing.

[0353] Example 27: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more polymeric, lipidic, and / or inorganic compounds A recombinant nucleic acid as described in any one or more of Examples 7-12 is designed in one or more polymeric, lipidic, and / or inorganic compounds as a non-viral gene delivery vehicle. The one or more polymeric, lipidic, and / or inorganic compounds are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0354] Example 28: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more natural polymers, synthetic polymers, biopolymers, biodegradable polymers, cationic polymers, protein polymers, and / or polysaccharide polymers The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more natural polymers, synthetic polymers, biopolymers, biodegradable polymers, cationic polymers, protein polymers, and / or polysaccharide polymers as non-viral gene delivery vehicles. The one or more natural polymers, synthetic polymers, biopolymers, biodegradable polymers, cationic polymers, protein polymers, and / or polysaccharide polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0355] Example 29: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more natural polymers A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more natural polymers as a non-viral gene delivery vehicle. The one or more natural polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0356] Example 30: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more synthetic polymers A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more synthetic polymers as a non-viral gene delivery vehicle. The one or more synthetic polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0357] Example 31: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more biopolymers The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more biopolymers as non-viral gene delivery vehicles. The one or more biopolymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0358] Example 32: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more biodegradable polymers A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more biodegradable polymers as non-viral gene delivery vehicles. The one or more biodegradable polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0359] Example 33: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more cationic polymers A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more cationic polymers as non-viral gene delivery vehicles. The one or more cationic polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0360] Example 34: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more protein polymers The recombinant nucleic acids described in any one or more of Examples 7-12 are designed into one or more protein polymers as non-viral gene delivery vehicles. The one or more protein polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0361] Example 35: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more polysaccharide polymers The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more polysaccharide polymers as non-viral gene delivery vehicles. The one or more polysaccharide polymers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0362] Example 36: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more nanoparticles, lipid nanoparticles, liposomes, cationic liposomes, solid lipid nanoparticles, lipid emulsions, lipidoids, cytofectins, surfactants, and / or gemini surfactants A recombinant nucleic acid as described in any one or more of Examples 7-12 is designed in one or more nanoparticles, lipid nanoparticles, liposomes, cationic liposomes, solid lipid nanoparticles, lipid emulsions, lipidoids, cytofectins, surfactants, and / or gemini surfactants as non-viral gene delivery vehicles. The one or more nanoparticles, lipid nanoparticles, liposomes, cationic liposomes, solid lipid nanoparticles, lipid emulsions, lipidoids, cytofectins, surfactants, and / or gemini surfactants are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. Results are not available at the time of filing.

[0363] Example 37: Recombinant nucleic acid of any one or more of Examples 7-12 in one or more nanoparticles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed into one or more nanoparticles as a non-viral gene delivery vehicle. The one or more nanoparticles are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0364] Example 38: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more lipid nanoparticles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more lipid nanoparticles as a non-viral gene delivery vehicle. The one or more lipid nanoparticles are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0365] Example 39: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more liposomes A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more liposomes as a non-viral gene delivery vehicle. The one or more liposomes are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0366] Example 40: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more cationic liposomes A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more cationic liposomes as a non-viral gene delivery vehicle. The one or more cationic liposomes are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0367] Example 41: Recombinant nucleic acid described in any one or more of Examples 7-12 in one or more solid lipid nanoparticles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more solid lipid nanoparticles as a non-viral gene delivery vehicle. The one or more solid lipid nanoparticles are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0368] Example 42: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more lipid emulsions The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more lipid emulsions as non-viral gene delivery vehicles. The one or more lipid emulsions are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0369] Example 43: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more lipidoids The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more lipidoids as non-viral gene delivery vehicles. The one or more lipidoids are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0370] Example 44: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more cytofectins The recombinant nucleic acids described in any one or more of Examples 7-12 are designed into one or more cytofectins as non-viral gene delivery vehicles. The one or more cytofectins are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0371] Example 45: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more surfactants A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more surfactants as a non-viral gene delivery vehicle. The one or more surfactants are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0372] Example 46: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more gemini surfactants The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more gemini surfactants as non-viral gene delivery vehicles. The one or more gemini surfactants are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0373] Example 47: Recombinant nucleic acids of any one or more of Examples 7-12 in one or more nanocarriers, DNA nanoparticles, gold nanoparticles, carbon nanotubes, graphene, quantum dots, upconversion nanoparticles, silica nanoparticles, iron oxide, and / or ferritin The recombinant nucleic acids described in any one or more of Examples 7-12 are designed in one or more nanocarriers, DNA nanoparticles, gold nanoparticles, carbon nanotubes, graphene, quantum dots, upconversion nanoparticles, silica nanoparticles, iron oxide, and / or ferritin as non-viral gene delivery vehicles. The one or more nanocarriers, DNA nanoparticles, gold nanoparticles, carbon nanotubes, graphene, quantum dots, upconversion nanoparticles, silica nanoparticles, iron oxide, and / or ferritin are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. Results of these tests were not available at the time of filing.

