WO2024168347A1

WO2024168347A1 - Treatment of cancer using combined tusc2/tusc4 gene therapy

Info

Publication number: WO2024168347A1
Application number: PCT/US2024/015417
Authority: WO
Inventors: Mark S. Berger; Hemant Kumar
Original assignee: Genprex, Inc.
Priority date: 2023-02-10
Filing date: 2024-02-12
Publication date: 2024-08-15

Abstract

Provided herein are nucleic acid constructs comprising a polynucleotide sequence encoding a TUSC2 protein and/or a TUSC4 (NPRL2) protein. Also contemplated are non-viral and viral vectors comprising nucleic acid constructs disclosed herein. Non-viral vectors include, but are not limited, to DOTAP:cholesterol liposomes. Further contemplated herein are pharmaceutical compositions comprising the nucleic acid constructs or vectors disclosed herein and methods of using the nucleic acid constructs, vectors, or pharmaceutical compositions for the treatment of cancer in a subject in need thereof. These methods can further include administering an additional anti-cancer therapy to the subject in need thereof.

Description

TREATMENT OF CANCER USING COMBINED TUSC2/TUSC4 GENE THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of the earlier filing date of U.S. Provisional Patent Application Serial No. 63/484,306, filed on February 10, 2023, which is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (198628_46476_SeqList.xml; Size: 27,300 bytes; and Date of Creation: February 1, 2024) is herein incorporated by reference in its entirety.

FIELD

[0003] The present disclosure relates to nucleic acid constructs, vectors, and pharmaceutical compositions for the expression of TUSC2 and TUSC4. Also contemplated are methods of treating cancer in a human subject including administration of one or more nucleic acid constructs, vectors, or pharmaceutical compositions disclosed herein to a subject in need thereof.

BACKGROUND

[0004] TUSC2 (TUmor Suppressor Candidate 2, also known as FUS1) is a tumor suppressor gene originally described as a member of the tumor suppressor gene cluster from human 3p21.3 chromosomal region that is frequently deleted in lung cancer. Human TUSC2 is a small protein (110 amino acids) with an estimated MW of 12 kD. According to computer modeling, TUSC2 lacks transmembrane domains, is highly hydrophobic and contains helixcoil domain secondary structures. At the N-terminus, TUSC2 contains a myristoylation signal (Met-Gly-X-X-X-Ser/Thr) (SEQ ID NO: 18), and experiments confirm that TUSC2 is myristoylated. In normal tissues, TUSC2 is ubiquitously expressed. TUSC2 has been demonstrated to act as tumor suppressor gene in lung, breast, bone, and other cancers.

[0005] The nitrogen permease regulator-like 2 gene (NPRL2), also known as TUmor Suppressor Candidate 4 (TUSC4) encodes a protein that is associated with the Gap activity toward rags 1 (GATOR1) complex. The TUSC4 protein regulates pathways including the mTOR signaling pathway and the PI3K/Akt signaling pathway. TUSC4 also acts as a tumor suppressor by regulating signaling pathways and other processes that are frequently associated with cancer progression. For example, the PDK1 signaling pathway is involved in promoting cell proliferation, and cancer cells frequently develop mechanisms to increase proliferation by increasing PDK1 signaling. TUSC4 plays a tumor inhibiting role by inhibiting PDK1 signaling. As another example, the Breast Cancer Gene (BRCA1) encodes a protein that functions to maintain genomic stability by regulating pathways responsible for detecting and repairing damaged DNA. TUSC4 can also inhibit cancer progression by enhancing stabilization of the protein encoded by the BRCA1 gene. Since TUSC4 has tumor suppressing activities, its expression is often downregulated in cancer cells.

[0006] Development of an effective therapy, which activates or restores TUSC2 and TUSC4 in cancer cells by increasing TUSC2 and TUSC4 expression or by overcoming TUSC2 and/or TUSC4 inhibition and which is less toxic than available therapies, would provide a great advance.

SUMMARY

[0007] Provided herein are nucleic acid constructs, vectors, and pharmaceutical compositions for the expression of TUSC2 and/or TUSC4 as well as methods of using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein for the benefit of patients.

[0008] Provided herein is a nucleic acid construct comprising (a) a nucleotide sequence encoding a TUmor Suppressor Candidate 2 (TUSC2) protein and (b) a nucleotide sequence encoding a TUmor Suppressor Candidate 4 (TUSC4) protein.

[0009] Provided herein is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome, (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, and (c) a pharmaceutically acceptable excipient.

[0010] In one embodiment, the TUSC2 protein is human TUSC2. In embodiments, the TUSC2 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. In embodiments, the TUSC2 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In one embodiment, the TUSC2 protein comprises SEQ ID NO: 14. In one embodiment, the nucleotide sequence encoding the TUSC2 protein is codon- optimized. In embodiments, the nucleotide sequence encoding the TUSC2 protein comprises a sequence that is at least 80% identical to SEQ ID NO: 1 or 2. In embodiments, the nucleotide sequence encoding TUSC2 protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2. In embodiments, the nucleotide sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2.

[0011] In one embodiment, the TUSC4 protein is human TUSC4. In embodiments, the TUSC4 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 15. In embodiments, the TUSC4 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In one embodiment, the TUSC4 protein comprises SEQ ID NO: 15. In one embodiment, the nucleotide sequence encoding the TUSC4 protein is codon- optimized. In embodiments, the nucleotide sequence encoding the TUSC4 protein comprises a sequence that is at least 80% identical to SEQ ID NO:3 or 4. In embodiments, the nucleotide sequence encoding the TUSC4 protein comprises a sequence that is at least 90% identical to SEQ ID NO:3 or 4. In embodiments, the nucleotide sequence encoding the TUSC4 protein comprises SEQ ID NO:3 or 4.

[0012] In embodiments, the nucleic acid construct further comprises (a) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein and/or (b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein. In embodiments, the CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:21. In one embodiment, the CMV promoter comprises SEQ ID NO:21. In one embodiment, the nucleic acid construct further comprises a CMV enhancer. In embodiments, the CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 19 or 20. In embodiments, the CMV enhancer comprises SEQ ID NO: 19 or 20.

[0013] In one embodiment, the nucleic acid construct further comprises a Human T-cell leukemia virus type I (HTLV-I) regulatory sequence. In embodiments, the HTLV-I regulatory sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8. In one embodiment, the HTLV-I regulatory sequence comprises SEQ ID NO:8.

[0014] In one embodiment, the nucleic acid construct further comprises a bovine growth hormone polyadenylation (BGH poly A) sequence. In embodiments, the BGH polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NOV. In one embodiment, the BGH polyA sequence comprises SEQ ID NOV.

[0015] In one embodiment, the nucleic acid construct further comprises a splicing enhancer sequence. In embodiments, the splicing enhancer sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 12. In one embodiment, the splicing enhancer sequence comprises SEQ ID NO: 12. [0016] In embodiments, the nucleic acid construct further comprises at least one intron. In one embodiment, the at least one intron is a P-globin intron. In embodiments, the P-globin intron sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In one embodiment, the P-globin intron sequence comprises SEQ ID NO: 10.

[0017] In one embodiment, the nucleic acid construct further comprises a bacterial backbone sequence. In embodiments, the bacterial backbone sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11. In one embodiment, the bacterial backbone sequence comprises SEQ ID NO: 11. In one embodiment, the bacterial backbone sequence comprises a R6K origin sequence. In one embodiment, the bacterial backbone sequence comprises at least one selectable marker.

[0018] In embodiments, the nucleic acid construct comprises a sequence that is at least 80% identical to SEQ ID NO: 13. In embodiments, the nucleic acid construct comprises a sequence that is at least 90% identical to SEQ ID NO: 13. In one embodiment, the nucleic acid construct comprises SEQ ID NO: 13.

[0019] Provided herein is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome, (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, and (c) a pharmaceutically acceptable excipient, wherein the first nucleic acid construct further comprises a first CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein, and/or the second nucleic acid construct further comprises a second CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein. In embodiments, the first CMV promoter and/or the second CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:7. In one embodiment, the first CMV promoter and/or the second CMV promoter comprises SEQ ID NO:7. In embodiment, the first nucleic acid construct further comprises a first CMV enhancer; and/or the second nucleic acid construct further comprises a second CMV enhancer. In embodiments, the first CMV enhancer and/or the second CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 19 or 20. In embodiments, the first CMV enhancer and/or the second CMV enhancer comprises SEQ ID NO: 19 or 20. In embodiments, the first CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 19 and the second CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO:20. In one embodiment, the first CMV enhancer comprises SEQ ID NO: 19 and the second CMV enhancer comprises SEQ ID NO:20. [0020] In embodiments, the first and/or the second nucleic acid construct further comprises a HTLV-I regulatory sequence. In embodiments, the HTLV-I regulatory sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8. In one embodiment, the HTLV-I regulatory sequence comprises SEQ ID NO:8.

[0021] In embodiments, the first and/or the second nucleic acid construct further comprises a BGH polyA sequence. In embodiments, the BGH polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NOV. In one embodiment, the BGH polyA sequence comprises SEQ ID NOV.

[0022] In embodiments, the first and/or the second nucleic acid construct further comprises a splicing enhancer sequence. In embodiments, the splicing enhancer sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 12. In one embodiment, the splicing enhancer sequence comprises SEQ ID NO: 12.

[0023] In embodiments, the first and/or the second nucleic acid construct further comprises at least one intron. In one embodiment, the at least one intron is a P-globin intron. In embodiments, the P-globin intron sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In one embodiment, the P-globin intron sequence comprises SEQ ID NO: 10.

[0024] In embodiments, the first and/or the second nucleic acid construct further comprises a bacterial backbone sequence. In embodiments, the bacterial backbone sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11. In one embodiment, the bacterial backbone sequence comprises SEQ ID NO: 11. In one embodiment, the bacterial backbone sequence comprises a R6K origin sequence. In one embodiment, the bacterial backbone sequence comprises at least one selectable marker.

[0025] Provided herein is a nucleic acid construct that is complexed with a liposome. Provided herein is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome, (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, and (c) a pharmaceutically acceptable excipient. In one embodiment, the liposome is a l,2-dioleoyl-3-trimethylammonium-propane (DOTAP): cholesterol liposome. In embodiments, the DOTAP: cholesterol ratio is between about 3: 1 and about 1 :3. In embodiments, the DOTAP: cholesterol liposome has a particle size range of about 40 to about 250 nanometers.

[0026] Provided herein is a pharmaceutical composition comprising a nucleic acid construct disclosed herein and a pharmaceutically acceptable excipient. [0027] In embodiments, a pharmaceutical composition disclosed herein further comprises approximately 5% dextrose, 0.9% sodium chloride, or a combination of both agents.

[0028] Provided herein is a viral vector comprising a nucleic acid construct disclosed herein. In one embodiment, the viral vector is an Adeno- Associated Virus (AAV) viral vector. [0029] Provided herein is a pharmaceutical composition comprising a viral vector disclosed herein.

