Methods and compositions for treating mesothelioma

Abstract

Provided herein are nucleic acid constructs comprising a polynucleotide sequence encoding a TUSC2 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 mesothelioma in a subject in need thereof. These methods can further include administering an additional anti-cancer therapy to the subject in need thereof.

Description

Docket No. 198628.46676METHODS AND COMPOSITIONS FOR TREATING MESOTHELIOMACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. Application No. 63 / 690,608, filed on September 4, 2024, the disclosure of which is incorporated by reference herein in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on July 31, 2025, is named SeqList- 198628-46676. xml and is 16,232 bytes in size.FIELD

[0003] The present disclosure relates to nucleic acid constructs, vectors, and pharmaceutical compositions for the expression of TUSC2 to treat mesothelioma. Also contemplated are methods of treating mesothelioma 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 multiple cancers. At least one allele is absent in 36% of Malignant Pleural Mesothelioma (MPM) cases. MPM is a rare, highly aggressive, asbestos-associated neoplasm with a median survival of only 10-12 months. MPM is poorly responsive to current therapies. When the TUSC2 gene is absent, an autoimmune disorder with an inflammatory background develops, resulting in tumors such as haemangiosarcomas.

[0005] 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 helix-coil domain secondary structures. At the N-terminus, TUSC2 contains a myristoylation signal (Met-Gly-X-X-X-Ser / Thr) and experiments have confirmed1175716766.2Docket No. 198628.46676 that TUSC2 is myristoyl ated. TUSC2 protein resides mostly in mitochondria and plays an important role in energy metabolism.

[0006] Accordingly, effective therapies that activate or restore TUSC2 in MPM cells, e.g., by increasing TUSC2 expression or by overcoming TUSC2 inhibition, and that are less toxic than available therapies are urgently needed.SUMMARY

[0007] Provided herein are nucleic acid constructs, vectors, and pharmaceutical compositions for the expression of TUSC2 as well as methods of using the nucleic acid constructs, vectors, and pharmaceutical compositions disclosed herein for treating mesothelioma (including MPM).

[0008] In one aspect, provided is a method for treating mesothelioma in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a nucleic acid construct comprising a nucleic acid sequence encoding a TUSC2 protein.

[0009] In one embodiment, the nucleic acid construct is complexed with a liposome. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. 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 nucleic acid sequence encoding the TUSC2 protein is codon-optimized. In embodiments, the nucleic acid sequence encoding the TUSC2 protein comprises a sequence that is at least 80% identical to SEQ ID NO: 1 or 2. In embodiments, the nucleic acid sequence encoding TUSC2 protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2. In embodiments, the nucleic acid sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2.

[0010] In embodiments, the nucleic acid construct further comprises a cytomegalovirus (CMV) promoter operably linked to the nucleic acid sequence encoding the TUSC2 protein. In embodiments, the CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:6. In one embodiment, the CMV promoter comprises SEQ ID NO:6.

[0011] 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: 16. In one embodiment, the CMV enhancer comprises SEQ ID NO: 16.2175716766.2Docket No. 198628.46676

[0012] 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: 7. In one embodiment, the HTLV-I regulatory sequence comprises SEQ ID NO:7.

[0013] 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 NO:8. In one embodiment, the BGH polyA sequence comprises SEQ ID NO:8.

[0014] 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: 11. In one embodiment, the splicing enhancer sequence comprises SEQ ID NO: 11.

[0015] 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 comprises a sequence that is at least 90% identical to SEQ ID NOV. In one embodiment, the P-globin intron comprises SEQ ID NOV.

[0016] 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: 10. In one embodiment, the bacterial backbone sequence comprises SEQ ID NO: 10. 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.

[0017] In one embodiment, the nucleic acid construct is complexed with a liposome, wherein the liposome is a l,2-bis(oleoyloxy)-3-(trimethyl ammonio) 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.

[0018] In embodiments, the pharmaceutical composition further comprises about 5% dextrose, about 0.9% sodium chloride, or a combination of both agents. In embodiments, the pharmaceutical composition is administered intravenously or intranasally.

[0019] In one embodiment, the subject is a human.

[0020] In embodiments, the mesothelioma is MPM or Peritoneal Mesothelioma.

[0021] In embodiments, the method further comprises administering a second anti-cancer therapy to the subject. In some embodiments, the second anti-cancer therapy comprises at least3175716766.2Docket No. 198628.46676 one of: chemotherapy, radiation treatment, and immunotherapy. In one embodiment, the subject is undergoing or has undergone surgery to remove a tumor from the subject.