[0374] Example 48: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more nanocarriers The recombinant nucleic acids described in any one or more of Examples 7-12 are designed into one or more nanocarriers as non-viral gene delivery vehicles. The one or more nanocarriers are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0375] Example 49: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more DNA nucleic acid clones The recombinant nucleic acids described in any one or more of Examples 7-12 are designed into one or more DNA nucleic acid clones as non-viral gene delivery vehicles. The one or more DNA nucleic acid clones are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0376] Example 50: Recombinant nucleic acid of any one or more of Examples 7-12 in one or more gold nanoparticles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed into one or more gold nanoparticles as a non-viral gene delivery vehicle. The one or more gold nanoparticles are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0377] Example 51: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more carbon nanotubes A recombinant nucleic acid described in any one or more of Examples 7-12 is designed into one or more carbon nanotubes as a non-viral gene delivery vehicle. The one or more carbon nanotubes are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0378] Example 52: Recombinant nucleic acids of any one or more of Examples 7-12 in one or more graphenes A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more graphenes as a non-viral gene delivery vehicle. The one or more graphenes are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0379] Example 53: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more quantum dots The recombinant nucleic acids described in any one or more of Examples 7-12 are designed into one or more quantum dots as non-viral gene delivery vehicles. The one or more quantum dots are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0380] Example 54: Recombinant nucleic acid described in any one or more of Examples 7-12 in one or more upconversion nanoparticles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed into one or more upconversion nanoparticles as non-viral gene delivery vehicles. The one or more upconversion nanoparticles are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0381] Example 55: Recombinant nucleic acid described in any one or more of Examples 7-12 in one or more silica nanoparticles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more silica nanoparticles as a non-viral gene delivery vehicle. The one or more silica nanoparticles are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0382] Example 56: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more iron oxides A recombinant nucleic acid described in any one or more of Examples 7-12 is designed in one or more iron oxides as a non-viral gene delivery vehicle. The one or more iron oxides are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0383] Example 57: Recombinant nucleic acids described in any one or more of Examples 7-12 in one or more ferritins The recombinant nucleic acids described in any one or more of Examples 7-12 are designed into one or more ferritins as non-viral gene delivery vehicles. The one or more ferritins are tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as cancer therapeutics. These results are not available at the time of filing.

[0384] Example 58: Recombinant nucleic acids of any one or more of Examples 7-12 delivered by one or more of electroporation, gene gun, jet gun, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, ballistic DNA injection, sonoporation, photoporation, laser pulse, magnetofection, magnetoporation, magnetic particles, and / or hydroporation. The recombinant nucleic acids described in any one or more of Examples 7-12 are designed to be delivered by one or more of electroporation, gene guns, jet guns, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, ballistic DNA injection, sonoporation, photoporation, laser pulses, magnetofection, magnetoporation, magnetic particles, and / or hydroporation as non-viral gene delivery vehicles. Delivery by one or more of electroporation, gene guns, jet guns, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, ballistic DNA injection, sonoporation, photoporation, laser pulses, magnetofection, magnetoporation, magnetic particles, and / or hydroporation is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as cancer therapeutics. Results are not available at the time of filing.