[0030] Provided is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition disclosed herein. In one embodiment, the subject is a human. In embodiments, the cancer is selected from the group consisting of colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, neuroblastoma, leukemia, lung cancer, renal cancer, and rectal cancer. In one embodiment, the cancer is positron emission tomography (PET) positive cancer. In embodiments, the cancer is lung cancer, including, for example, Small Cell Lung Cancer (SCLC). In embodiments, the lung cancer is Non-Small Cell Lung Cancer (NSCLC). In further embodiments, the lung cancer is an adenocarcinoma. In embodiments, the pharmaceutical composition is administered intravenously or intranasally. In one embodiment, the method for treating cancer in a subject in need thereof further comprises administering a second anti-cancer therapy to the subject. In embodiments, the second anti-cancer therapy comprises at least one of chemotherapy, radiation treatment, and surgery. In one embodiment, the second anti-cancer therapy is a checkpoint inhibitor or a BRAF inhibitor. In one embodiment, the checkpoint inhibitor is pembrolizumab. In one embodiment, the BRAF inhibitor is encorafenib. In one embodiment, the second anti-cancer therapy is an EGFR inhibitor. In one embodiment, the EGFR inhibitor is cetuximab or nivolumab. In one embodiment, the second anti-cancer therapy is a KRAS inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

[0031] Fig. 1A and Fig. IB illustrate that a combination of TUSC2 expression and TUSC4 expression inhibits tumor growth in mice bearing LLC2 tumors more effectively that TUSC2 or TUSC4 alone (Fig. 1A), and more effectively than an anti-PDl antibody or a combination of TUSC4 (NPRL2) and an anti-PD-Ll antibody (Fig. IB). 10⁶LLC2-luc cells were injected subcutaneously into syngeneic mice (n = 9 per each treatment group and control) and treated with (7) a control empty plasmid complexed with DOTAP/cholesterol liposome (25 pg i.v., every 3 days for 3 weeks), (2) a plasmid expressing TUSC4 (NPRL2) complexed with DOTAP/cholesterol liposome (25 pg i.v., every 3 days for 3 weeks), (3) a plasmid expressing TUSC2 complexed with DOTAP/cholesterol liposome (25 pg i.v., every 3 days for 3 weeks), (4) a plasmid expressing TUSC4 (NPRL2) complexed with a DOTAP/cholesterol liposome and a plasmid expressing TUSC2 complexed with a DOTAP/cholesterol liposome (25 pg i.v. each, every 3 days for 3 weeks), (5) an anti-PDl antibody (“aPDl”) (250 pg i.p., two times a week for 3 weeks); or (6) a plasmid expressing TUSC4 (NPRL2) complexed with a DOTAP/cholesterol liposome (25 pg i.v., every 3 days for 3 weeks) and an anti-PDl antibody (250 pg i.p., two times a week for 3 weeks). Fig. 1A: traces from top to bottom: Control, TUSC2 alone, TUSC4 (NPRL2) alone, TUSC2 + TUSC4 (NPRL2). Fig. IB: traces from top to bottom: Control, anti-PDl antibody alone, TUSC2 alone, TUSC4 (NPRL2) and anti-PDl antibody, TUSC4 (NPRL2) alone, TUSC2 + TUSC4 (NPRL2).

[0032] Figs. 2A-2C demonstrate the TUSC2 +NPRL2 dual gene therapy combination effects on inflamed H841 tumors in humanized NSG mice. Fig. 2A is a schematic of the treatment strategy. Fig. 2B is a graph that illustrates that a combination of a liposome- complexed TUSC2 expression construct and a liposome-complexed TUSC4 expression construct inhibits tumor growth in mice bearing H841 SCLC cells more effectively than liposome-complexed expression constructs expressing either TUSC2 or TUSC4 (NPRL2) alone. Humanized mice were injected subcutaneously with 5 million H841 SCLC cells. After three weeks, mice were treated every three days for three weeks with (1) a control empty plasmid complexed with DOTAP/cholesterol liposome, (2) a plasmid expressing TUSC2 complexed with DOTAP/cholesterol liposome, (3) a plasmid expressing TUSC4 complexed with DOTAP/cholesterol liposome, or (4) a plasmid expressing TUSC2 complexed with a DOTAP/cholesterol liposome and a plasmid expressing TUSC4 complexed with a DOTAP/cholesterol liposome, respectively, for three weeks. Traces from top to bottom: Control, TUSC2 alone, TUSC4 (NPRL2) alone, TUSC2 + TUSC4 (NPRL2). Fig. 2C are graphs of the individual mouse response towards the indicated treatments.

[0033] Fig. 3 shows an illustration of an expression construct comprising a codon- optimized sequence encoding TUSC2 (“TUSC2co”) and a codon-optimized sequence encoding TUSC4 (“TUSC4co”). The TUSC2 and TUSC4 protein encoding sequences are under the control of separate CMV promoter/enhancers.

[0034] Figs. 4A, 4B, and 4C illustrate that TUSC2/TUSC4 treatment reduces tumor growth in mice bearing luciferase expressing A549-luc tumors. Mice were treated every three days for three weeks (n=50 per group) with (1) DOTAP/cholesterol-liposome-complexed empty vector (control), (2) Quaratusugene Ozeplasmid (Reqorsa®), a DOTAP/cholesterol liposome-complexed TUSC2 expression plasmid (50 pg/mouse), (3) a DOTAP/cholesterol liposome-complexed TUSC4 expression plasmid (50 pg/mouse), (4) a DOTAP/cholesterol liposome-complexed TUSC2/TUSC4 expression plasmid (50 pg/mouse) (“TUSC2-TUSC4”), and (5) a combination of Quaratusugene Ozeplasmid and a DOTAP/cholesterol liposome- complexed TUSC4 expression plasmid (50 pg each/mouse) (“TUSC2+TUSC4”). Total luciferase expression at week four after start of the treatment is shown (Figs. 4A and 4B). Fig. 4C provides IVIS images at week four after start of the treatment showing metastasis.

[0035] Figs. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, and 51 illustrate the effect of TUSC2/ TUSC4 (NPRL2) treatment on the LLC2 tumor microenvironment (TME) in mice bearing LLC2 tumors. Fig. 5A shows the gating strategy used for flow cytometry analysis of CD8⁺ T cells. Fig. 5B is a graph of the percent of CD8⁺ T cells in the LLC2 TME. Fig. 5C is a graph of the percent of CD4⁺ T cells in the LLC2 TME. Fig. 5D shows the gating strategy used for flow cytometry analysis of CD69⁺CD8⁺ T cells. Fig. 5E is a graph of the percent of CD69⁺CD8⁺ T cells in the LLC2 TME. Fig. 5F shows the gating strategy used for flow cytometry analysis of MHCII+ dendritic cells (DCs). Fig. 5G is a graph of the percent of MHCII+ DCs in the LLC2 TME. Fig. 5H shows the gating strategy used for flow cytometry analysis of myeloid-derived suppressor cells (MDSCs). Fig. 51 is a graph of the percent of MDSCs in the LLC2 TME.

DETAILED DESCRIPTION

[0036] Provided herein are nucleic acid constructs comprising nucleotide sequences encoding TUSC2 and/or TUSC4. Also provided are non-viral and viral vectors comprising a nucleic acid construct disclosed herein. In embodiments, the non-viral vectors are DOTAP: cholesterol liposomes. Further contemplated herein are pharmaceutical compositions comprising the nucleic acid constructs, the non-viral vectors, or the viral vectors disclosed herein. Also provided herein are methods of using the nucleic acid constructs, vectors, or pharmaceutical compositions disclosed herein for the treatment of cancer in a human subject in need thereof. These methods can further include administering an additional anti-cancer therapy to the subjects in need thereof (z.e., a combination therapy in addition to the TUSC2/TUSC4 therapy). [0037] Nucleic Acid Constructs

[0038] In one aspect, provided is a nucleic acid construct comprising (a) a nucleotide sequence encoding a TUSC2 protein and (b) a nucleotide sequence encoding a TUSC4 protein. In embodiments, the nucleotide sequence encoding the TUSC2 protein and/or the nucleotide sequence encoding the TUSC4 protein is codon-optimized. In another aspect, nucleotide sequence encoding a TUSC2 protein and the nucleotide sequence encoding a TUSC4 protein are on separate nucleic acid constructs.

[0039] In one aspect, provided is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome and (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome. In one aspect, provided is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome, (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, and (c) a pharmaceutically acceptable excipient.

[0040] In one embodiment, the nucleic acid construct is used for recombinant production of human TUSC2 and/or TUSC4 in cancer cells (e.g., in a patient’s cancer cells). Nucleic acid constructs include expression constructs such as plasmids. The term “expression construct” refers to a recombinant polynucleotide construct that includes a nucleic acid coding for an RNA capable of being transcribed in a cell. Methods for constructing expression constructs and plasmids through standard recombinant techniques are known in the art. Methods for designing expression constructs/plasmids for gene therapy applications, including antibiotic- free vector production, are also known. Various sequences and elements have been reported to increase and sustain therapeutic protein production (e.g., introns, Kozak consensus and other control/regulatory sequences discussed herein). Expression constructs constructs/plasmids for inclusion in the non-viral vectors described herein can be produced in a suitable host producer cells (e.g., E. coif) using suitable methods, e.g., fed-batch fermentation, batch fermentation, etc. For example, the HyperGRO™ inducible fed-batch fermentation process may be used to manufacture plasmid DNA. The HyperGRO™ process yields plasmid productivity of up to 2,600 mg/L with low levels of nicking or multimerization. High yield of plasmid per gram of bacteria improves final product purity since plasmid is enriched relative to host cell impurities. Boehringer Ingelheim (Vienna, Austria) has developed an alternative high yield fermentation process which is commercially available for cGMP production of plasmid DNA-based vectors. Plasmid DNA can be extracted from fermentation cells using alkaline lysis. Following plasmid production, the plasmid can be purified by processes, such as anion exchange chromatography followed by hydrophobic interaction chromatography, that isolate plasmid DNA away from impurities (e.g., endotoxin, bacterial RNA, genomic DNA).

[0041] In embodiments, the TUSC2 protein is human TUSC2. In embodiments, the TUSC4 protein is human TUSC4. In embodiments, the TUSC2 protein comprises an amino acid sequence that is 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%, or at least 99% identical to SEQ ID NO: 14. In embodiments, the TUSC2 protein comprises SEQ ID NO: 14. In one embodiment, the TUSC2 protein consists of SEQ ID NO: 14. In embodiments, the TUSC4 protein comprises an amino acid sequence that is 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%, or at least 99% identical to SEQ ID NO: 15. In embodiments, the TUSC4 protein comprises SEQ ID NO: 15. In one embodiment, the TUSC4 protein consists of SEQ ID NO: 15. As used herein, the term “sequence identity” refers to the degree of which two sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits.

[0042] In embodiments, the nucleotide sequence encoding the TUSC2 protein comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 1 or 2. In embodiments, the nucleotide sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2.

[0043] In embodiments, the nucleotide sequence encoding the TUSC4 protein comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO:3 or 4. In embodiments, the nucleotide sequence encoding the TUSC2 protein.

[0044] In embodiments, the nucleotide sequence encoding the TUSC2 protein and/or nucleotide sequence encoding the TUSC4 protein are flanked by a 5' untranslated region (UTR) and a 3' UTR.