[0022] In one embodiment, the pharmaceutical composition is administered at about 0.12 mg / kg about every 3 weeks.

[0023] In one embodiment, the method further comprises administering pembrolizumab to the subject.

[0024] In one embodiment, the method further comprises administering 2-4 cycles of pememtrexed and cisplatinum to the subject.

[0025] In one embodiment, the method further comprises administering ipilimumab and nivolumab to the subject.BRIEF DESCRIPTION OF THE FIGURES

[0026] FIGS. 1A and IB illustrate that expression of TUSC2 using a TUSC2 vector increases apoptotic activity and decreases cell invasion and cell proliferation in H2595, HP1, H2373, and H2591 mesothelial cells. FIG. 1A depicts graphs of the apoptotic activity, cell invasion, and cell proliferation in the indicated cell lines treated with PBS (negative control), empty vector (negative control), or a TUSC2-containing vector. FIG. IB is a Western blot of the TUSC2 protein levels in the five indicated cell lines in response to treatment with PBS, empty vector or a TUSC2-containing vector. GAPDH is a loading control.

[0027] FIGS. 2A and 2B illustrate that REQORSA® (Quaratusugene Ozeplasmid) increases apoptotic activity and decreases cell invasion and cell proliferation in H2595, HP1, H2373, and H2591 cells. FIG. 2A depicts graphs of the apoptotic activity, cell invasion, and cell proliferation in the indicated cell lines treated with PBS (negative control), liposomes (negative control), or REQORSA®. FIG. 2B is a Western blot of the TUSC2 protein levels in the five indicated cell lines in response to treatment with PBS, liposomes or REQORSA®. GAPDH served as a loading control.

[0028] FIG. 3 shows a Western blot of TUSC2 protein levels in LP9, H2595, HP1, H2591, and H2373 cells treated with PBS (negative control), liposomes (negative control), or REQORSA®. LP9 is a non-malignant mesothelial cell line. P-actin served as a loading control.DETAILED DESCRIPTION

[0029] Provided herein are nucleic acid constructs comprising nucleotide sequences encoding TUSC2. Also provided are non-viral and viral vectors comprising a nucleic acid4175716766.2Docket No. 198628.46676 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 mesothelioma 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 TUSC2 therapy is administered as part of a combination therapy).

[0030] Nucleic Acid Constructs

[0031] In one aspect, provided is a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein. In embodiments, the nucleotide sequence encoding the TUSC2 protein is codon-optimized.

[0032] In one aspect, provided is a pharmaceutical composition comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome. In one aspect, provided is a pharmaceutical composition comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome and a pharmaceutically acceptable excipient.

[0033] In one embodiment, the nucleic acid construct is used for recombinant production of human TUSC2 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 fermentation5175716766.2Docket No. 198628.46676 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).

[0034] In embodiments, the TUSC2 protein is human TUSC2. 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. 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.

[0035] 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.

[0036] In embodiments, the nucleotide sequence encoding the TUSC2 protein is flanked by a 5' untranslated region (UTR) and a 3' UTR.

[0037] The nucleic acid constructs disclosed herein can include control and regulatory sequences that are operably linked to the polynucleotide sequence encoding the TUSC2 protein. The nucleic acid constructs disclosed herein can include appropriate control sequences for expression of TUSC2 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 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 protein, the relationship is such that the control element modulates expression of the TUSC2 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),6175716766.2Docket No. 198628.46676 origins of replication, Kozak sequences (e.g., GCCACCATG), posttranslational regulatory elements, introns, nuclear targeting sequences, etc. For example, with respect to origins of replication, the high copy number pUC replication origin can be reduced to 700 bp without loss of high copy number replication. Alternatively, e.g., a non-pUC mini-origin R6K Nanoplasmid™ from Nature Technology Corporation, Lincoln, NE, can be used.

[0038] 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 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.

[0039] 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 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 I7175716766.2Docket No. 198628.46676(HLTV-I) R Region Exon 1, HTLV-I R element, P globin intron, splicing enhancer, Kozak sequence, BGH polyA signal, trpA terminator, and origin.

[0040] In embodiments, the expression construct comprising the polynucleotide sequence encoding a TUSC2 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 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 cleavagejoining 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.

[0041] In embodiments, the nucleic acid construct comprises a promoter operably linked to the nucleotide sequence encoding the TUSC2 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: 5 or SEQ ID NO:6. In embodiments, the CMV promoter comprises SEQ ID NO:5. In embodiments, the CMV promoter comprises SEQ ID NO:6. 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:3, 4, or 16. In embodiments, the CMV enhancer comprises SEQ ID NO:3, 4, or 16. In embodiments, the nucleic acid construct comprises a CMV promoter and a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein.