[0385] Example 59: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by electroporation A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by electroporation as a non-viral gene delivery vehicle. The electroporation delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0386] Example 60: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by gene gun A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by a gene gun as a non-viral gene delivery vehicle. The gene gun delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0387] Example 61: Recombinant nucleic acid described in any one or more of Examples 7-12 delivered by jet gun A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by jet gun as a non-viral gene delivery vehicle. The jet gun delivery will be tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0388] Example 62: Recombinant nucleic acid described in any one or more of Examples 7-12 delivered by ultrasound A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by ultrasound as a non-viral gene delivery vehicle. The ultrasound delivery will be tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0389] Example 63: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by nucleofection A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by nucleofection as a non-viral gene delivery vehicle. The nucleofection-mediated delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0390] Example 64: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by hydrodynamic gene delivery A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by hydrodynamic gene delivery as a non-viral gene delivery vehicle. The hydrodynamic gene delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0391] Example 65: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by needle injection A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by needle injection as a non-viral gene delivery vehicle. The needle injection delivery will be tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and for efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0392] Example 66: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by microinjection A recombinant nucleic acid as described in any one or more of Examples 7-12 is designed to be delivered by microinjection as a non-viral gene delivery vehicle. The microinjection delivery will be tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0393] Example 67: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by ballistic DNA injection A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by ballistic DNA injection as a non-viral gene delivery vehicle. The ballistic DNA injection delivery will be tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0394] Example 68: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by sonoporation A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by sonoporation as a non-viral gene delivery vehicle. The sonoporation delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0395] Example 69: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by photoporation A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by photoporation as a non-viral gene delivery vehicle. The photoporation delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0396] Example 70: Recombinant nucleic acid described in any one or more of Examples 7-12 delivered by laser pulse A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by laser pulse as a non-viral gene delivery vehicle. The laser pulse delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0397] Example 71: Recombinant nucleic acids described in any one or more of Examples 7 to 12 delivered by magnetofection A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by magnetofection as a non-viral gene delivery vehicle. The magnetofection-mediated delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0398] Example 72: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by magnetoporation A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by magnetoporation as a non-viral gene delivery vehicle. The magnetoporation delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0399] Example 73: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by magnetic particles A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by magnetic particles as a non-viral gene delivery vehicle. The magnetic particle delivery is tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

[0400] Example 74: Recombinant nucleic acids described in any one or more of Examples 7-12 delivered by hydroporation A recombinant nucleic acid described in any one or more of Examples 7-12 is designed to be delivered by hydroporation as a non-viral gene delivery vehicle. The hydroporation delivery will be tested for in vitro and / or in vivo expression of IL-12 and / or IL-2, and efficacy as a cancer therapeutic. These results are not available at the time of filing.

Claims

1. A composition comprising a first recombinant polynucleotide encoding an interleukin (IL)-2 polypeptide and a second recombinant polynucleotide encoding an IL-12 polypeptide.

2. A pharmaceutical composition comprising the first recombinant polynucleotide and the second recombinant polynucleotide of claim 1.

3. 3. The pharmaceutical composition of claim 2, comprising a recombinant nucleic acid comprising the first polynucleotide and a recombinant nucleic acid comprising the second polynucleotide.

4. 4. The pharmaceutical composition of claim 3, wherein the recombinant nucleic acid comprising the first polynucleotide and the recombinant nucleic acid comprising the second polynucleotide are the same recombinant nucleic acid.

5. 4. The pharmaceutical composition of claim 3, wherein the recombinant nucleic acid comprising the first polynucleotide and the recombinant nucleic acid comprising the second polynucleotide are different recombinant nucleic acids.

6. 6. The pharmaceutical composition of any one of claims 2 to 5, comprising one or more gene delivery vehicles comprising said first polynucleotide and said second polynucleotide.