[0045] The nucleic acid constructs disclosed herein can include control and regulatory sequences that are operably linked to the polynucleotide sequence encoding the TUSC2 and/or the TUSC4 protein. The nucleic acid constructs disclosed herein can include appropriate control sequences for expression of TUSC2 and/or TUSC4 in human cancer cells. “Control sequences” include nucleic acid sequences necessary for replication of a vector in a producer cell (e.g., E. coli cell), as well as nucleic acid sequences necessary for, or involved in, transcription and/or translation of an operably linked TUSC2 and/or TUSC4 coding sequence in a target cell (e.g., a human cancer cell). As used herein, the term “operably linked” refers to a physical or functional juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a polynucleotide sequence encoding a TUSC2 and/or TUSC4 protein, the relationship is such that the control element modulates expression of the TUSC2 and/or TUSC4 protein encoding sequence. Examples of control/regulatory sequences include promoters, enhancers, translation initiation signals, termination signals, polyadenylation sequences (e.g., polyA signals derived from bovine growth hormone, SV40, rabbit P-globin), origins of replication (e.g. , the high copy number pUC replication origin which can be reduced to 700 bp without loss of high copy number replication, a non-pUC mini-origin R6K Nanoplasmid™ from Nature Technology Corporation, Lincoln, NE, etc.), Kozak sequences (e.g., GCCACCATG, SEQ ID NO: 17), posttranslational regulatory elements, introns, nuclear targeting sequences, etc. A suitable promoter may be used in the nucleic acid constructs described herein. In embodiments, the CMV promoter/enhancer is used, and serves a dual role as a promoter and an enhancer. In other embodiments, chimeric promoters that are a fusion of two different promoter sequences or a fusion of a promoter sequence and an inducible element can be used. For example, a chicken P-actin/CMV enhancer combination can be used. Promoters, in addition to the CMV promoter, that can be used to promote transcription of the TUSC2 and/or TUSC4 transgene include simian virus 40 (SV40) early promoter, elongation factor-la, (EFla), phosphoglycerate kinase (PGK), and human P-actin promoter (ACTB). In some embodiments, a tissue-specific promoter can be used. In some embodiments, a nucleic acid construct as described herein includes one or more (e.g., 1, 2, 3, 4, 5, etc.) introns. For example, in a nucleic acid construct as disclosed herein, the 5' UTR, 3' UTR, and/or the TUSC2 and/or TUSC4 coding sequence can include an intron. As another example, a chimeric intron (e.g., from the P-globulin and/or immunoglobulin heavy chain genes) upstream of the coding sequence can be used. Additionally or alternatively, the 5' UTR can include a Human T-cell leukemia virus type I (HTLV-I) R element for enhancement of mRNA translation efficiency and increasing transgene expression. Nuclear targeting sequences, which promote shuttling of the nucleic acid construct into the nucleus, can be included the nucleic acid construct as described herein. MicroRNA target sites that mediate transgene expression in specific tissues or cell lineages and S/MAR regions that promote replication and long-term episomal transgene expression can also be included in some embodiments of a nucleic acid construct as described herein. In embodiments, a P-globin intron is included for its efficient splice acceptor, and in further embodiments, the splice donor is derived from the upstream HTLV-I R. However, any strong splice acceptor and splice donor could be used. In embodiments, HTLV-I R is included as a translational enhancer. However, any suitable translational enhancer can be used. In embodiments transgene expression through increased intron splicing. In embodiments, a splicing enhancer is included within the intron and/or a flanking exon to increase transgene expression through increased intron splicing. In embodiments, provided are nucleic acid constructs comprising one or more of the following sequences: RNA-OUT, CMV enhancer/promoter, CMV-human T-lymphotropic virus type I (HLTV-I) R Region Exon 1, HTLV-I R element, P globin intron, splicing enhancer, Kozak sequence, BGH polyA signal, trpA terminator, and origin.

[0046] In embodiments, the expression construct comprising the polynucleotide sequence encoding a TUSC2 and/or a TUSC4 protein is a covalently closed linear DNA (“doggybone DNA” or dbDNA”). dbDNA may be created starting with a circular double-stranded DNA molecule (e.g., a plasmid) containing a TUSC2 and/or a TUSC4 encoding sequence flanked on each side by 56 bp palindromic protelomerase recognition sequences. The DNA starting material is then denatured and Phi29 DNA polymerase is primed. Phi29 initiates rolling circle amplification of the template, creating double-stranded concatameric repeats of the original construct. Protelomerase is added, which binds to the recognition sites flanking the TUSC2 encoding sequence and performs a cleavage-joining reaction that results in monomeric double-stranded, linear, covalently closed DNA constructs. One of a panel of common restriction enzymes is added to cut undesired backbone DNA sequences, exposing open ended DNA that can be removed through digestion with exonuclease. dbDNA is purified from small fragments and reaction components with size separation to leave only the dbDNA construct comprising the TUSC2 encoding sequence. The resulting dbDNA construct can be used as a starting material for further amplification reactions. dbDNA constructs and methods of making them are disclosed in W02010086626 (PCT/GB2010/000165), incorporated herein by reference in its entirety.

[0047] In embodiments, the nucleic acid construct comprises (a) a promoter operably linked to the nucleotide sequence encoding the TUSC2 protein and/or (b) a promoter operably linked to the nucleotide sequence encoding the TUSC4 protein. In embodiments, the promoter is a CMV promoter. In embodiments, the CMV promoter comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 7 or SEQ ID NO:21. In embodiments, the CMV promoter comprises SEQ ID NO:7. In embodiments, the CMV promoter comprises SEQ ID NO:21. In embodiments, the nucleic acid construct comprises one or more enhancer sequences. In some embodiments, the nucleic acid construct comprises one or more CMV enhancer sequences. In embodiments, the CMV enhancer comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO:5, 6, 19, or 20. In embodiments, the CMV enhancer comprises SEQ ID NO:5, 6, 19, or 20. In embodiments, the nucleic acid construct comprises

(a) a CMV promoter and a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein and/or (b) a CMV promoter and a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein.

[0048] In embodiments, the nucleic acid construct comprises:

(a) a nucleotide sequence encoding a TUSC2 protein;

(b) a promoter operably linked to the nucleotide sequence encoding the TUSC2 protein;

(c) an enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein;

(d) a nucleotide sequence encoding a TUSC4 protein;

(e) a promoter operably linked to the nucleotide sequence encoding the TUSC4 protein; and/or

(f) an enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein.

[0049] In embodiments, the nucleic acid construct comprises:

(a) a nucleotide sequence encoding a TUSC2 protein;

(b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein;

(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein;

(d) a nucleotide sequence encoding a TUSC4 protein;

(e) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein; and/or

(f) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein.

[0050] In embodiments, the nucleic acid construct comprises:

(a) a nucleotide sequence encoding a TUSC2 protein, wherein the nucleotide sequence encoding the TUSC2 protein comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 1 or 2;

(b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV promoter comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 7;

(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV enhancer comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 5;

(d) a nucleotide sequence encoding a TUSC4 protein, wherein the nucleotide sequence encoding the TUSC4 protein comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 3 or 4;

(e) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV promoter comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 7; and/or

(f) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV enhancer comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO:6.

[0051] In embodiments, the nucleic acid construct comprises:

(a) a nucleotide sequence encoding a TUSC2 protein, wherein the nucleotide sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2;

(b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV promoter comprises SEQ ID NO:7;

(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV enhancer comprises SEQ ID NO:5;

(d) a nucleotide sequence encoding a TUSC4 protein, wherein the nucleotide sequence encoding the TUSC4 protein comprises SEQ ID NO: 3 or 4;

(e) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV promoter comprises SEQ ID NO:7; and/or (f) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV enhancer comprises SEQ ID NO:6.

[0052] In embodiments, the nucleic acid construct comprises:

(b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV promoter comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO:21;

(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV enhancer comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 19;

(e) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV promoter comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO:21; and/or

(g) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV enhancer comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO:20.

[0053] In embodiments, the nucleic acid construct comprises:

(b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV promoter comprises SEQ ID NO:21; (c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV enhancer comprises SEQ ID NO: 19;

(e) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV promoter comprises SEQ ID NO:21; and/or

(f) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC4 protein, wherein the CMV enhancer comprises SEQ ID NO:20.

[0054] In embodiments, the nucleic acid construct comprises a Human T-cell leukemia virus type I (HTLV-I) regulatory sequence. In embodiments, the HTLV-I regulatory sequence comprises a sequence 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%, or at least 99% identical to SEQ ID NO:8. In embodiments, the HTLV-I regulatory sequence comprises SEQ ID N0:8.

[0055] In embodiments, the nucleic acid construct comprises a bovine growth hormone polyadenylation (BGH polyA) sequence. In embodiments, the BGH polyA sequence comprises a sequence that is 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%, or at least 99% identical to SEQ ID NOV. In embodiments, the BGH polyA sequence comprises SEQ ID NOV.

[0056] In embodiments, the nucleic acid construct comprises a splicing enhancer sequence. In embodiments, the splicing enhancer sequence comprises SEQ ID NO: 16 (GAAGAAGAC). In embodiments, the splicing enhancer sequence comprises one or more repeats of SEQ ID NO: 16 (GAAGAAGAC). In embodiments, the splicing enhancer sequence comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 12. In embodiments, the splicing enhancer sequence comprises SEQ ID NO: 12.

[0057] In embodiments, the nucleic acid construct comprises at least one intron. In embodiments, the at least one intron is a P-globin intron. In embodiments, the P-globin intron sequence comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 10. In embodiments, the P-globin intron sequence comprises SEQ ID NO: 10. [0058] The nucleic acid construct can also include a bacterial plasmid backbone for production of the plasmid in bacterial cells (in some tissues, bacterial regions of approximately 1,000 bp or more promote transgene silencing). In embodiments, the plasmid is derived from a NTC9385R plasmid (J. A. Williams, Vaccines 2013 1 :225-249; Borggren et al., Hum Vaccin Immunother. 2015 11(8): 1983-1990), which is commercially available (Nature Technologies Corporation, Lincoln, NE, US). In embodiments, the bacterial backbone sequence comprises a sequence that is 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%, or at least 99% identical to SEQ ID NO: 11. In embodiments, the bacterial backbone sequence SEQ ID NO: 11. In embodiments, the bacterial backbone sequence comprises a R6K origin sequence. In embodiments, the nucleic acid constructs as disclosed herein include a selectable marker. In embodiments, the nucleic acid construct comprises a bacterial backbone sequence comprising at least one selectable marker. A “selectable marker” as used herein is a nucleic acid sequence that confers a trait suitable for selection for a cell containing the nucleic acid construct. Selectable markers can include RNA selectable markers such as RNA-OUT (Luke et al., Vaccine 2009 vol. 27(46): 6454-6459; Luke et al. Methods Mol Biol. 2014 vol. 1143:91-111), RNAI (US Patent No. 9297014), and suppressor tRNAs (Soubrier et al., Gene Therapy 1999 vol. 6: 1482-1488). RNA selectable markers are useful in applications where use of antibioticresistance markers is undesirable, including in production of non-viral vectors. For example, some regulatory agencies recommend avoiding inclusion of antibiotic resistance markers in DNA therapies administered to humans due to risk of unintended immune response and transmission of the antibiotic-resistant genes to the patient’s enteric bacteria. Thus, in some embodiments of a nucleic acid construct, the selectable marker is not an antibiotic resistance gene. In other embodiments, selectable markers can include an antibiotic resistance gene, for example, genes encoding resistance to ampicillin, chloramphenicol, tetracycline or kanamycin.