[0042] In embodiments, the nucleic acid construct comprises:(a) a nucleotide sequence encoding a TUSC2 protein;8175716766.2Docket No. 198628.46676(b) a promoter operably linked to the nucleotide sequence encoding the TUSC2 protein; and(c) an enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein;

[0043] 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; and(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein.

[0044] 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: 5; or(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:3.

[0045] 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:5; or(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV enhancer comprises SEQ ID NO:3.

[0046] 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;9175716766.2Docket No. 198628.46676(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: 6; or(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: 16.

[0047] 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:6; or(c) a CMV enhancer operably linked to the nucleotide sequence encoding the TUSC2 protein, wherein the CMV enhancer comprises SEQ ID NO: 16.

[0048] 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:7. In embodiments, the HTLV-I regulatory sequence comprises SEQ ID N0:7.

[0049] In embodiments, the nucleic acid construct comprises a bovine growth hormone polyadenylation (BGH poly A) 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 NO:8. In embodiments, the BGH polyA sequence comprises SEQ ID NO:8.

[0050] In embodiments, the nucleic acid construct comprises a splicing enhancer sequence. In embodiments, the splicing enhancer sequence comprises GAAGAAGAC. In embodiments, the splicing enhancer sequence comprises one or more repeats of 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: 11. In embodiments, the splicing enhancer sequence comprises SEQ ID NO: 11.10175716766.2Docket No. 198628.46676

[0051] 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 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:9. In embodiments, the P-globin intron comprises SEQ ID NO:9.

[0052] 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 (Williams, Vector Design for Improved DNA Vaccine Efficacy, Safety and Production, Vaccines (Basel). 2013 Jun 25;l(3):225-49; Borggren et al., Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans, Hum Vaccin Immunother. 2015; 11(8): 1983- 90), which is commercially available (Nature Technologies Corporation, Lincoln, NE, US).

[0053] 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: 10. In embodiments, the bacterial backbone sequence SEQ ID NO: 10. 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., Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system, Vaccine. 2009 Oct 30;27(46):6454-9; Luke et al., Development of antibiotic-free selection system for safer DNA vaccination, Methods Mol Biol. 2014;1143:91-111), RNAI (U.S. Patent No. 9,297,014), and suppressor tRNAs (Soubrier et al., pCOR: a new design of plasmid vectors for nonviral gene therapy, Gene Ther. 1999 Aug;6(8): 1482-8). RNA selectable markers are useful in applications where use of antibiotic-resistance 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 an11175716766.2Docket No. 198628.46676 antibiotic resistance gene, for example, genes encoding resistance to ampicillin, chloramphenicol, tetracycline or kanamycin.

[0054] Vectors

[0055] The term “vector” as used herein refers to a vehicle for delivering genetic material (e.g., RNA or DNA) to a cell. The term vector includes, 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.

[0056] 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 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, about12175716766.2Docket No. 198628.46676270 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 pg, about 350 pg, about 360 pg, about 370 pg, about 375 pg, about 400 pg, about 425 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about or 1000 ig 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.

[0057] 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.

[0058] 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., about 1 mM, about 2 to about 7 mM, about 3 to about 6 mM, about 4 to about 5 mM, about 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 (c.g, about 0.1 mM, about 0.2 to about 1 mM, about 2 to about 7 mM, about 3 to about 6 mM, about 4 to about 5 mM, or about 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.13175716766.2Docket No. 198628.46676

[0059] Provided herein are liposomes comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein.

[0060] In embodiments, provided is a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein, wherein the nucleic acid construct is complexed with a liposome. In embodiments, provided is a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein, wherein the nucleic acid construct is complexed with a liposome, and wherein the liposome is a DOTAP:cholesterol liposome. In embodiments, provided is a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 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.

[0061] In one embodiment the vector, or the pharmaceutical composition comprising the vector, comprises Quaratusugene ozeplasmid (REQORSA®). REQORSA® is a positively charged non-viral lipid nanoparticle (z.e., a DOTAP: cholesterol liposome as described herein) that encapsulates a DNA expression plasmid comprising SEQ ID NO:2, which encodes for a TUSC2 polypeptide comprising SEQ ID NO: 14.

[0062] In embodiments, provided is a pharmaceutical composition comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 protein complexed with a liposome, wherein the liposome is a DOTAP:cholesterol liposome. In embodiments, provided is a pharmaceutical composition comprising a nucleic acid construct comprising a nucleotide sequence encoding a TUSC2 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.

