WO2022056488A9 - Nonviral vectors for increasing fas expression in cancer cells and methods of use thereof - Google Patents

Abstract

The disclosure provides nucleic acid constructs including a polynucleotide sequence encoding a FAS protein (e.g., human FAS isoform 1). Also contemplated are nonviral vectors including the nucleic acid constructs and DOT AP : cholesterol liposomes. Further contemplated herein are compositions including the nonviral vectors and methods of using the compositions for the treatment of cancer in a human subject in need thereof. These methods can further include administering a second anti-cancer therapy to the subjects in need thereof.

Description

NONVIRAL VECTORS FOR INCREASING FAS EXPRESSION IN CANCER CELLS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63/078,145, filed September 14, 2020, entitled “NONVIRAL VECTORS FOR INCREASING FAS EXPRESSION IN CANCER CELLS AND METHODS OF USE THEREOF”, the disclosure of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 13, 2021, is named 198628-46076-sequence listing_ST25.txt and is 4.49 kilobytes in size.

FIELD

[0003] The present disclosure relates to vectors, in particular, nonviral vectors, that include a nucleic acid sequence encoding a FAS (CD95) protein. Also contemplated are compositions including nonviral vectors for increasing FAS expression in cancer cells. Further contemplated are methods of treating cancer in a human subject including administration of the nonviral vectors and compositions to the subject.

BACKGROUND

[0004] A hallmark of cancer cells is apoptosis evasion. One of the mechanisms by which apoptosis of cancer cells may be triggered is via binding of death ligands to death receptors. The FAS gene (also known as CD95, Fas, and FAS receptor) encodes the tumor necrosis factor receptor (TNFR) superfamily member 6 protein (TNFRSF6, referred to herein as the FAS protein), a type I transmembrane glycoprotein and a death receptor. FAS harbors extracellular amino-terminal cysteine-rich domains (CRDs) that define its specificity for its cognate ligand (CD95L, also known as FasL and FasL/CD178), and a conserved 80 amino acid sequence located in the cytoplasmic tail called the death domain (DD) that is necessary for the induction of apoptosis. Binding of FAS to its cognate ligand FasL via the TNF homology domain (THD) region of FasL induces receptor oligomerization and triggers pro- apoptotic signals through its DD, leading to activation of the caspase protease family which is

SUBSTITUTE SHEET (RULE 26) ultimately responsible for dismantling the cell. Specifically, interaction of FAS with FasL leads to recruitment of the adaptor protein Fas-associated death domain (FADD) via homotypic DD-mediated interactions. FADD recruits the proteases caspase-8 and caspase- 10 and the regulator of apoptosis cellular FADD-like interleukin- 1-P-converting enzyme- inhibitory protein (c-FLIP) via death effector domain (DED)-mediated interactions.

Together, these proteins form the death-inducing signaling complex (DISC). Oligomerization of procaspase-8 promotes its autocatalytic activation and the release of a mature tetramer to the cytosol, leading to the apoptotic response. An apoptotic signal can also be induced by the mitochondria (intrinsic pathway). The intrinsic apoptosis pathway starts from within the cell either by direct activation of caspases or through intracellular changes such as DNA damage resulting in the release of a number of pro-apoptotic factors such as bcl2 from the intermembrane space of mitochondria. Cancer cells are often characterized by deregulation of both pathways.

[0005] Many cancer cells develop mechanisms to evade apoptosis and become resistant to cancer therapy by manipulating the levels of antiapoptotic molecules or inactivating proapoptotic cell death components. Several malignancies downregulate FAS to evade apoptotic signaling, and some mutations in FAS allow tumors to evade immune surveillance. FAS is silenced by epigenetic mechanisms (e.g., histone deacetylation, DNA hypermethylation) as a means of evading host immunosurveillance. In addition, cancer cells can increase the level of certain proteins to inhibit FAS-induced apoptosis. For example, Protein Tyrosine Phosphatase Non-Receptor Type 13 (PTPN13), also called Fas-associated protein-tyrosine phosphatase 1 (FAP-1), is a tyrosine phosphatase that negatively regulates FAS-induced apoptosis and FAS cell surface expression. High expression levels of PTPN13 inversely correlate with FAS cell surface levels. PTPN13 expression retains FAS in cytoplasmic pools within the cytoskeleton network, thereby blocking FAS trafficking from the Golgi compartment to the cell surface. Inhibition of FAS surface expression by PTPN13 is mediated by association of the PDZ2 domain of PTPN13 with the C terminus of FAS.