7. 7. The pharmaceutical composition of any one of claims 2 to 6, wherein the first polynucleotide and the second polynucleotide are contiguous.

8. 7. The pharmaceutical composition of any one of claims 2 to 6, wherein the first polynucleotide and the second polynucleotide are not contiguous.

9. 9. The pharmaceutical composition of any one of claims 2 to 8, wherein the first polynucleotide and / or the second polynucleotide comprises deoxyribonucleic acid (DNA).

10. The pharmaceutical composition of any one of claims 2 to 9, wherein the DNA is linear DNA, circular DNA, or close-ended DNA (ceDNA).

11. 9. The pharmaceutical composition of any one of claims 2 to 8, wherein the first polynucleotide and / or the second polynucleotide comprises ribonucleic acid (RNA), messenger RNA (mRNA), or modified mRNA (mmRNA).

12. The pharmaceutical composition of claim 11, wherein the mRNA and / or the mmRNA further comprises a 5' untranslated region (UTR), a 3' UTR, a polyadenylation (poly(A)) tail, and / or a 5' cap analog.

13. 13. The pharmaceutical composition of claim 12, wherein the 5' cap analog is 7mG(5')ppp(5')NlmpNp.

14. The pharmaceutical composition of any one of claims 11 to 13, wherein the mRNA and / or the mmRNA further comprises 1-methylpseudouridine modifications and / or 1-ethylpseudouridine modifications.

15. 15. The pharmaceutical composition of any one of claims 2 to 14, wherein said first polynucleotide and / or said second polynucleotide is codon-optimized.

16. 16. The pharmaceutical composition of any one of claims 2 to 15, wherein said first polynucleotide and / or said second polynucleotide is codon-optimized for human codon usage.

17. The pharmaceutical composition of any one of claims 2 to 16, wherein the IL-2 polypeptide is a human IL-2 polypeptide.

18. 18. The pharmaceutical composition of any one of claims 2 to 17, wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

1.

19. The pharmaceutical composition of any one of claims 2 to 18, wherein the IL-12 polypeptide is a human IL-12 polypeptide.

20. 20. The pharmaceutical composition of any one of claims 2-19, wherein the IL-12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs:2-4.

21. 21. The pharmaceutical composition of any one of claims 6 to 20, wherein said one or more gene delivery vehicles are one or more viral gene delivery vehicles and / or one or more non-viral gene delivery vehicles.

22. 22. The pharmaceutical composition of claim 21, wherein the one or more viral gene delivery vehicles comprise a recombinant viral genome.

23. 23. The pharmaceutical composition of claim 22, wherein the recombinant viral genome comprises the first polynucleotide and / or the second polynucleotide.

24. 24. The pharmaceutical composition of claim 22 or claim 23, wherein the recombinant viral genome is replication-competent or replication-defective.

25. The pharmaceutical composition of any one of claims 22 to 24, wherein the recombinant viral genome is a recombinant oncolytic viral genome or the recombinant viral genome is not a recombinant oncolytic viral genome.

26. 26. The pharmaceutical composition of any one of claims 22 to 25, wherein the recombinant viral genome is selected from the group consisting of a recombinant adenovirus genome, a recombinant retrovirus genome, a recombinant adeno-associated virus genome, a recombinant herpesvirus genome, a recombinant poxvirus genome, a recombinant bacteriophage genome, a recombinant alphavirus genome, a recombinant picornavirus genome, a recombinant iridovirus genome, a recombinant Newcastle disease virus genome, a recombinant baculovirus genome, a recombinant geminivirus genome, a recombinant caulimovirus genome, a recombinant anellovirus genome, and combinations thereof.

27. 27. The pharmaceutical composition of claim 26, wherein the recombinant adenovirus genome is selected from the group consisting of a recombinant atadenovirus genome, a recombinant aviadenovirus genome, a recombinant ichtadenovirus genome, a recombinant mastadenovirus genome, a recombinant siadenovirus genome, a recombinant testadenovirus genome, and any derivative thereof.