[0063] 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., Improved DNA: liposome complexes for increased systemic delivery and gene expression, Nat Biotechnol. 1997 Jul;15(7):647-52 and U.S. 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,14175716766.2Docket No. 198628.46676DOTAP (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.

[0064] 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 U.S. 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 exponential15175716766.2Docket No. 198628.46676 enlargement of the interface between the fluids ensuring rapid mixing. In embodiments, a postfiltration step may be completed to reduce visible particles. In such embodiments, particles greater than 1 pm may be filtered out.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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 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 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.

[0069] A “recombinant AAV vector” or “rAAV vector” comprises an 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.16175716766.2Docket No. 198628.46676Where an 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 AAV 1, 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.

[0070] An 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 et al., Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells, J Virol. 2006 Feb;80(4): 1874-85; Kotin, Large-scale recombinant adeno- associated virus production, Hum Mol Genet. 2011 Apr 15;20(Rl):R2-6; Kohlbrenner et al., Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system, Mol Ther. 2005 Dec; 12(6): 1217-25; Mietzsch et al., OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy, Hum Gene Ther. 2014 Mar;25(3):212-22; and U.S. Patent Nos. 6,436,392, 7,241,447, and 8,236,557.

[0071] Pharmaceutical Compositions17175716766.2Docket No. 198628.46676

[0072] 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.

[0073] 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.

[0074] 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 aqueous18175716766.2Docket No. 198628.46676 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 about 10-60% w / w of propylene glycol or the like.

[0075] 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.

[0076] Administration and Methods of Treatment

[0077] The present disclosure provides a method of treating cancer by administering to a patient in need thereof a nucleic acid construct for the expression of TUSC2 (a TUSC2 expression construct), e.g., by administering to the patient a pharmaceutical composition disclosed herein. In one embodiment, the cancer is mesothelioma. In one embodiment, the cancer is MPM. In further embodiments, the cancer is peritoneal mesothelioma. The TUSC2 expression constructs can be administered to the patient in a pharmaceutical composition comprising the TUSC2 expression constructs.

[0078] 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 a nucleic acid construct for the expression of TUSC2 (a TUSC2 expression construct), e.g., by administering to the patient a pharmaceutical composition disclosed herein. In embodiments, the tumor is mesothelioma. In one embodiment, the cancer is MPM. In one embodiment, the cancer is peritoneal mesothelioma. The TUSC2 expression constructs can be administered to the patient in a pharmaceutical composition comprising the TUSC2 expression constructs. In19175716766.2Docket No. 198628.46676 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 the TUSC2 protein.

[0079] In embodiments, administration of the nucleic acid construct described herein to a subject results in decreased cell proliferation, decreased cell invasion, and / or increased apoptotic activity of tumor cells.

[0080] 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 expression / signaling to TUSC2-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.

[0081] 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, z.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.

[0082] In some embodiments, the pharmaceutical composition described herein is administered at about 0.1 to about 2.0 mg / kg every 3 week such as about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, or any20175716766.2Docket No. 198628.46676 amount in-between. In one embodiment, the pharmaceutical composition described herein is administered at about 0.12 mg / kg every 3 weeks.

[0083] 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 to TUSC2 -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 signaling / expression to cancer cells that have reduced TUSC2 levels or inhibition of TUSC2 signaling. 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.

[0084] 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 (e.g., from the codon optimized TUSC2 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 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.

[0085] 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. In21175716766.2Docket No. 198628.46676 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.

[0086] 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 (z.e., for expression of TUSC2), and an additional anti-cancer therapy. In embodiments, the additional anti-cancer 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 U.S. 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), Yervoy® (ipilimumab), and Opdivo® (nivolumab). A further example of an additional anti-cancer therapy is a BRAF 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. In22175716766.2Docket No. 198628.46676 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 anti -cancer 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 anticancer 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.), z.e., admixed in the same injection or infusion volume.

[0087] 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 (e.g., for treatment of cancer). Human subjects suffering from cancer include individuals suffering from mesothelioma (e.g., MPM). 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.

[0088] In some methods of treating mesothelioma in a subject, the method includes selecting a subject having mesothelioma 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.

[0089] Molecular markers are known for mesothelioma. For example, calretinin, WT-1, Osteopontin (OPN), fibulin-3, High Mobility Group Box 1 protein (HMGB1), soluble23175716766.2Docket No. 198628.46676 mesothelin-related peptides (SMRPs), MPF, BAP1, BARD1, Epithelial Membrane Antigen, NF2, SETD2, LATS2 and podoplanin can serve as markers for mesothelioma. A human subject having one or more of any of these markers can be selected for treatment with the pharmaceutical compositions, nucleic acid constructs, vectors, and methods described herein.