[0059] In embodiments, provided is a nucleic acid construct comprising a sequence that is 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%, or at least 99% identical to SEQ ID NO: 13. In embodiments, provided is a nucleic acid construct comprising SEQ ID NO: 13. In one embodiment, provided is a nucleic acid construct consisting of SEQ ID NO: 13. [0060] Vectors

[0061] The term “vector” as used herein refers to a vehicle for delivering genetic material (e.g., RNA or DNA) to a cell, including for example, viral vectors (such as AAV and lentiviral vectors) and non-viral vectors. The term “non-viral vector” is used herein to refer to a non- viral vehicle for delivering genetic material to a cell.

[0062] In embodiments, the non-viral vector comprises one or more carrier molecules (e.g. , DOTAP: cholesterol liposome) complexed with a nucleic acid construct (e.g., a plasmid) as disclosed herein. In embodiments, a nucleic acid construct disclosed herein is complexed with a liposome. The liposome formulations described herein are useful for delivering nucleic acid constructs into the target cell. Specifically, the liposome formulations described herein can enter target cells via endocytosis pathways to avoid lysosomal degradation. Once a liposome particle binds to a negatively-charged cancer cell, the nucleic acid construct is transfected into the cell (e.g., via endocytosis) and TUSC2 and/or TUSC4 is expressed. In embodiments, the non-viral vectors described herein result in a high level of transfection efficiency with a low level of toxicity. The non-viral vectors display specificity and protect against degradation of the nucleic acid construct by the target cell during transfection. The liposome formulations are designed for stability, increased half-life of the polynucleotide construct and the prevention of aggregation of the lipid particles. In the liposomal non-viral vectors disclosed herein, the nucleic acid constructs can be added to liposomes in a range of concentrations. The ratio of the nucleic acid construct to lipids (liposomes) can be optimized for transfection efficiency. In embodiments, nucleic acid constructs are added to the liposomes at a concentration of 20, 25, 50, 75, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 275, 300, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pg per 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, or 10,000 pl, as well as 15, 20, 25, 50 ml final volume. These concentrations may vary depending upon the ratio of the liposome components (e.g., DOTAP to cholesterol, cholesterol derivative or cholesterol mixture) in the particular liposome preparation. In some embodiments, equal volumes of nucleic acid construct and lipids (e.g., DOTAP: cholesterol liposome), at a concentration to obtain about 25 pg, about 50 pg, about 75 pg, about 100 pg, about 110 pg, about 120 pg, about 125 pg, about 130 pg, about 140 pg, about 150 pg, about 160 pg, about 170 pg, about 180 pg, about 190 pg, about 200 pg, about 210 pg, about 220 pg, about 225 pg, about 230 pg, about 240 pg, about 250 pg, about 260 pg, about 270 pg, about 275 pg, about 280 pg, about 290 pg, about 300 pg, about 310 pg, about 320 pg, about 325 pg, about 330 pg, about 340 ig, about 350 pig, about 360 pig, about 370 pig, about 375 pig, about 400 pig, about 425 pig, about 450 pig, about 500 pig, about 550 pig, about 600 pig, about 650 pig, about 700 pig, about 750 pig, about 800 pig, about 850 pig, about 900 pig, about 950 pig, about or 1000 pig of nucleic acid per 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, 30 mM, 32 mM, 34 mM, 36 mM, 38 mM, or 40 mM lipids per 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, or 10,000 pl, as well as 15, 20, 25, or 50 ml, are mixed by adding the nucleic acid construct rapidly to the surface of the lipid (e.g., DOTAP:cholesterol) solution followed by mixing.

[0063] The non-viral vectors disclosed herein are typically of an average particle size of between about 40 nm and about 250 nm (e.g., 39 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 250 nm, 251 nm). In some embodiments, the average mean particle size of the non-viral vector is between about 300 and about 325 nm.

[0064] DOTAP:cholesterol liposomes are nanoparticle liposomal formulations composed of l,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP) and cholesterol. Nanoparticle liposomal formulations are considered non-viral vectors herein. DOTAP:cholesterol liposomes form a stable structure and are efficient carriers of biologically active agents such as nucleic acid constructs. In embodiments, the liposomal formulation includes DOTAP in a concentration ranging from about 1 to 8 about millimolar (mM) (e.g., 1 mM, 2 to 7 mM, 3 to 6 mM, 4 to 5 mM, 8 mM). In embodiments, the liposomal formulation includes cholesterol or cholesterol derivative or cholesterol mixture in a concentration ranging from about 0.1 to about 8 mM (e.g., 0.1 mM, 0.2 to 1 mM, 2 to 7 mM, 3 to 6 mM, 4 to 5 mM, or 8 mM). In some embodiments of a non-viral vector, the DOTAP:cholesterol molar ratio is between about 3: 1 and about 1 :3 (e.g., about 3.1: 1, about 3: 1, about 2.5: 1, about 2:1, about 1.5: 1, about 1 : 1, about 1 : 1.5, about 1 :2, about 1 :2.5, about 1 :3, or about 1 :3.1). Methods of making DOTAP: cholesterol liposomes are known in the art. For example, extrusion, microfluidization, reverse phase evaporation, sonication, solvent (e.g., ethanol) injection, detergent dialysis, ether injection, and dehydration/rehydration may be utilized.

[0065] Provided herein are liposomes comprising a nucleic acid construct comprising (a) a nucleotide sequence encoding a TUSC2 protein and (b) a nucleotide sequence encoding a TUSC4 protein. Provided herein are liposomes comprising (a) a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein and/or (b) a nucleic acid construct comprising a nucleotide sequence encoding a TUSC4 protein. Provided herein is a mixture of (a) liposomes comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein and (b) liposomes comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC4 protein.

[0066] In embodiments, provided is a nucleic acid construct comprising (a) a nucleotide sequence encoding a TUSC2 protein and (b) a nucleotide sequence encoding a TUSC4 protein, wherein the nucleic acid construct is complexed with a liposome. In embodiments, provided is a nucleic acid construct comprising (a) a nucleotide sequence encoding a TUSC2 protein and (b) a nucleotide sequence encoding a TUSC4 protein, wherein the nucleic acid construct is complexed with a liposome, wherein the liposome is a DOTAP: cholesterol liposome. In embodiments, provided is a nucleic acid construct comprising (a) a nucleotide sequence encoding a TUSC2 protein and (b) a nucleotide sequence encoding a TUSC4 protein, wherein the nucleic acid construct is complexed with a liposome, wherein the liposome is a DOTAP: cholesterol liposome and wherein the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3.

[0067] In embodiments, provided is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome and (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, wherein the liposome is a DOTAP: cholesterol liposome. In embodiments, provided is a pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome and (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, wherein the liposome is a DOTAP: cholesterol liposome and wherein the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3.

[0068] The DOTAP: cholesterol liposomes described herein may be prepared, for example, by an extrusion method including the steps of heating, sonicating, and sequential extrusion of the lipids through filters of decreasing pore size, thereby resulting in the formation of small, stable liposome structures. In such methods, the production of liposomes often is accomplished by sonication or serial extrusion of liposomal mixtures after (i) reverse phase evaporation (ii) dehydration-rehydration (iii) detergent dialysis and (iv) thin film hydration. Methods of producing liposomes via extrusion are described in Templeton et al. (Nat. Biotechnol., 1997 15(7):647-52) and US Patent No. 10,293,056. In these methods, DNAlipid complexes are prepared by diluting a given nucleic acid and lipids in 5% dextrose in water to obtain an appropriate concentration of nucleic acid and lipids in an isotonic solution. For example, DOTAP (cationic lipid) is mixed with cholesterol (neutral lipid) at about equimolar concentrations. This mixture of powdered lipids is then dissolved with a solvent such as chloroform. The lipid solution is dried to a thin film at 30 °C for 30 minutes (using, e.g., a rotary evaporator). The thin film is further freeze dried under vacuum for 15 minutes. The film is hydrated with water containing 5% dextrose (w/v) to give a final concentration of about 20 mM DOTAP and about 20 mM cholesterol. The hydrated lipid film is rotated in a 50 °C water bath for 45 minutes and then at 37 °C for an additional 10 minutes. The mixture is left standing at room temperature overnight. The following day the mixture is sonicated for 5-8 minutes at 50 °C. The sonicated mixture is transferred to a new vessel and is heated for 10 minutes at 50° C. This mixture is sequentially extruded through filters (e.g., syringe filters) of decreasing pore size (e.g., 1 pm, 0.45 pm, 0.2 pm, and 0.1 pm). The 0.2 pm and 0.1 pm filters can be, e.g., Whatman Anotop filters (Cat. #: 6809-2122 or equivalent). The filtrate can be stored at, e.g., 4 °C or lower under argon or other inert gas.

[0069] In other embodiments, the DOTAP: cholesterol liposomes are produced using a microfluidization method. Microfluidization can be used when consistently small (e.g, 40 to 200 nm) and relatively uniform aggregates are desired. Large scale production of DOTAP:cholesterol liposomes by microfluidization are known in the art. Methods of manufacturing liposomes using microfluidization are described, for example, in US Patent Application No. 16/098619. In certain microfluidization methods, the liposomal suspension is pumped at high velocity through an inlet that is divided into two streams and progressively bifurcates. These streams eventually collide within an interaction chamber leading to the formation of smaller particles due to turbulence and pressure. Generally, in microfluidization methods, DOTAP: cholesterol liposomes are formed by a quick increase in polarity of the environment induced by rapid mixing of the two miscible phases. This rapid mixing induces supersaturation of lipid molecules which leads to the self-assembly of DOTAP:cholesterol liposomes. Microfluidic mixing methods may include: microfluidic mixing using a staggered herringbone mixer (SHM), in-line T-junction mixing, and microfluidic hydrodynamic mixing (MHF). MHF is a continuous-flow technique where, in the case of liposome production, lipids dissolved in an organic solvent are hydrodynamically focused using an aqueous phase. In T- junction mixing, rapid mixing occurs when the two input streams in the T-junction collide, resulting in a turbulent output flow. SHM is microfluidic mixing by chaotic advection. Similar to other microfluidic techniques, the main characteristic is controlled millisecond mixing of two miscible phases, for example, ethanol and an aqueous buffer. The structure of the SHM allows efficient wrapping of the two fluids around each other resulting in an exponential enlargement of the interface between the fluids ensuring rapid mixing. In embodiments, a post-filtration step may be completed to reduce visible particles. In such embodiments, particles greater than 1 pm may be filtered out.