28. 27. The pharmaceutical composition of claim 26, wherein the recombinant retroviral genome is selected from the group consisting of a recombinant Alpharetrovirus genome, a recombinant Betaretrovirus genome, a recombinant Gammaretrovirus genome, a recombinant Deltaretrovirus genome, a recombinant Epsilonretrovirus genome, a recombinant Lentivirus genome, a recombinant Bovispumavirus genome, a recombinant Equispumavirus genome, a recombinant Felispumavirus genome, a recombinant Prosimiispumavirus genome, a recombinant Simiispumavirus genome, and any derivative thereof.

29. 27. The pharmaceutical composition of claim 26, wherein the recombinant adeno-associated virus genome is selected from the group consisting of a recombinant adeno-associated dependoparvovirus A genome, a recombinant adeno-associated dependoparvovirus B genome, and any derivative thereof.

30. 30. The pharmaceutical composition of claim 29, wherein the recombinant adeno-associated virus (AAV) genome is a recombinant AAV serotype 1 genome, a recombinant AAV serotype 2 genome, a recombinant AAV serotype 3 genome, a recombinant AAV serotype 4 genome, a recombinant AAV serotype 5 genome, a recombinant AAV serotype 6 genome, a recombinant AAV serotype 7 genome, a recombinant AAV serotype 8 genome, a recombinant AAV serotype 9 genome, a recombinant AAV serotype 10 genome, a recombinant AAV serotype 11 genome, or a combination thereof.

31. 27. The pharmaceutical composition of claim 26, wherein the recombinant herpesvirus genome is selected from the group consisting of a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any derivative thereof.

32. 32. The pharmaceutical composition of claim 31, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof.

33. 27. The pharmaceutical composition of claim 26, wherein the recombinant poxvirus genome is selected from the group consisting of a recombinant smallpox virus genome, a recombinant vaccinia virus genome, a recombinant cowpox virus genome, a recombinant empox virus genome, a recombinant orf virus genome, a recombinant pseudocowpox virus genome, a recombinant bovine papular stomatitis virus genome, a recombinant tanapox virus genome, a recombinant yabasa tumor virus genome, a recombinant molluscum contagiosum virus genome, and any derivative thereof.

34. The recombinant bacteriophage genome is selected from the group consisting of a recombinant 186 phage genome, a recombinant Escherichia virus Lambda genome, a recombinant Pseudomonas virus Phi6 genome, a recombinant Bacillus virus Φ29 genome, a recombinant Escherichia virus ΦX174 genome, a recombinant bacteriophage ΦCb5 genome, a recombinant Escherichia virus G4 genome, a recombinant Escherichia virus M13 genome, a recombinant Emesvirus zinderi genome, a recombinant N4 phage genome, a recombinant Escherichia virus P1 genome, a recombinant Escherichia virus P2 genome, a recombinant Enterobacteriaceae phage genome, a recombinant Escherichia virus P4 genome, a recombinant Escherichia virus P2 genome, a recombinant Enterobacteriaceae phage genome, a recombinant Escherichia virus P1 ...2 genome, a recombinant Escherichia virus P1 genome, a recombinant Escherichia virus P2 genome, a recombinant Escherichia virus P2 genome, a recombinant Escherichia virus P 27. The pharmaceutical composition of claim 26, wherein the recombinant bacteriophage T4 genome, the recombinant bacteriophage T4 genome, the recombinant bacteriophage T7 genome, the recombinant bacteriophage T12 genome, the recombinant bacteriophage T4 ...

35. The recombinant alphavirus genome is selected from the group consisting of a recombinant Aura virus genome, a recombinant Barmah Forest virus genome, a recombinant Bebaru virus genome, a recombinant Caaingua virus genome, a recombinant Cabassou virus genome, a recombinant Chikungunya virus genome, a recombinant Eastern Equine Encephalitis virus genome, a recombinant Eilat virus genome, a recombinant Everglades virus genome, a recombinant Fort Morgan virus genome, a recombinant Getah virus genome, a recombinant Highlands J virus genome, a recombinant Madariaga virus genome, a recombinant Mayaro virus genome, a recombinant Middelburg virus genome, a recombinant Moso das Pedras virus genome, a recombinant E. coli ...