[0090] In one embodiment of a combination therapy as described herein, the additional anti-cancer therapy is specific for a cancer associated with a particular mutation.

[0091] Provided herein are methods of treating mesothelioma that has 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-[N-(7-nitrobenz-2-oxa-l, 3 -diaxol-4-yl)amino]-2-deoxy glucose (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).

[0092] Table 1 provide an overview of the nucleic acid sequences (see also Table 2) and amino acid sequences (see also Table 3) disclosed herein.

[0093] Table 1. Summary of sequences disclosed herein.24175716766.2Docket No. 198628.46676

[0094] Table 2 Nucleic acid sequences.25175716766.2Docket No. 198628.4667626175716766.2Docket No. 198628.46676

[0095] Table 3 Amino acid sequences.

[0096] The following numbered embodiments merely serve to illustrate, but not limit, the present disclosure.

[0097] Embodiment 1. A method for treating mesothelioma in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a nucleic acid construct comprising a nucleic acid sequence encoding a TUmor Suppressor Candidate 2 (TUSC2) protein.27175716766.2Docket No. 198628.46676

[0098] Embodiment 2. The method of embodiment 1, wherein the nucleic acid construct is complexed with a liposome.

[0099] Embodiment 3. The method of embodiment 1 or 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0100] Embodiment 4. The method of any one of embodiments 1-3, wherein the TUSC2 protein is human TUSC2.

[0101] Embodiment 5. The method of any one of embodiments 1-4, wherein the TUSC2 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

[0102] Embodiment 6. The method of embodiment 5, wherein the TUSC2 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.

[0103] Embodiment 7. The method of embodiment 6, wherein the TUSC2 protein comprises SEQ ID NO: 14.

[0104] Embodiment 8. The method of any one of embodiments 1-7, wherein the nucleic acid sequence encoding the TUSC2 protein is codon-optimized.

[0105] Embodiment 9. The method of any one of embodiments 1-6, wherein the nucleic acid sequence encoding the TUSC2 protein comprises a sequence that is at least 80% identical to SEQ ID NO: 1 or 2.

[0106] Embodiment 10. The method of embodiment 9, wherein the nucleic acid sequence encoding TUSC2 protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

[0107] Embodiment 11. The method of embodiment 10, wherein the nucleic acid sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2.

[0108] Embodiment 12. The method of any one of embodiments 1-11, wherein the nucleic acid construct further comprises a CMV promoter operably linked to the nucleic acid sequence encoding the TUSC2 protein.

[0109] Embodiment 13. The method of embodiment 12, wherein the CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:6.

[0110] Embodiment 14. The method of embodiment 13, wherein the CMV promoter comprises SEQ ID NO:6.[OHl] Embodiment 15. The method of any one of embodiments 12-14, the nucleic acid construct further comprising a CMV enhancer.

[0112] Embodiment 16. The method of embodiment 15, wherein the CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 16.28175716766.2Docket No. 198628.46676

[0113] Embodiment 17. The method of embodiment 16, wherein the CMV enhancer comprises SEQ ID NO: 16.

[0114] Embodiment 18. The method of any one of embodiments 1-17, wherein the nucleic acid construct further comprising a Human T-cell leukemia virus type I (HTLV-I) regulatory sequence.

[0115] Embodiment 19. The method of embodiment 18, wherein the HTLV-I regulatory sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 7.

[0116] Embodiment 20. The method of embodiment 19, wherein the HTLV-I regulatory sequence comprises SEQ ID NO:7.

[0117] Embodiment 21. The method of any one of embodiments 1-20, the nucleic acid construct further comprising a bovine growth hormone polyadenylation (BGH polyA) sequence.

[0118] Embodiment 22. The method of embodiment 21, wherein the BGH polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8.

[0119] Embodiment 23. The method of embodiment 22, wherein the BGH polyA sequence comprises SEQ ID NO:8.

[0120] Embodiment 24. The method of any one of embodiments 1-23, wherein the nucleic acid construct further comprises a splicing enhancer sequence.

[0121] Embodiment 25. The method of embodiment 24, wherein the splicing enhancer sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11.

[0122] Embodiment 26. The method of embodiment 25, wherein the splicing enhancer sequence comprises SEQ ID NO: 11.

[0123] Embodiment 27. The method of any one of embodiments 1-26, wherein the nucleic acid construct further comprises at least one intron.

[0124] Embodiment 28. The method of embodiment 27, wherein the at least one intron is a P-globin intron.