[0070] Once manufactured, DOTAP: cholesterol liposomes can be used to encapsulate nucleic acids (e.g., a nucleic acid construct as described herein) resulting in the non-viral vectors described herein. In some embodiments, a non-viral vector is prepared by diluting nucleic acid constructs and lipids (DOTAP:cholesterol) in 5% dextrose in water to obtain an appropriate concentration of nucleic acid constructs and lipids (DOTAP:cholesterol). The nucleic acid constructs can be added to the DOTAP:cholesterol liposomes in a range of concentrations as indicated above. For example, equal volumes of nucleic acid construct and DOTAP: cholesterol, at a concentration to obtain about 100 pg of nucleic acid construct/about 0.1 to 4 mM lipids/about 100 pl, can be mixed by adding the nucleic acid construct rapidly to the DOTAP:cholesterol solution followed by rapid mixing.

[0071] In other methods, non-viral vectors can be produced using the heating, sonicating, and sequential extrusion methods described above. In some embodiments, non-viral vectors are produced using the microfluidization methods described above.

[0072] Once non-viral vectors are produced, they can be characterized using any suitable method. For example, mean particle size can be determined by dynamic light scattering using a particle size analyzer (e.g., a Malvern Zetasizer or Coulter N4 particle size analyzer).

[0073] Also provided herein are viral vector comprising one or more nucleic acid constructs disclosed herein. The term “viral vector” is used herein to refers to a recombinant viral vector for delivering genetic material (e.g., a polynucleotide sequence encoding a TUSC2 and/or a TUSC4 protein) into a cell. A recombinant viral vector comprises capsid or envelope proteins and a recombinant viral genome, which is a nucleic acid construct comprising components derived from a viral genome (e.g., AAV) and heterologous polynucleotide sequences (e.g., a polynucleotide sequence encoding a TUSC2 and/or a TUSC4 protein or other therapeutic nucleic acid expression cassette). Examples of viral vectors include, but are not limited to, AAV vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, herpesvirus vectors, alphavirus vectors, and the like.

[0074] A “recombinant AAV vector” or “rAAV vector” comprises a rAAV genome derived from the wildtype genome of AAV. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild type AAV genome are retained in the rAAV vector. A recombinant viral genome can be packaged into a virus (also referred to herein as a “particle” or “virion”) for subsequent infection (transformation) of a cell, ex vivo, in vitro, or in vivo. Where a rAAV genome is encapsidated or packaged into an AAV particle, the particle can be referred to as a “rAAV.” Such particles or virions include proteins that encapsidate or package the viral genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins (VP1, VP2, VP3). As used herein, the term “serotype” refers to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). Recombinant AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8, and variants thereof. Examples of rAAV can include capsid proteins of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8, or a capsid variant of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8, or a capsid variant of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8. Particular capsid variants include a capsid sequence with an amino acid substitution, deletion or insertion/addition.

[0075] A rAAV vector can comprise a genome derived from an AAV serotype distinct from the AAV serotype of one or more of the capsid proteins that package the recombinant viral genome. rAAV particles (vectors) can include one or more capsid proteins from a different serotype, a mixture of serotypes, or hybrids or chimeras of different serotypes, such as a VP1, VP2 or VP3 capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8 serotype. In some embodiments, an AAV serotype having a specific tissue tropism is used. rAAV can be produced using any suitable methods. Methods for large-scale production of rAAV are known and are described in, e.g., Urabe M. J. (2006) Virol. 80: 1874-1885; Kotin R.M. (2011) Hum. Mol. Genet. 20:R2- 6; Kohlbrenner E. et al. (2005) Mol. Ther. 12: 1217-1225; Mietzsch M. (2014) Hum. Gene Ther. 25:212-222; and U.S. Patent Nos. 6,436,392, 7,241,447, and 8,236,557.

[0076] Pharmaceutical Compositions

[0077] Provided are pharmaceutical compositions including the nucleic acid constructs, non-viral vectors, or viral vectors are described herein. In some embodiments, the pharmaceutical composition includes a nucleic acid construct or a vector as described herein and dextrose, e.g., about 5% dextrose in water or saline. In other embodiments, the pharmaceutical composition includes a nucleic acid construct or a vector as described herein and about 0.9% (e.g., 0.8%, 0.9%, 1.0%, etc.) sodium chloride. In additional embodiments, the pharmaceutical composition includes a nucleic acid construct or a vector comprising a nucleic acid construct described herein and a combination of about 5% dextrose and about 0.9% sodium chloride. The vector can be a non-viral vector.

[0078] The pharmaceutical compositions, nucleic acid constructs, non-viral vectors, and viral vectors described herein may be administered to mammals (e.g., rodents, humans, nonhuman primates, canines, felines, ovines, bovines) in a suitable formulation according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2000) and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (1988-1999)). A description of illustrative pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington. Other substances may be added to the pharmaceutical compositions to stabilize and/or preserve the pharmaceutical compositions. As used herein the terms “pharmaceutically acceptable” means a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A pharmaceutically acceptable excipient is a material that is not biologically or otherwise undesirable, e.g, the material may be administered to a subject without causing substantial undesirable biological effects. In embodiments, the pharmaceutical composition may comprise components that are generally regarded as safe.

[0079] The pharmaceutical compositions described herein may be in a form suitable for sterile injection or infusion. To prepare such a pharmaceutical composition, the active therapeutic(s) (e.g, a nucleic acid construct or a vector disclosed herein) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles, diluents and solvents that may be employed are water; water adjusted to a suitable pH by addition of an appropriate amount of a pH modifier (e.g., acid or base) or a suitable buffer; Ringer’s solution; isotonic sodium chloride solution; and dextrose solution. For example, in one embodiment, the vectors may be administered over 0.5 to several hours by infusion with a pharmaceutically acceptable diluent such as 5% dextrose in water, Ringer’s, and/or 0.9% NaCl. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the therapeutics is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

[0080] In other embodiments, the pharmaceutical compositions described herein may be in a form suitable for intranasal administration. In one embodiment, the intranasal formulation is an aqueous formulation including a nucleic acid construct, viral vector, or pharmaceutical composition as described herein, a pH modifying agent, and a thickening agent. In the intranasal formulation, the pH modifying agent may provide or adjust the pH of the formulation to a suitable pH, e.g., a pH that assists in solubilizing an active agent in solution. In some embodiments, the intranasal formulation is administered as a stable intranasal spray that provides sufficient residence time on the nasal mucosa to allow trans-nasal absorption of the active agent(s). The thickening agent of the intranasal formulations described herein may modify the viscosity of the formulation to provide improved adherence of the formulation to the nasal mucosa without adversely affecting the ease of administration as an intranasal spray. The thickening agent may additionally increase the residence time of the formulation on the nasal mucosa, reduce loss of the formulation via mucociliary clearance of the nasal passages and/or improve the trans-nasal absorption. Such intranasal formulations may provide a sustained or controlled release of a nucleic acid construct or a vector as described herein.

[0081] Administration and Methods of Treatment

[0082] The present disclosure provides a method of treating cancer by administering to a patient in need thereof (i) a nucleic acid construct for the expression of TUSC2 (a TUSC2 expression construct) and (ii) a nucleic acid construct for the expression of TUSC4 (a TUSC4 expression construct), e.g., by administering to the patient a pharmaceutical composition disclosed herein. . In embodiments, the cancer is lung cancer, including, for example, Small Cell Lung Cancer (SCLC). In embodiments, the lung cancer is Non-Small Cell Lung Cancer (NSCLC). In further embodiments, the lung cancer is an adenocarcinoma. The TUSC2 and TUSC4 expression constructs can be administered to the patient in a pharmaceutical composition comprising both the TUSC2 and TUSC4 expression constructs. Alternatively, the TUSC2 and TUSC4 expression constructs can be administered to the patient in separate pharmaceutical compositions. In other embodiments, the disclosure provides a method of treating cancer by administering to a patient in need thereof, a nucleic acid construct for the expression of both the TUSC2 and TUSC4 proteins.

[0083] In embodiments, the present disclosure provides a method of suppressing or inhibiting the growth of a tumor in a patient in need thereof by administering to the patient (i) a nucleic acid construct for the expression of TUSC2 (a TUSC2 expression construct) and (ii) a nucleic acid construct for the expression of TUSC4 (a TUSC4 expression construct), e.g., by administering to the patient a pharmaceutical composition disclosed herein. In embodiments, the tumor is lung cancer, including, for example, Small Cell Lung Cancer (SCLC). In embodiments, the lung cancer is Non-Small Cell Lung Cancer (NSCLC). In further embodiments, the lung cancer is an adenocarcinoma. The TUSC2 and TUSC4 expression constructs can be administered to the patient in a pharmaceutical composition comprising both the TUSC2 and TUSC4 expression constructs. Alternatively, the TUSC2 and TUSC4 expression constructs can be administered to the patient in separate pharmaceutical compositions. In other embodiments, the disclosure provides a method of treating cancer by administering to a patient in need thereof, a nucleic acid construct for the expression of both the TUSC2 and TUSC4 proteins.

[0084] The nucleic acid constructs, non-viral vectors, viral vectors, and pharmaceutical compositions described herein are preferably administered to a mammal (e.g., human) in a therapeutically effective amount. By the phrases “therapeutically effective amount”, “effective amount” and “effective dosage” is meant an amount sufficient to produce a therapeutically (e.g., clinically) desirable result; for example, the result can include increasing or restoring TUSC2 and/or TUSC4 express! on/signaling to TUSC2 and/or TUSC4-deficient cancer cells, inducing apoptosis of cancer cells, decreasing tumor size, eliminating a tumor, or preventing or reducing metastasis in a subject. Dosage for a subject may depend on multiple factors, including the subject’s size, body surface area, creatine clearance, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. A delivery dose of a nucleic acid construct, non-viral vector, viral vector or composition as described herein is determined based on preclinical efficacy and safety.

[0085] In some embodiments, a therapeutically effective amount of nucleic acid construct or vector as described herein or a pharmaceutical composition containing a therapeutically effective amount of the nucleic acid construct or vector is injected intravenously. In other embodiments, a therapeutically effective amount of a nucleic acid construct or a vector as described herein or a composition containing a therapeutically effective amount of a nucleic acid construct or vector is administered intranasally. The nucleic acid constructs, non-viral vectors, viral vectors, and pharmaceutical compositions can be administered, for example, as a “unit dose.” A unit dose as used herein is defined as containing a predetermined quantity of the therapeutic agent calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. A unit dose as described herein may be described in terms of nucleic acid mass (pg) of the nucleic acid construct in the lipid complex. Unit doses range from 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1000 pg and higher.

[0086] Provided herein are methods of treating cancer in a subject. As used herein, the term “treating cancer” means administration of a therapeutic agent (e.g., nucleic acid constructs, vectors, or pharmaceutical compositions as described herein) to a patient having cancer with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, one or more symptoms of the disease, or predisposition toward disease. The treatment methods described herein inhibit, decrease or reduce one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases or complications caused by or associated with cancer, including for example, increasing or restoring TUSC2 and/or TUSC4 to TUSC2 and/or TUSC4-deficient cancer cells, inducing apoptosis of cancer cells, decreasing tumor size or eliminating a tumor in a subject, and/or reducing or preventing metastasis. Methods of treating cancer generally include increasing or restoring TUSC2 and/or TUSC4 signaling/expression to cancer cells that have reduced TUSC2 and/or TUSC4 levels or inhibition of TUSC2 and/or TUSC4 signaling. In embodiments, the cancer is colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, leukemia, neuroblastoma, lung cancer, prostate cancer, renal cancer, or rectal cancer. In embodiments, the cancer is positron emission tomography (PET) positive cancer. In one embodiment of a method of treating cancer, a pharmaceutical composition including a nucleic acid construct as described herein is administered to a human subject in need thereof. In another embodiment of a method of treating cancer, a pharmaceutical composition including a vector as described herein is administered to a human subject in need thereof. In an embodiment of a method of treating cancer, a pharmaceutical composition comprising a non-viral vector described herein is administered to a human subject in need thereof. In an embodiment of a method of treating cancer, a pharmaceutical composition comprising a viral vector described herein is administered to a human subject in need thereof.