27. The pharmaceutical composition of claim 26, wherein the recombinant human encephalitis virus is selected from the group consisting of a recombinant Pedras virus genome, a recombinant Mucambo virus genome, a recombinant Ndumu virus genome, a recombinant O'nyong'nyong virus genome, a recombinant Pixuna virus genome, a recombinant Rio Negro virus genome, a recombinant Ross River virus genome, a recombinant salmon pancreatic disease virus genome, a recombinant Semliki Forest virus genome, a recombinant Sindbis virus genome, a recombinant southern elephant seal virus genome, a recombinant Tonate virus genome, a recombinant Trocara virus genome, a recombinant Una virus genome, a recombinant Venezuelan equine encephalitis virus genome, a recombinant Western equine encephalitis virus genome, a recombinant Whataroa virus genome, and any derivative thereof.

36. The recombinant picornavirus genome is selected from the group consisting of a recombinant Aalivirus genome, a recombinant Ailurivirus genome, a recombinant Ampivirus genome, a recombinant Anativirus genome, a recombinant Aphthovirus genome, a recombinant Aquamavirus genome, a recombinant Avihepatovirus genome, a recombinant Avisivirus genome, a recombinant Boosepivirus genome, a recombinant Bopivirus genome, a recombinant Caecilivirus genome, a recombinant Cardiovirus genome, a recombinant Cosavirus genome, a recombinant Crahelivirus genome, a recombinant Crohivirus genome, a recombinant Danipivirus genome, a recombinant Dicipivirus genome, a recombinant Diresapi ... virus genome, recombinant Enterovirus genome, recombinant Erbovirus genome, recombinant Felipivirus genome, recombinant Fipivirus genome, recombinant Gallivirus genome, recombinant Gruhelivirus genome, recombinant Grusopivirus genome, recombinant Harkavirus genome, recombinant Hemipivirus genome, recombinant Hepatovirus genome, recombinant Hunnivirus genome, recombinant Kobuvirus genome, recombinant Kunsagivirus genome, recombinant Limnipivirus genome, recombinant Livupivirus genome, recombinant Ludopivirus genome, recombinant Malagasivirus genome, recombinant Marsupivirus genome,Recombinant Megrivirus genome, Recombinant Mischivirus genome, Recombinant Mosavirus genome, Recombinant Mupivirus genome, Recombinant Myrropivirus genome, Recombinant Orivirus genome, Recombinant Oscivirus genome, Recombinant Parabovirus genome, Recombinant Parechovirus genome, Recombinant Pasivirus genome, Recombinant Passerivirus genome, Recombinant Pemapivirus genome, Recombinant Poecivirus genome, Recombinant Potamipivirus genome, Recombinant Pygoscepivirus genome, Recombinant Rabovirus genome, Recombinant Rafivirus genome 27. The pharmaceutical composition of claim 26, wherein the virus is selected from the group consisting of a recombinant Rajidapivirus genome, a recombinant Rohelivirus genome, a recombinant Rosavirus genome, a recombinant Sakobuvirus genome, a recombinant Salivirus genome, a recombinant Sapelovirus genome, a recombinant Senecavirus genome, a recombinant Shanbavirus genome, a recombinant Sicinivirus genome, a recombinant Symapivirus genome, a recombinant Teschovirus genome, a recombinant Torchivirus genome, a recombinant Tottorivirus genome, a recombinant Tremovirus genome, a recombinant Tropivirus genome, and any derivative thereof.

37. 27. The pharmaceutical composition of claim 26, wherein the recombinant iridovirus genome is a recombinant invertebrate iridescent virus 6 (IIV-6) genome or a recombinant invertebrate iridescent virus 31 (IIV-31) genome.