[0125] Embodiment 29. The method of embodiment 28, wherein the P-globin intron comprises a sequence that is at least 90% identical to SEQ ID NOV.

[0126] Embodiment 30. The method of embodiment 29, wherein the P-globin intron comprises SEQ ID NOV.

[0127] Embodiment 31. The method of any one of embodiments 1-20, wherein the nucleic acid construct further comprises a bacterial backbone sequence.

[0128] Embodiment 32. The method of embodiment 31, wherein the bacterial backbone sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 10.29175716766.2Docket No. 198628.46676

[0129] Embodiment 33. The method of embodiment 32, wherein the bacterial backbone sequence comprises SEQ ID NO: 10.

[0130] Embodiment 34. The method of any one of embodiments 31 or 32, wherein the bacterial backbone sequence comprises a R6K origin sequence.

[0131] Embodiment 35. The method of any one of embodiments 31-34, wherein the bacterial backbone sequence comprises at least one selectable marker.

[0132] Embodiment 36. The method of any one of embodiments 2-35, wherein the liposome is a l,2-bis(oleoyloxy)-3 -(trimethyl ammonio) propane (DOTAP):cholesterol liposome.

[0133] Embodiment 37. The method of embodiment 36, wherein the ratio ofDOTAP:cholesterol is between about 3: 1 and about 1 :3.

[0134] Embodiment 38. The method of embodiment 36 or 37, wherein theDOTAP:cholesterol liposome has a particle size range of about 40 to about 250 nanometers.

[0135] Embodiment 39. The method of any one of embodiments 1-38, wherein the pharmaceutical composition further comprises about 5% dextrose, about 0.9% sodium chloride, or a combination of both agents.

[0136] Embodiment 40. The method of any one of embodiments 1-39, wherein the subject is a human.

[0137] Embodiment 41. The method of any one of embodiments 1-40, wherein the mesothelioma is Malignant Pleural Mesothelioma (MPM) or Peritoneal Mesothelioma.

[0138] Embodiment 42. The method of any one of embodiments 1-41, wherein the pharmaceutical composition is administered intravenously or intranasally.

[0139] Embodiment 43. The method of any one of embodiments 1-42, wherein the method further comprises administering a second anti-cancer therapy to the subject.

[0140] Embodiment 44. The method of embodiment 43, wherein the second anti-cancer therapy comprises at least one of: chemotherapy, radiation treatment, immunotherapy, and surgery.

[0141] Embodiment 45. The method of embodiment 43 or 44, wherein the pharmaceutical composition is administered at about 0.12 mg / kg every 3 weeks.

[0142] Embodiment 46. The method of embodiment 45, wherein the method further comprises administering pembrolizumab.

[0143] Embodiment 47. Th method of embodiment 45, wherein the method further comprises administering 2-4 cycles of pememtrexed and cisplatinum.30175716766.2Docket No. 198628.46676

[0144] Embodiment 48. The method of embodiment 45, wherein the method further comprises administering ipilimumab and nivolumab.

[0145] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.

[0146] In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0147] It is to be understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is further to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0148] All other referenced patents, patent publications, book chapters, scientific articles, etc. are incorporated herein by reference in their entireties, whether explicitly stated or not. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0149] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention.EXAMPLES

[0150] Example 1: Materials and Methods

[0151] This Example describes the materials and methods used in Example 2.

[0152] Cell Lines31175716766.2Docket No. 198628.46676

[0153] Four MPM cell lines (HP1, H2595, H2373 and H2591) and a tert-immortalized mesothelial LP9 cells were maintained in IX Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) containing 10% Fetal Bovine Serum (FBS, Invitrogen) and Penicillin / Streptomycin (Invitrogen).

[0154] In vitro Transfections

[0155] MPM cells and LP9 cells were seeded in a six well plate in triplicates at 5 X 105cells / well and were transfected with 2.5 pg of REQORSA® and control liponanoparticles in serum-free medium for 3 hours. Following transfection, cells were replenished with complete medium and incubated at 37 °C for 48 hours. Cells were harvested for downstream protein, gene expression and tumorigenic assays.

[0156] RNA extractions for sene expression analysis

[0157] RNA was extracted from post-transfected cells using Direct-zol™ RNA miniprep Kit (Zymoresearch) for RT-PCR gene expression.

[0158] Western Blot Analysis

[0159] The protein lysates were prepared by lysing the cells in M-PER™ reagent (Thermofisher Scientific) and analyzed for TUSC2 protein expression.