[0087] In embodiments, provided is a method for generating or augmenting an anti-tumor immune response in a human subject, comprising administering to the human subject in need thereof a pharmaceutical composition comprising a nucleic acid construct disclosed herein that expresses TUSC2 and/or TUSC4 (e.g., from the codon optimized TUSC2 and/or TUSC4 coding sequences disclosed herein). In embodiments, the method for generating or augmenting an anti-tumor therapeutic immune response comprises administering to the human subject in need thereof a pharmaceutical composition comprising a viral vector or a non-viral vector disclosed herein (e.g., DOTAP/cholesterol liposomes with a TUSC2 and/or TUSC4 expression construct). In embodiments, the method for generating or augmenting an anti -tumor immune response comprises administering to the human subject in need thereof the pharmaceutical compositions disclosed herein.

[0088] Any suitable methods of administering nucleic acid constructs, non-viral vectors, viral vectors, and pharmaceutical compositions to a subject in need thereof may be used. In these methods, the nucleic acid constructs, non-viral vectors, viral vectors, and pharmaceutical compositions can be administered to the human subject by any suitable route. In some embodiments, for example, they are administered intravenously (IV). If administered via IV injection, the nucleic acid constructs, vectors, and pharmaceutical compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously, pump infusion). In other embodiments, for example, they are administered intranasally. The nucleic acid constructs, vectors, and pharmaceutical compositions can be administered to the human subject once (at one time point), or more than one time (e.g., two times, three times, four times, five times, six times, seven times, eight times, nine times, 10 times, etc.), z.e., at multiple time points. When the nucleic acid constructs, vectors, or pharmaceutical compositions are administered multiple times, the administrations may be separated by one day, three days, one week, two weeks, three weeks, one month, two months, or six months.

[0089] Some methods of treatment described herein are combination therapies that include administering to the human subject one or more nucleic acid constructs, non-viral vectors, viral vectors, or pharmaceutical compositions as described herein (i.e., for expression of TUSC2/TUSC4), and an additional anti-cancer therapy. In embodiments, the additional anticancer therapy is radiation therapy. In embodiments the additional anti-cancer therapy is chemotherapy, including, but not limited to, an alkylating agent (e.g., a platin — including carboplatin, cisplatin, or oxaliplatin — cyclophosphamide, melphalan, and temozolomide), an antimetabolite (e.g., 5 -fluorouracil (5-FU), 6-mercaptopurine, cytarabine, gemcitabine, and methotrexate), an antitumor antibiotic (e.g., actinomycin-D, bleomycin, daunorubicin, and doxorubicin), and topoisomerase inhibitors (e.g., etoposide, irinotecan, teniposide, and topotecan). Another example of an additional anti-cancer therapy is a checkpoint inhibitor. Use of checkpoint inhibitors as immunotherapy for treating cancer is known in the art (see US Patent Application Nos. 15/536718; 15/216585; 15/648423; 16/144549). Examples of checkpoint inhibitors include PD-L1 inhibitors and PD-1 inhibitors such as pembrolizumab, Bavencio® (avelumab) and Tecentriq® (atezolizumab). Other examples of checkpoint inhibitors include Keytruda® (pembrolizumab) and Opdivo® (nivolumab). A further example of an additional anti-cancer therapy is aBRAF inhibitor such as encorafenib. Another example of an additional anti-cancer therapy is an EGFR inhibitor. Examples of EGFR inhibitors include cetuximab, osimertinib, Tarceva® (erlotinib), and nivolumab. In embodiments, the additional anti-cancer therapy is a KRAS inhibitor. In embodiments, the additional anti-cancer therapy is additional nucleic acid construct. In embodiments of a combination therapy as described herein, the nucleic acid constructs, non-viral vectors, viral vectors, or pharmaceutical compositions are administered to the human subject before the additional anticancer therapy is administered to the human subject (z.e., at two different time points). In another embodiment, the nucleic acid constructs, non-viral vectors, viral vectors, or pharmaceutical compositions are administered to the human subject at the same time that (concurrently with) the additional anti-cancer therapy is administered. In another embodiment, the nucleic acid constructs, non-viral vectors, viral vectors, or pharmaceutical compositions are administered to the human subject after the additional anti-cancer therapy is administered to the human subject (z.e., at two different time points). In some embodiments, a pharmaceutical composition as described herein can comprise one or more nucleic acid constructs, non-viral vectors, or viral vectors as described herein and an additional anti-cancer therapy (e.g., a checkpoint inhibitor, a BRAF inhibitor, an EGFR inhibitor, etc.), i.e., admixed in the same injection or infusion volume.

[0090] The terms “patient,” “subject,” and “individual” are used interchangeably herein, and mean a mammalian (e.g., human) subject in need of treatment with a nucleic acid construct, a vector, or a pharmaceutical composition comprising a sequence encoding TUSC2 and/or TUSC4 (e.g., for treatment of cancer). Human subjects suffering from cancer include individuals suffering from various types of cancers, such as colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, rectal cancer, lung cancer (e.g., small cell or nonsmall cell lung cancer), leukemia, and neuroblastoma. In the methods described herein, the subject can be undergoing surgery for any reason, such as for removal of diseased tissue, and/or radiation treatment. For example, in some embodiments of the methods described herein, the subject is undergoing, or has undergone, surgical resection of a tumor. As another example, in some embodiments of the methods described herein, the subject is undergoing, or has undergone, radiation treatment. As another example, in some embodiments of the methods described herein, the subject is undergoing, or has undergone, chemotherapy. In some embodiments of the methods described herein, the subject is undergoing, or has undergone, surgery (e.g., resection of a tumor) and/or radiation treatment and/or chemotherapy.

[0091] In some methods of treating cancer in a subject, the method includes selecting a subject having a cancer for administration of a therapy as described herein (e.g., administration of one or more pharmaceutical compositions containing one or more nucleic acid constructs or vectors disclosed herein). A subject may be selected for therapy based on the presence of one or more mutations or other molecular markers for cancer in the subject’s cancer cells. For example, a patient’s tumor may be screened for having one or more mutations associated with a particular type of cancer, or types of cancer, including, e.g., BRAF mutations found in melanoma and colorectal cancer. Thus, a subject having cancer cells with the BRAF V600E mutation can be selected for receiving a therapy as described herein. Molecular markers associated with colon cancer are listed below in Table 1.

Table 1. Gene mutations and molecular markers associated with colon cancer.

[0092] Molecular markers are known for several other cancers. For example, several EGFR mutations are associated with non-small cell lung cancer (NSCLC), adenocarcinoma, and squamous cell carcinoma. These mutations include: exon 19 deletion, exon 21 L858R substitution, exon 20 T790M mutation, exon 19 deletion and T790M, and exon 21 (L858R) and T790M. Several KRAS mutations are associated with NSCLC, adenocarcinoma and squamous cell carcinoma, including G12C, G12D and G12V. Other mutations associated with NSCLC, adenocarcinoma and squamous cell carcinoma include mutations in ALK, MET exon 14, PIK3CA, BRAF (V600E) and ROS 1. A human subject having one or more of any of these mutations can be selected for treatment with the pharmaceutical compositions, nucleic acid constructs, vectors, and methods described herein.

[0093] In some embodiments, subjects with a cancer having microsatellite instability (MSI) are selected for treatment with the therapies described herein. MSI is an important factor in the occurrence and development of tumors (e.g., gastric cancer, colon cancer, breast cancer) and molecular marker for cancer. MSI tumors may be characterized by high MSI (MSI-H) or low MSI (MSI-L). In some embodiments, a tumor characterized by MSI contains cells with MSI-H. A cell with high MSI is typically a cell having MSI at a level higher than a reference value or a control cell, e.g., a non-cancerous cell of the same tissue type as the cancer. In embodiments, nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein are administered to a patient having a cancer with MSI. In embodiments of a method for treating cancer in a human subject as described herein, a pharmaceutical composition, nucleic acid construct, or vector as described herein is administered to a human subject having a tumor characterized by MSI alone, or in combination with administration of a checkpoint inhibitor (e.g., pembrolizumab, nivolumab, etc.).

[0094] In embodiments, subjects with a cancer that is deficient in mismatch repair (dMMR) are selected for treatment with the therapies described herein. MMR deficiency is most common in colorectal cancer, other types of gastrointestinal cancer, and endometrial cancer, but may also be found in cancers of the breast, prostate, bladder, and thyroid.

[0095] Human subjects having cancer cells with a particular mutation (e.g., BRAF V600E) and/or having MSI and/or dMMR can be treated with one or more nucleic acid constructs, non-viral vectors, viral vectors, or pharmaceutical compositions as described herein (i.e., for expression of TUSC2/TUSC4), or can be treated with a combination therapy as described herein. In one embodiment of a combination therapy as described herein, the additional anticancer therapy is specific for a cancer associated with a particular mutation. For example, if a human subject has a BRAF V600E mutation, the additional anti-cancer therapy may be a BRAF inhibitor (e.g., encorafenib), or may be a combination of drugs including, for example, a BRAF inhibitor, e.g., encorafenib, cetuximab, and/or Mektovi® (binimetinib). In another embodiment of a combination therapy as described herein, the additional anti-cancer therapy is specific for a cancer with MSI. For example, if a human subject has an MSI tumor, the additional anti-cancer therapy may be a checkpoint inhibitor such as pembrolizumab or nivolumab. As another example, if the human subject has cancer cells with a KRAS mutation (e.g. a G12C mutation), the additional anti-cancer therapy may be a KRAS inhibitor (e.g. sotorasib). In yet another embodiment of a combination therapy as described herein, the additional anti-cancer therapy is specific for colon cancer. For example, if a human subject has colon cancer, the additional anti-cancer therapy may be a cyclin dependent kinase (CDK) inhibitor. [0096] Provided herein are methods of treating cancers that have elevated glucose uptake due to the Warburg Effect. The cancer cells utilize aerobic glycolysis deriving most of their energy from glycolysis (glucose converted to lactate followed by lactate fermentation) even when oxygen is available, rather than utilizing oxidative respiration. The enhanced glucose demand of these cancers can be detected using [¹⁸F] 2-fluoro-2-deoxy-D-glucose (¹⁸F-FDG) PET imaging (e.g., PET/computerized tomography (CT) imaging). ¹⁸F-FDG (a glucose analog) is administered to the patient and is taken up by cells via glucose transporter proteins. The glucose analog then undergoes phosphorylation by hexokinase to FDG-6 phosphate. Unlike glucose, FDG-6 phosphate does not undergo further metabolism and so becomes trapped in the cell as the cell membrane is impermeable to FDG-6 phosphate following phosphorylation. PET positive cancers are those that are identified as having increased glucose demand and thus increased accumulation of the radiolabeled glucose analog using PET imaging. Other methods for detecting elevated glucose demand of cancer cells that are utilizing aerobic glycolysis can be used in the disclosed methods as an alternative to ¹⁸F-FDG PET imaging. For example, glucose uptake by cancer cells may be measured using labeled glucose or glucose analogs including 2-deoxy-D-[l,2-³H] -glucose, 2-deoxy-D-[l-¹⁴C]- glucose, and 2-[7V-(7-nitrobenz-2-oxa-l,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). In addition, glycolytic flux can be determined by measuring metabolites of glycolysis and in particular, lactate production. Other methods for detecting elevated glucose demand of cancer cells that are utilizing aerobic glycolysis include bioanalytic methods such as glycolytic rate assay (e.g., Agilent Seahorse XF Glycolytic Rate Assay; see also the Agilent Seahorse XF Cell Mito Stress Test).