38. 27. The pharmaceutical composition of claim 26, wherein the recombinant baculovirus genome is selected from the group consisting of a recombinant alphabaculovirus genome, a recombinant betabaculovirus genome, a recombinant deltabaculovirus genome, a recombinant gammabaculovirus genome, and any derivative thereof.

39. 27. The pharmaceutical composition of claim 26, wherein the recombinant Geminivirus genome is selected from the group consisting of a recombinant Becurtovirus genome, a recombinant Begomovirus genome, a recombinant Capulavirus genome, a recombinant Citlodavirus genome, a recombinant Curtovirus genome, a recombinant Eragrovirus genome, a recombinant Grablovirus genome, a recombinant Maldovirus genome, a recombinant Mastrevirus genome, a recombinant Mulcrilevirus genome, a recombinant Opunvirus genome, a recombinant Topilevirus genome, a recombinant Topocuvirus genome, a recombinant Turncurtovirus genome, and any derivative thereof.

40. 27. The pharmaceutical composition of claim 26, wherein the recombinant caulimovirus genome is selected from the group consisting of a recombinant Angelica bushy stunt virus genome, a recombinant Atractylodes mild mottle virus genome, a recombinant Carnation etched ring virus genome, a recombinant Cauliflower mosaic virus genome, a recombinant Dahlia mosaic virus genome, a recombinant Figwort mosaic virus genome, a recombinant Horseradish latent virus genome, a recombinant Lamium malformed leaf virus genome, a recombinant Mirabilis mosaic virus genome, a recombinant Soybean Putnam virus genome, a recombinant Strawberry vein banding virus genome, a recombinant Thistle mottle virus genome, and any derivative thereof.

41. 22. The pharmaceutical composition of any one of claims 6 to 21, wherein said gene delivery vehicle is said one or more non-viral gene delivery vehicles.

42. 42. The pharmaceutical composition of claim 41, wherein the one or more non-viral gene delivery vehicles are chemical compounds, bacteria, mammalian cells, or physical delivery systems.

43. 43. The pharmaceutical composition of claim 42, wherein the chemical compound is selected from the group consisting of a polymeric compound, a lipid compound, an inorganic compound, and combinations thereof.

44. 44. The pharmaceutical composition of claim 43, wherein the polymeric compound is a natural polymer, a synthetic polymer, a biopolymer, a biodegradable polymer, a cationic polymer, a protein polymer, a polysaccharide polymer, or any combination thereof.

45. The polymer compound may be poly-L-lactide (PLA), chitosan, pectin, polyethyleneimine (PEI), poly-L-lysine (PLL), poly-2-N-dimethylaminoethyl methacrylate (PDMAEMA), polyaminoamine, polyaminoester, polyamidoamine, polyvinylimidazole (PVI), polybeta-aminoester (PBAE), gelatin, albumin, chitosan, beta-cyclodextrin, dextran, hyaluronic acid, polyester, alpha-pyrrolidinohexiophenone (PHP), poly-2-acrylamide 45. The pharmaceutical composition of claim 43 or claim 44, wherein the polymer is selected from the group consisting of polyaminoglycolic acid (PAGA), polydelta-valerolactone (PVL), polyphenyl ether (PPE), polybeta-amino-ester (PBAE), polyamine-co-ester (PACE), polylactic-co-glycolic acid (PLGA), polycarbonate, polyurethane, micelles, cationic emulsions, exosomes, Reb blood cell membranes, DNA-protein complexes, plasmid DNA, naked DNA, pBR322, pUC, and any derivatives thereof.

46. 44. The pharmaceutical composition of claim 43, wherein the lipid compound is selected from the group consisting of nanoparticles, lipid nanoparticles, liposomes, cationic liposomes, solid lipid nanoparticles, lipid emulsions, lipidoids, cytofectins, surfactants, gemini surfactants, and any derivatives thereof.