[0160] Caspase-8 Activity Assay

[0161] Caspase-8 activity assay was performed as described by the manufacturer (Genscript, Piscatway, NJ) to measure apoptosis. Then 3 * 105post-transfected cells were collected and washed twice with l x PBS. The cells were incubated in lysis buffer for 60 minutes on ice and centrifuged at 10,000 rpm at 4 °C for 10 minutes. Clear lysate supernatant containing 200 pg of protein was assayed using caspase-8 substrate. Caspase-8 activity was measured at 405 nM using a spectrophotometer.

[0162] Cell-Proliferation Assay

[0163] Malignant Mesothelioma (MM) cells and LP9 cells transfected with REQORSA® and control liposomes were seeded onto a 96-well plate at a density of 4 x 104cells per well in 100 pl of DMEM / 10% FBS and incubated at 37 °C for 48 hours. Twenty microliters of CellTiter-Blue® reagent (Promega, Madison, WI) was added to each well and incubated for 1 hour. Optical density was measured at 560 of 590 X using the Universal Reader Victor (PerkinElmer, Waltham, MA) (Gaoparaju et al. Journal of Thoracic Oncology. 2013. 8(9): 1203-1211).

[0164] Matri el Invasion Assay

[0165] The BD BioCoat Matrigel invasion chambers (BD Sciences, Franklin Lakes, NJ) were used according to the manufacturer's protocol. 5 x 104post-transfected cells were32175716766.2Docket No. 198628.46676 transferred onto a 8 pM pore-size polycarbonate membrane inserts coated with a thin layer Matrigel basement membrane matrix without phenol-red (BD Sciences) and were incubated for 48 hours at 37 °C and 5% CO2. The inserts were removed and non-invading cells on the upper surface were removed with a cotton swab. The cells that migrated through the Matrigel to the lower surface were fixed and stained with Giemsa solution and the cells were counted in five individual high-power fields for each membrane under a light microscope. Assays were performed in triplicate for each treatment group.

[0166] Statistical Analysis

[0167] The REQORSA® dependent changes for all the functional assays were analyzed for statistical differences by standard Student's / test, with p value less than 0.05 considered as significant. All the experiments were performed in triplicates.

[0168] Example 2: TUSC2 suppresses tumorigenic properties in Malignant Pleural Mesothelioma (MPM) cells

[0169] A study was conducted in four different MPM cell lines (z.e., H2595, HP1, H2373, and H2591) to determine the anti-tumorigenic role of TUSC2 re-expression in MPM.

[0170] Four MPM cell lines (H2595, HP1, H2373, and H2591) and tert-transformed mesothelial LP9 cells were treated with Quar Oze and control liposomes for 48 h. Post-treated cells were then evaluated for TUSC2 expression by semi -quantitative RT-PCR, Western blot analysis, and functional assays including cell proliferation, invasion, and apoptosis as described in Example 1.

[0171] All cell lines except LP9 demonstrated the absence of TUSC2 by Western blot (FIG. 2B) All MPM cell lines treated with quaratusugene ozeplasmid (Quar Oze) demonstrated a high transfection efficiency, confirmed by semi-quantitative RT-PCR and Western blot analysis. Quar Oze treatment resulted in a significant decrease in cell proliferation, cell invasion, and a significant increase in cell apoptosis. No effect was seen with LP9 cells (Table 5 and FIG. 2A).Table 5. Summary of % change in proliferation, invasion, and apoptosis in cells treated with Quar Oze.33175716766.2Docket No. 198628.46676

[0172] The foregoing results demonstrate potent tumor suppressive activity of the TUSC2 gene delivered by Quar Oze, and its re-expression can serve as a therapeutic strategy for the treatment of MPM.34175716766.2

Claims

Docket No. 198628.46676CLAIMSWe Claim:

1. A method for treating mesothelioma in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a nucleic acid construct comprising a nucleic acid sequence encoding a TUmor Suppressor Candidate 2 (TUSC2) protein.

2. The method of claim 1, wherein the nucleic acid construct is complexed with a liposome.

3. The method of claim 1 or 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

4. The method of any one of claims 1-3, wherein the TUSC2 protein is human TUSC2.

5. The method of any one of claims 1-4, wherein the TUSC2 protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

6. The method of claim 5, wherein the TUSC2 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.

7. The method of claim 6, wherein the TUSC2 protein comprises SEQ ID NO: 14.

8. The method of any one of claims 1-7, wherein the nucleic acid sequence encoding the TUSC2 protein is codon-optimized.

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

10. The method of claim 9, wherein the nucleic acid sequence encoding TUSC2 protein comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or 2.35175716766.2Docket No. 198628.4667611. The method of claim 10, wherein the nucleic acid sequence encoding the TUSC2 protein comprises SEQ ID NO: 1 or 2.