Lowering PTPN13 levels by expression of dominant-negative forms of PTPN13, or inhibition of PTPN13 expression by short interfering RNA, has been shown to efficiently up-regulate surface expression of FAS. As another example, cancer cells can increase the expression of cFLIP to inhibit FAS-induced apoptosis. At low expression levels, cFLIP enhances caspase- 8 activation as part of the death-inducing signaling complex (DISC). But when expressed at high levels, cFLIP interferes with apoptosis by blocking the FAS receptor from activating caspases and inhibiting FAS-induced apoptosis. [0006] Additionally, tumor cells have been shown to downregulate FAS expression to evade T cells and NK cells, as these cells express FasL on their surface and upon FAS/FasL recognition, the apoptotic pathway is activated. Cytotoxic T lymphocytes (CTLs) use the FAS-FasL pathway as an effector mechanism to suppress tumor growth, and NK cells eliminate tumor cells through FAS/FasL engagement and the release of cytotoxic granules containing granzymes and perforin. The FAS/FasL apoptosis pathway plays an important role in host cancer immune surveillance and in eradication of established tumors.

[0007] High levels of FAS expression in cancer cells have been found to be a positive prognostic marker for cancer patients, indicating higher survival rates. FAS expression levels are significantly downregulated in tumor tissues from colorectal cancer (CRC) patients as compared to non-neoplastic colon. FAS was found to be expressed in only 23.4% (30/128) of CRC specimens (Strater, et al, 2005. Gut. 54:661-665) and is positively correlated with CRC patient survival. The loss of FAS expression is a hallmark of human CRC. Given the prominent role of FAS in cancer, drugs targeting FAS such as FAS agonistic monoclonal antibodies have been explored as a potential treatment for cancer. However, attempts to treat cancer by administering FAS agonistic monoclonal antibodies have partly failed due to a lack of specificity, including binding to healthy liver cells causing liver damage. Moreover, the agonistic antibodies are not effective when the tumor is circumventing apoptotic signals by downregulating FAS on the tumor cell surface.

[0008] Thus, development of an effective therapy for activating or restoring the FAS/FasL apoptotic pathway in cancer cells by increasing FAS expression and/or overcoming FAS inhibition that is less toxic than available therapies would provide a great advance.

SUMMARY

[0009] In one aspect, the disclosure provides a nucleic acid construct including (i) a polynucleotide sequence encoding a FAS protein (e.g., human FAS isoform 1) flanked by a 5' untranslated region (UTR) and a 3' UTR; (ii) a promoter (e.g., a CMV promoter) operably linked to the polynucleotide sequence encoding the FAS protein; and (iii) a selectable marker. In embodiments, the FAS protein is human FAS isoform 1 including the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having greater than 95% sequence identity to SEQ ID NO: 1. In embodiments, the polynucleotide sequence encodes an amino acid sequence including amino acids 26 to 335 of SEQ ID NO: 1, or an amino acid sequence having greater than 95% sequence identity to amino acids 26 to 335 of SEQ ID NO: 1. The polynucleotide sequence encoding human FAS isoform 1 can, for example, be SEQ ID NO: 2, or a polynucleotide sequence having greater than 90% sequence identity to SEQ ID NO: 2. In embodiments, the polynucleotide sequence encoding the FAS protein is a codon optimized sequence. In some nucleic acid constructs, the selectable marker is an RNA selectable marker (e.g., RNA-OUT, RNAI, a suppressor tRNA, etc.). In the nucleic acid constructs, the 5' UTR can include, for example, at least one intron and/or a HTLV-I R element.

[0010] In another aspect, the disclosure provides a nonviral vector including a nucleic acid construct encoding a FAS protein as described herein. In embodiments the nonviral vector is a liposomal nonviral vector. Such liposomes can include l,2-dioleoyl-3- trimethylammonium-propane (DOTAP) and cholesterol. The DOTAP: cholesterol molar ratio can be between about 3: 1 and about 1 :3. In embodiments, the DOTAP:cholesterol molar ratio is 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. In embodiments, the DOTAP:cholesterol molar ratio is about 1 : 1.