47. 44. The pharmaceutical composition of claim 43, wherein the inorganic compound is selected from the group consisting of nanocarriers, DNA nanclews, gold nanoparticles, carbon nanotubes, graphene, quantum dots, upconversion nanoparticles, silica nanoparticles, iron oxide, ferritin, and any derivatives thereof.

48. 43. The pharmaceutical composition of claim 42, wherein the physical delivery system is selected from the group consisting of electroporation, a gene gun, a jet gun, ultrasound, nucleofection, hydrodynamic gene delivery, needle injection, microinjection, ballistic DNA injection, sonoporation, photoporation, laser pulses, magnetofection, magnetoporation, magnetic particles, hydroporation, and any combination thereof.

49. 49. Use of the pharmaceutical composition of any one of claims 2 to 48 in the manufacture of a medicament for treating cancer.

50. The cancer is acute myeloid leukemia, acute lymphoblastic leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, brain stem glioma, brain low-grade glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma, childhood cancer, bile duct cancer, chordoma , chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor, Gestational trophoblastic tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, heart cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leptomeningeal cancer, lip cancer, liver cancer, lymphoid neoplasms, diffuse large B-cell lymphoma, malignant fibrous histiocytoma, bone cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous cell neck cancer of occult primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oralcavity cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma, rectal cancer, kidney cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, salivary gland 50. The use of claim 49, wherein the cancer is selected from the group consisting of cancer, sarcoma, Sezary syndrome, cutaneous melanoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, stomach cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, testicular germ cell tumor, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

51. 49. A method of expressing, enhancing, increasing, augmenting and / or supplementing the levels of an IL-2 polypeptide and / or an IL-12 polypeptide in one or more cells of a subject, the method comprising administering to said subject an effective amount of the pharmaceutical composition of any one of claims 2 to 48.

52. 52. The method of claim 51, wherein said one or more cells are one or more respiratory, airway epithelial, and / or pulmonary cells.

53. 49. A method of providing prophylactic, symptomatic, or therapeutic alleviation of one or more signs or symptoms of cancer in a subject in need thereof, comprising administering to said subject an effective amount of the pharmaceutical composition of any one of claims 2-48.

54. 49. A method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of the pharmaceutical composition of any one of claims 2-48.

55. The cancer is acute myeloid leukemia, acute lymphoblastic leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, brain stem glioma, brain low-grade glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma of unknown primary site, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, cervical squamous cell carcinoma, cervical adenocarcinoma, childhood cancer, bile duct cancer, chordoma , chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor, Gestational trophoblastic tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, heart cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leptomeningeal cancer, lip cancer, liver cancer, lymphoid neoplasms, diffuse large B-cell lymphoma, malignant fibrous histiocytoma, bone cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous cell neck cancer of occult primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oralCavity cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pheochromocytoma and paraganglioma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, primary hepatocellular carcinoma, prostate cancer including prostate adenocarcinoma, rectal cancer, kidney cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, 55. The method of claim 53 or claim 54, wherein the cancer is selected from the group consisting of melanoma, Sezary syndrome, cutaneous melanoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, stomach cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, testicular germ cell tumor, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral cancer, urethral cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

56. 56. The method of any one of claims 53-55, wherein the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, and thyroid cancer.

57. 57. The method of any one of claims 53 to 56, wherein the cancer is small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, or squamous cell carcinoma of the lung.

58. 58. The method of any one of claims 53 to 57, wherein the cancer is non-small cell lung cancer.

59. The method of any one of claims 51 to 58, wherein the subject is a human.

60. 60. The method of any one of claims 51-59, wherein the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epidermally, intradermally, orally, sublingually, bucally, rectally, vaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, epichoroidally, intracranially, intrathecally, intraventricularly, intraarticularly, periarticularly, intratumorally, locally, or via inhalation.

61. 61. The method of any one of claims 51-60, wherein the pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation.

62. 61. The method of any one of claims 51 to 60, wherein the pharmaceutical composition is administered intratumorally to the subject.

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