12. The method of any one of claims 1-11, wherein the nucleic acid construct further comprises a cytomegalovirus (CMV) promoter operably linked to the nucleic acid sequence encoding the TUSC2 protein.

13. The method of claim 12, wherein the CMV promoter comprises a sequence that is at least 90% identical to SEQ ID NO:6.

14. The method of claim 13, wherein the CMV promoter comprises SEQ ID NO:6.

15. The method of any one of claims 12-14, the nucleic acid construct further comprising a CMV enhancer.

16. The method of claim 15, wherein the CMV enhancer comprises a sequence that is at least 90% identical to SEQ ID NO: 16.

17. The method of claim 16, wherein the CMV enhancer comprises SEQ ID NO: 16.

18. The method of any one of claims 1-17, wherein the nucleic acid construct further comprising a Human T-cell leukemia virus type I (HTLV-I) regulatory sequence.

19. The method of claim 18, wherein the HTLV-I regulatory sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 7.

20. The method of claim 19, wherein the HTLV-I regulatory sequence comprises SEQ ID NO:7.

21. The method of any one of claims 1-20, the nucleic acid construct further comprising a bovine growth hormone polyadenylation (BGH poly A) sequence.36175716766.2Docket No. 198628.4667622. The method of claim 21, wherein the BGH polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NO:8.

23. The method of claim 22, wherein the BGH polyA sequence comprises SEQ ID NO:8.

24. The method of any one of claims 1-23, wherein the nucleic acid construct further comprises a splicing enhancer sequence.

25. The method of claim 24, wherein the splicing enhancer sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11.

26. The method of claim 25, wherein the splicing enhancer sequence comprises SEQ ID NOT E27. The method of any one of claims 1-26, wherein the nucleic acid construct further comprises at least one intron.

28. The method of claim 27, wherein the at least one intron is a P-globin intron.

29. The method of claim 28, wherein the P-globin intron comprises a sequence that is at least 90% identical to SEQ ID NO:9.

30. The method of claim 29, wherein the P-globin intron comprises SEQ ID NO:9.

31. The method of any one of claims 1-20, wherein the nucleic acid construct further comprises a bacterial backbone sequence.

32. The method of claim 31, wherein the bacterial backbone sequence comprises a sequence that is at least 90% identical to SEQ ID NOTO.

33. The method of claim 32, wherein the bacterial backbone sequence comprises SEQ ID NOTO.37175716766.2Docket No. 198628.4667634. The method of any one of claims 31 or 32, wherein the bacterial backbone sequence comprises a R6K origin sequence.

35. The method of any one of claims 31-34, wherein the bacterial backbone sequence comprises at least one selectable marker.

36. The method of any one of claims 2-35, wherein the liposome is a l,2-bis(oleoyloxy)-3- (trimethyl ammonio) propane (DOTAP):cholesterol liposome.

37. The method of claim 36, wherein the ratio of DOTAP:cholesterol is between about 3 : 1 and about 1 :3.

38. The method of claim 36 or 37, wherein the DOTAP: cholesterol liposome has a particle size range of about 40 to about 250 nanometers.

39. The method of any one of claims 1-38, wherein the pharmaceutical composition further comprises about 5% dextrose, about 0.9% sodium chloride, or a combination of both agents.

40. The method of any one of claims 1-39, wherein the subject is a human.

41. The method of any one of claims 1-40, wherein the mesothelioma is Malignant Pleural Mesothelioma (MPM) or Peritoneal Mesothelioma.

42. The method of any one of claims 1-41, wherein the pharmaceutical composition is administered intravenously or intranasally.

43. The method of any one of claims 1-42, wherein the method further comprises administering a second anti-cancer therapy to the subject.

44. The method of claim 43, wherein the second anti-cancer therapy comprises at least one of: chemotherapy, radiation treatment, and immunotherapy.38175716766.2Docket No. 198628.4667645. The method of claim 43 or 44, wherein the pharmaceutical composition is administered at about 0.12 mg / kg every 3 weeks to the subject.

46. The method of claim 45, wherein the method further comprises administering pembrolizumab to the subject.

47. Th method of claim 45, wherein the method further comprises administering 2-4 cycles of pememtrexed and cisplatinum to the subject.

48. The method of claim 45, wherein the method further comprises administering ipilimumab and nivolumab to the subject.

49. The method of any one of claims 1-48, wherein the subject is further undergoing surgery to remove a tumor from the subject.39175716766.2

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