[0097] Table 2 provide an overview of the nucleic acid sequences (see also Table 3) and amino acid sequences (see also Table 4) disclosed herein.

[0098] Table 2. Summary of sequences disclosed herein.

[0099] Table 3. Nucleic acid sequences.

[0100] Table 4. Amino acid sequences.

[0101] It is to be understood and expected that variations of the compositions of matter and methods herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present disclosure. All references cited herein are hereby incorporated by reference in their entirety. EXAMPLES

[0102] Example 1: Effect of co-expression of TUSC2 and TUSC 4 (NPRL2) in mice bearing LLC2 tumors

[0103] Endogenous expression of TUSC2 and TUSC4 in LLC2-luc and H841 cells was examined by western blotting. LLC2-luc and H841 were seeded at 700,000 cells per well in 6-well plates in RPMI with 10% FBS. After three days the cells were harvested and lysed in Laemili lysis buffer. Total protein was quantified and samples run on a 4-20 % acrylamide gel followed by western blotting. Membrane was probed with anti-TUSC2 and anti-TUSC4 antibodies (1 : 1000) followed by secondary antibody (1 :4000) and detection using Clarity ECL (BioRad). Neither LLC2-luc cell nor H841 cells expressed detectable levels of TUSC2 or TUSC4.

[0104] To test the anti-cancer effect of a combination of a TUSC2 and a TUSC4 expression construct, a syngeneic mouse model with LLC2 (Lewis lung carcinoma) tumors, which are KRAS mutant and anti-PDl resistant, was used. Expression constructs for TUSC2 and TUSC4 expression were separately loaded in DOTAP/cholesterol liposomes (treatment nanovesicles). Nanovesicles loaded with empty plasmid served as a control. In addition, the anti-cancer effect of the TUSC2/TUSC4 combination treatment was compared to treatment with the anti-PDl antibody pembrolizumab and with a combination of anti-PDl antibody and the TUSC4 nanovesicles.

[0105] LLC2-luc cells (10⁶) were injected subcutaneously into syngeneic mice (n=9 per each treatment group and control). After 7 to 10 days, mice in the treatment groups received (i) anti-PDl antibody (250 pg i.p., two times a week for 3 weeks); (ii) TUSC4 expression construct in DOTAP/cholesterol liposomes (25 pg i.v., every 3 days for 3 weeks); (iii) TUSC2 expression construct in DOTAP/cholesterol liposomes (25 pg i.v., every 3 days for 3 weeks); (iv) combination of (i) and (ii); or (v) combination of (ii) and (iii). Tumor volume was assessed over time using in vivo imaging system (IVIS).

[0106] As shown in Fig. 1A, treatment with a combination of TUSC2 and TUSC4 results in increased inhibition of tumor growth in mice bearing LLC2 tumors as compared to treatment with either TUSC2 or TUSC4 alone. Further, as shown in Fig. IB, treatment with a combination of TUSC2 and TUSC4 inhibited tumor growth in mice more effectively than treatment with (1) an anti-PDl antibody or (2) a combination of TUSC4 (NPRL2) and an anti- PD-L1 antibody. [0107] Example 2: Antitumor effect of a combination of a liposome-complexed TUSC2 expression construct and a liposome-complexed TUSC4 expression construct in humanized mice bearing H841 small cell lung cancer (SCLC) tumors

[0108] To test the antitumor effect of a TUSC2/TUSC4 combination on H841 SCLC cells, mice were humanized. The humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice(hu-NSG). The mice were subcutaneously injected with H841 cancer cells (5 mil/inj ection) and tumors developed following 3-4 weeks (Fig. 5A). Nine weeks later, 5 million H841 SCLC cells were injected subcutaneously. After three weeks, mice (n = 8 per group) were treated every three days for three weeks with (1) a control empty plasmid complexed with DOTAP/cholesterol liposome, (2) TUSC2 expression plasmid complexed with DOTAP/cholesterol liposome (25 pg/mouse i.v.), (3) TUSC4 expression plasmid complexed with DOTAP/cholesterol liposome (25 pg/mouse i.v.), or (4) an expression plasmid expressing TUSC2 complexed with a DOTAP/cholesterol liposome and an expression plasmid expressing TUSC4 complexed with a DOTAP/cholesterol liposome (25 pg each/mouse i.v.). Tumor volume was assessed over time using I VIS.

[0109] As shown in Figs. 2B-2C, a combination of a liposome-complexed TUSC2 expression construct and a liposome-complexed TUSC4 expression construct inhibited tumor growth in mice bearing H841 SCLC cells more effectively than liposome-complexed expression constructs expressing either TUSC2 or TUSC4 alone.

[0110] Example 3: Antitumor effect of liposome-complexed TUSC2/TUSC4 expression construct on A549 lung metastasis in non-humanized mice bearing A549 tumors

[OHl] To test the effect of a liposome-complexed expression construct expressing both TUSC2 and TUSC4 on anti-PDl resistant A549 human lung adenocarcinoma cells (KRAS/STK11 mutant), a TUSC2/TUSC4 expression construct (Fig. 3) was generated based on a plasmid derived from a NTC9385R plasmid (J. A. Williams, Vaccines 2013 1 :225-249; Borggren et al., Hum Vaccin Immunother. 2015 11(8): 1983-1990), which is commercially available (Nature Technologies Corporation, Lincoln, NE, US).

[0112] Mice were injected intravenously with one million A549-luc cells. This cell line constitutively expresses high levels of enzymatically active luciferase protein, which can be detected via in vitro and in vivo bioluminescence assays. After 8-10 days, the mice were imaged with an in vivo imaging system (IVIS) for a pre-treatment baseline. Mice (n = 10 per group) were treated every three days for three weeks with (1) DOTAP/cholesterol liposome- complexed empty vector (control); (2) DOTAP/cholesterol liposome-complexed TUSC2 expression plasmid (Quaratusugene Ozeplasmid, Reqorsa®) (50 pg/mouse i.v.); (3) a DOTAP/cholesterol liposome-complexed TUSC4 expression plasmid (50 pg/mouse i.v.); (4) a DOTAP/cholesterol liposome-complexed TUSC2/TUSC4 expression plasmid (50 pg/mouse i.v.); and (5) a combination of Quaratusugene Ozeplasmid and a liposome-complexed TUSC4 expression plasmid (50 pg each/mouse i.v.). After the treatment, mice were imaged with IVIS. [0113] Figs. 4A-4C show the results of in vivo imaging at week 4 after the start of treatment.

[0114] Example 4: Effect of TUSC2 and NPRL2 (TUSC4) dual gene therapy effect in the tumor microenvironment (TME)

[0115] To examine the effect of TUSC2 and NPRL2 (TUSC4) dual gene therapy in the tumor microenvironment (TME), mice bearing LLC2 tumors were intravenously treated with the dual gene therapy, control, TUSC2 gene therapy, or NPRL2 gene therapy. After T cells were harvested from the mice, CD8⁺ T cells were confirmed using flow cytometry analysis based on CD8⁺ FITC and mCD3-PerCp-Cy5.5 staining (Fig. 5A). CD4+ T cells were also confirmed using flow cytometry. LLC2 tumors treated with TUSC2 or NPRL2 (TUSC4) gene therapies alone or in combination exhibited an increase in the percent of CD8+ T cells (Fig. 5B). Additionally, LLC2 tumors treated with TUSC2/ NPRL2 (TUSC4) combination therapy exhibited a significant increase in CD4+ T cells (helper T cells) compared to LLC tumors treated only with TUSC2 gene therapy (Fig. 5C).

[0116] The percentage of CD8+CD69+ T cells were also evaluated. CD8+CD69+ T cells were identified with flow cytometry based on mCD8-FITX and mCD69-APC-Cy7 staining (Fig. 5D). CD69 is a marker used to identify tissue resident memory T cells. LLC2 tumors treated with TUSC2/ NPRL2 (TUSC4) combination therapy exhibited a significant increase in the percent of CD8⁺CD69⁺ T cells (Fig. 5E).

[0117] To further examine the effect of TUSC2 and NPRL2 (TUSC4) dual gene therapy in the TME, the percent of MHCII⁺ dendritic cells (DCs) were also determined. After dendritic cells were harvested from the mice, MHCII⁺ DCs were identified with flow cytometry based on mMHCII-PerCP-Cy5.5 staining (Fig. 5F). LLC2 tumors treated with TUSC2/NPRL2 (TUSC4) combination therapy exhibited a significant increase in the percent of MHCII DCs compared to control -treated tumors (Fig. 5G).

[0118] Additionally, LLC2 tumors treated with TUSC2/ NPRL2 (TUSC4) combination therapy exhibited a significant decrease in the percent of myeloid-derived suppressor cells ( MDSCs) (Fig. 51). MDSCs were identified using flow cytometry based on Gr-l-APC-Cy7 and mMHCII-PerCP-Cy5.5 staining (Fig. 5H). Collectively, these results indicate that the antitumor tumor effect of the TUSC2/NPRL2 (TUSC4) dual gene therapy was associated with increased infiltration of CD8⁺ T cells, CD4⁺ T cells, CD8⁺CD69⁺ T cells, MHCII⁺ DCs, and a decreased infiltration of MDSCs.

Claims

CLAIMS We Claim:

1. A nucleic acid construct comprising (a) a nucleotide sequence encoding a TUmor Suppressor Candidate 2 (TUSC2) protein and (b) a nucleotide sequence encoding a TUmor Suppressor Candidate 4 (TUSC4) protein.

2. A pharmaceutical composition comprising (a) a first nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome, (b) a second nucleic acid construct comprising nucleotide sequence TUSC4 protein complexed with a liposome, and (c) a pharmaceutically acceptable excipient.

3. The nucleic acid construct of claim 1 or the pharmaceutical composition of claim 2, wherein the TUSC2 protein is human TUSC2.

4. The nucleic acid construct of claim 1 or the pharmaceutical composition of claim 2, wherein the TUSC2 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

5. The nucleic acid construct or the pharmaceutical composition of claim 4, wherein the TUSC2 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.