[0011] In another aspect, the disclosure provides a pharmaceutical composition including a nonviral vector as described herein. In embodiments, the pharmaceutical composition comprises about 0.1 mg/ml to about 0.5 mg/ml of the nucleic acid construct (e.g., FAS expression construct) and about 3 mM to about 5 mM DOTAP: cholesterol. In embodiments, the pharmaceutical composition comprises about 5% dextrose, about 0.9% sodium chloride, or a combination thereof. Typically, the DOTAP:cholesterol liposome has a particle size range of about 40 to 250 nanometers.

[0012] In another aspect, the disclosure provides a viral vector including a nucleic acid construct encoding a FAS protein as described herein. In embodiments, the viral vector is, for example, an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, an adenoviral vector, etc.

[0013] In another aspect, the disclosure provides a method for treating cancer in a human subject. The method includes administering to the human subject in need thereof a composition including a nucleic acid construct as described herein. In embodiments, the method for treating cancer in a human subject includes administering to the human subject in need thereof a composition including a nonviral vector or a viral vector as described herein. In embodiments, the method for treating cancer in a human subject includes administering to the human subject in need thereof a composition as described herein comprising a nonviral vector comprising a FAS expression construct disclosed herein. In embodiments, the method for treating cancer includes reduction or inhibition of metastasis. In embodiments, the disclosure provides a method for reducing or inhibiting metastasis of a cancer in a human subject in need thereof comprising administering to the human subject a composition including a nucleic acid construct/nonviral vector as described herein. The nonviral vectors and compositions can be administered intravenously, intratumorally, or intranasally.

[0014] In these methods, the cancer can be, for example, colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, rectal cancer, lung cancer (e.g., small cell or nonsmall cell lung cancer), leukemia, or neuroblastoma. In embodiments, the cancer is colorectal cancer (CRC).

[0015] The methods can further include administering a second anti -cancer therapy to the subject. The second anti-cancer therapy can be one or more of: radiation therapy; chemotherapy; a checkpoint inhibitor, e.g., Keytruda® (pembrolizumab) and Opdivo® (nivolumab); a BRAF inhibitor, e.g., Braftovi® (encorafenib); CAR-T cell immunotherapy, and/or an EGFR inhibitor, e.g., Erbitux® (cetuximab) and Tagrisso® (osimertinib). In the methods of treatment described herein, typically FAS expression (including, e.g., expression on the cell surface) is increased in, or restored to, FAS-deficient cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figs. 1 A-1C show FAS expression and function in human CRC. Fig. 1 A: FAS mRNA data in non-neoplastic human colon and colorectal tumor were extracted from TCGA database and plotted. Fig. IB: FAS mRNA data in human colorectal tumor were extracted from TCGA (top panel) and GEPIA (bottom panel) databases and grouped based on tumor stages. Fig. 1C: FAS mRNA datasets and CRC patient survival data were mined from TCGA database and analyzed for Kaplan-Meier survival curve.