6. The nucleic acid construct or the pharmaceutical composition of claim 5, wherein the TUSC2 protein comprises SEQ ID NO: 14.

7. The nucleic acid construct of any one of claims 1 or claims 3-6 or the pharmaceutical composition of any one of claims 2-6, wherein the nucleotide sequence encoding the TUSC2 protein is codon-optimized.

8. The nucleic acid construct of any one of claims 1 or claims 3-6 or the pharmaceutical composition of any one of claims 2-6, wherein the nucleotide sequence encoding the TUSC2 protein comprises a sequence that is at least 80% identical to SEQ ID NO: 1 or 2.

9. The nucleic acid construct or the pharmaceutical composition of claim 8, wherein the nucleotide sequence encoding TUSC2 protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

10. The nucleic acid construct or the pharmaceutical composition of claim 9, wherein the nucleotide sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2.

11. The nucleic acid construct of any one of claims 1 or 3-10 or the pharmaceutical composition of any one of claims 2-10, wherein the TUSC4 protein is human TUSC4.

12. The nucleic acid construct of any one of claims 1 or 3-10 or the pharmaceutical composition of any one of claims 2-10, wherein the TUSC4 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 15.

13. The nucleic acid construct or the pharmaceutical composition of claim 12, wherein the TUSC4 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15.

14. The nucleic acid construct or the pharmaceutical composition of claim 13, wherein the TUSC4 protein comprises SEQ ID NO: 15.

15. The nucleic acid construct of any one of claims 1 or 3-14 or the pharmaceutical composition of any one of claims 2-14, wherein the nucleotide sequence encoding the TUSC4 protein is codon-optimized.

16. The nucleic acid construct of any one of claims 1 or 3-14 or the pharmaceutical composition of any one of claims 2-14, wherein the nucleotide sequence encoding the TUSC4 protein comprises a sequence that is at least 80% identical to SEQ ID NO:3 or 4.

17. The nucleic acid construct or the pharmaceutical composition of claim 16, wherein the nucleotide sequence encoding the TUSC4 protein comprises a sequence that is at least 90% identical to SEQ ID NO:3 or 4.

18. The nucleic acid construct or the pharmaceutical composition of claim 17, wherein the nucleotide sequence encoding the TUSC4 protein comprises SEQ ID NO:3 or 4.

19. The nucleic acid construct of any one of claims 1 or 3-18, the nucleic acid construct further comprising (a) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein and/or (b) a CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein.

20. The nucleic acid construct of claim 19, wherein the CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:21.

21. The nucleic acid construct of claim 20, wherein the CMV promoter comprises SEQ ID NO:21.

22. The nucleic acid construct of any one of claims 19-21, the nucleic acid construct further comprising a CMV enhancer.

23. The nucleic acid construct of claim 21, wherein the CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 19 or 20.

24. The nucleic acid construct of claim 23, wherein the CMV enhancer comprises SEQ ID NO: 19 or 20.

25. The nucleic acid construct of any one of claims 1 or 3-24, the nucleic acid construct further comprising a Human T-cell leukemia virus type I (HTLV-I) regulatory sequence.

26. The nucleic acid construct of claim 25, wherein the HTLV-I regulatory sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8.

27. The nucleic acid construct of claim 26, wherein the HTLV-I regulatory sequence comprises SEQ ID NO:8.

28. The nucleic acid construct of any one of claims 1 or 3-27, the nucleic acid construct further comprising a bovine growth hormone polyadenylation (BGH poly A) sequence.

29. The nucleic acid construct of claim 28, wherein the BGH polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NO:9.

30. The nucleic acid construct of claim 29, wherein the BGH polyA sequence comprises SEQ ID N0:9.

31. The nucleic acid construct of any one of claims 1 or 3-30, wherein the nucleic acid construct further comprises a splicing enhancer sequence.

32. The nucleic acid construct of claim 31, wherein the splicing enhancer sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 12.

33. The nucleic acid construct of claim 32, wherein the splicing enhancer sequence comprises SEQ ID NO: 12.

34. The nucleic acid construct of any one of claims 1 or 3-33, wherein the nucleic acid construct further comprises at least one intron.

35. The nucleic acid construct of claim 34, wherein the at least one intron is a P-globin intron.

36. The nucleic acid construct of claim 35, wherein the P-globin intron sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 10.

37. The nucleic acid construct of claim 36, wherein the P-globin intron sequence comprises SEQ ID NO: 10.

38. The nucleic acid construct of any one of claims 1 or 3-37, wherein the nucleic acid construct further comprises a bacterial backbone sequence.

39. The nucleic acid construct of claim 38, wherein the bacterial backbone sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11.

40. The nucleic acid construct of claim 39, wherein the bacterial backbone sequence comprises SEQ ID NO: 11.

41. The nucleic acid construct of any one of claims 38-40, wherein the bacterial backbone sequence comprises a R6K origin sequence.

42. The nucleic acid construct of any one of claims 38-41, wherein the bacterial backbone sequence comprises at least one selectable marker.

43. The nucleic acid construct of claim 1, wherein the nucleic acid construct comprises a sequence that is at least 80% identical to SEQ ID NO: 13.

44. The nucleic acid construct of claim 43, wherein the nucleic acid construct comprises a sequence that is at least 90% identical to SEQ ID NO: 13.

45. The nucleic acid construct of claim 44, wherein the nucleic acid construct comprises SEQ ID NO: 13.

46. The nucleic acid construct of any one of claims 1 or 3-45, wherein the nucleic acid construct is complexed with a liposome.

47. The pharmaceutical composition of any one of claims 2-18, wherein:

(a) the first nucleic acid construct further comprises a first CMV promoter operably linked to the nucleotide sequence encoding the TUSC2 protein; and/or

(b) the second nucleic acid construct further comprises a second CMV promoter operably linked to the nucleotide sequence encoding the TUSC4 protein.

48. The pharmaceutical composition of claim 47, wherein the first CMV promoter and/or the second CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:21.

49. The pharmaceutical composition of claim 48, wherein the first CMV promoter and/or the second CMV promoter comprises SEQ ID NO:21.

50. The pharmaceutical composition of any one of claims 47-49, wherein:

(a) the first nucleic acid construct further comprises a first CMV enhancer; and/or

(b) the second nucleic acid construct further comprises a second CMV enhancer.

51. The pharmaceutical composition of claim 50, wherein the first CMV enhancer and/or the second CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 19 or 20.

52. The pharmaceutical composition of claim 51, wherein the first CMV enhancer and/or the second CMV enhancer comprises SEQ ID NO: 19 or 20.

53. The pharmaceutical composition of claim 50, wherein:

(a) the first CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 19; and

(b) the second CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NOVO.

54. The pharmaceutical composition of claim 53, wherein the first CMV enhancer comprises SEQ ID NO: 19 and the second CMV enhancer comprises SEQ ID NO:20.

55. The pharmaceutical composition of any one of claims 2-18 or 47-54, wherein the first and/or the second nucleic acid construct further comprises a HTLV-I regulatory sequence.

56. The pharmaceutical composition of claim 55, wherein the HTLV-I regulatory sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8.

57. The pharmaceutical composition of claim 56, wherein the HTLV-I regulatory sequence comprises SEQ ID NO:8.

58. The pharmaceutical composition of any one of claims 2-18 or 47-57, wherein the first and/or the second nucleic acid construct further comprises a BGH polyA sequence.

59. The pharmaceutical composition of claim 58, wherein the BGH polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NOV.

60. The pharmaceutical composition of claim 59, wherein the BGH polyA sequence comprises

SEQ ID NOV.

61. The pharmaceutical composition of any one of claims 2-18 or 47-60, wherein the first and/or the second nucleic acid construct further comprises a splicing enhancer sequence.

62. The pharmaceutical composition of claim 61, wherein the splicing enhancer sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 12.

63. The pharmaceutical composition of claim 62, wherein the splicing enhancer sequence comprises SEQ ID NO: 12.

64. The pharmaceutical composition of any one of claims 2-18 or 47-63, wherein the first and/or the second nucleic acid construct further comprises at least one intron.

65. The pharmaceutical composition of claim 64, wherein the at least one intron is a P-globin intron.

66. The pharmaceutical composition of claim 65, wherein the P-globin intron sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 10.

67. The nucleic acid construct of claim 66, wherein the P-globin intron sequence comprises SEQ ID NO: 10.

68. The pharmaceutical composition of any one of claims 2-18 or 47-67, wherein the first and/or the second nucleic acid construct further comprises a bacterial backbone sequence.

69. The pharmaceutical composition of claim 68, wherein the bacterial backbone sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11.

70. The pharmaceutical composition of claim 69, wherein the bacterial backbone sequence comprises SEQ ID NO: 11.

71. The pharmaceutical composition of any one of claims 68-70, wherein the bacterial backbone sequence comprises a R6K origin sequence.

72. The pharmaceutical composition of any one of claims 68-71, wherein the bacterial backbone sequence comprises at least one selectable marker.

73. The nucleic acid construct of claim 46 or the pharmaceutical composition of any one of claims 2-18 or 47-72, wherein the liposome is a l,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP):cholesterol liposome.

74. The nucleic acid construct or the pharmaceutical composition of claim 73, wherein the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3.

75. The nucleic acid construct or the pharmaceutical composition of claim 73 or 74, wherein the DOTAP: cholesterol liposome has a particle size range of about 40 to about 250 nanometers.

76. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 46 and 73-75 and a pharmaceutically acceptable excipient.

77. The pharmaceutical composition of any one of claims any one of claims 2-18 or 47-76, the pharmaceutical composition further comprising approximately 5% dextrose, 0.9% sodium chloride, or a combination of both agents.

78. A viral vector comprising a nucleic acid construct according to any one of claims 1 and 3- 45.

79. The viral vector of claim 78, wherein the viral vector is an Adeno- Associated Virus (AAV) viral vector.

80. A pharmaceutical composition comprising the viral vector of claim 78 or 79.

81. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition according to any one of claims 2-18, 47-77, or 80.

82. The method of claim 81, wherein the subject is a human.

83. The method according to any one of claims 81 or 82, wherein the cancer is selected from the group consisting of: colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, neuroblastoma, leukemia, lung cancer, renal cancer, and rectal cancer.

84. The method according to any one of claims 81-83, wherein the cancer is positron emission tomography (PET) positive cancer.

85. The method according to any one of claims 81-84, wherein the pharmaceutical composition is administered intravenously or intranasally.

86. The method according to any one of claims 81-85, the method further comprising administering a second anti-cancer therapy to the subject.

87. The method of claim 86, wherein the second anti-cancer therapy comprises at least one of: chemotherapy, radiation treatment, and surgery.

88. The method of claim 86, wherein the second anti-cancer therapy is a checkpoint inhibitor or a BRAF inhibitor.

89. The method of claim 88, wherein the checkpoint inhibitor is pembrolizumab.

90. The method of claim 88 or 89, wherein the BRAF inhibitor is encorafenib.

91. The method of claim 86, wherein the second anti-cancer therapy is an EGFR inhibitor.

92. The method of claim 91, wherein the EGFR inhibitor is cetuximab or nivolumab.

93. The method of claim 86, wherein the second anti-cancer therapy is a KRAS inhibitor.