[0017] Figs. 2A-2C show FAS is silenced by its promoter DNA hypermethylation in human CRC. Fig. 2A: The FAS promoter region showing CpG islands surrounding the FAS gene transcription start site. Fig. 2B: Top panel (Left): The FAS promoter CpG island and three CpG sites significantly differentially methylated in TCGA CRC samples. Bottom panel (Left): Heatmaps of 450K DNA methylation array data of three CpG sites (cg26478401, cg03111039, cgl3456138) and FAS expression (FPKM values) generated from TCGA human CRC datasets. The three columns on the left represent the beta values of the three CpG sites in primary CRC patient samples and normal colon tissues. The fourth column (on the right) shows the FPKM values of FAS RNA-seq data. The box plot on top of each heatmap summarizes the statistical difference between normal and tumor samples for CpG site methylation and FAS mRNA expression. The p-values indicate the Welch's t-test results. Fig. 2C: Negative correlation between methylation of the three CpG sites as shown in Fig. 2B (left three columns) and FAS mRNA expression as shown in Fig. 2B (right column). [0018] Figs. 3A-3C show FAS expression profiles in the single cell level in human CRC patients. Fig. 3 A: UMAP projection of human CRC scRNA-seq data. Original datasets were extracted from GSE146771 dataset. Cells are annotated according to dataset designation. Fig. 3B: UMAP projection of FAS expression. Cells were subsetted by tissue of origin (normal colon, peripheral blood, and colorectal tumor). Fig. 3C: FAS expression level in the indicated cell types in normal colon (N), peripheral blood (P), and colorectal tumor (T). [0019] Figs 4A-4D show restoring FAS expression enabled FasL-mediated elimination of FAS⁺ mouse colon tumor cells in vitro. Fig. 4A: DOTAP and cholesterol (1 : 1) cationic lipid (left panel) was used to encapsulate a codon usage optimized mouse FAS cDNA-expressing plasmid (middle panel) to produce the nanoparticle DOTAP-mFas. Fig. 4B: DOTAP - Cholesterol and the plasmid were formulated in different ratios as indicated (left table). The formulated DNA nanoparticles were then used to transfect mouse colon tumor cells for 24 hours. The transfected cells were analyzed for FAS expression on the tumor cell surface by flow cytometry. The FAS⁺ cells were quantified and presented on the right panel. Fig. C: Representative dot plot of flow cytometry data showing Fas expression in control and DOTAP -mFas-transfected cells as described in Fig. 4B. Fig. 4D: The transfected cells as shown in Fig. 4C were cultured with FasL for 24h, stained with anti-Fas mAb and propidium iodide (PI) and analyzed by flow cytometry. The indicated cell populations were quantified. [0020] Figs. 5A-5E show DOTAP-mFas gene immunotherapy suppresses colon tumor growth in immune competent mice. Fig. 5A: Study design in which CT26 tumor cells were injected into mice subcutaneously and the mice treated with DOTAP-mFas nanoparticles starting at day 4 as indicated. Fig. 5B: Tumors were analyzed for size and weight. Fig. 5C: Study design in which CT26 tumor cells were injected into mice and mice were treated as indicated. Fig. 5D: Tumor growth was analyzed at the start of treatment and at the end of the experiment. Fig. 5E: Tumors as shown in Fig. 5D were collected and analyzed for CD8⁺ and FasL⁺ T cells as shown.

[0021] Figs. 6A-6H show restoring FAS expression induces colon tumor cell autoapoptosis. Fig. 6A: FAS expression in human colon tumor cells. Fig. 6B: Tumor cells were transfected with codon optimized FAS cDNA-expressing plasmid. Shown are FAS protein dot plots and quantification of FAS+ cells. Fig. 6C: Tumor cells were transfected as in B, cultured in the presence of FasL for 24h, stained with FAS antibody and PI, and analyzed by flow cytometry. Shown are representative dot plots. Fig. 6D: The indicated cell populations were quantified. Figs. 6E & 6F: SW480 (Fig. 6E) and SW620 (Fig. 6F) cells were cultured in the presence of FasL (100 ng/ml), collected at 0, 2, 4, and 24h and analyzed by Western blotting. The blot was probed sequentially with anti-cleaved caspase 3, cleaved PARP, and P- actin. Fig. 6G: SW620 cells were transfected with hFAS and analyzed by Western blotting as in Fig. 6F. Fig. 6H: Control cells and hFAS-transfected SW620 and LS41 IN cells were analyzed by immunofluorescence for FAS protein (Green). Arrows point to FAS aggregations. Blue is nuclear staining.

[0022] Figs. 7A-7E show DOTAP-hFAS gene therapy suppresses human metastatic colon tumor xenograft growth in athymic mice. Fig. 7A: SW620 cells were injected subcutaneously into athymic nude mice. The tumor-bearing mice were treated as indicated. Tumors were collected for genomic DNA isolation. Fig. 7B: Codon-optimized FAS cDNA plasmid, human tumor cells genomic DNA, and human FAS cDNA plasmid were used as templates for PCR analysis using primers that are specific for the codon-optimized FAS cDNA. Fig. 7C: Genomic DNA from xenografts as shown in Fig. 7A was analyzed by PCR using primers that are specific for the codon-optimized FAS cDNA. The hFAS codon optimized cDNA plasmid was used as a positive control. Fig. 7D: SW620 cells were injected into athymic nude mice to establish xenografts. The tumor-bearing mice were treated as shown in the study design. Fig. 7E: the top panel shows the tumor xenografts. The size and weight of tumors as shown in the top panel were quantified and are provided in the bottom panel. DOTAP-hFAS gene therapy significantly reduced tumor size in a nude mouse model with human CRC SW620 xenografts.

[0023] Figs. 8A and 8B show a dose response of mouse colon tumor to DOTAP-Chol- mFas in tumor-bearing mice. Fig. 8A: the study design in which CT26 cells were injected into mice subcutaneously followed by treatment with DOTAP-Chol or DOTAP-Chol-hFAS. Fig. 8B: Tumor-bearing mice were randomized into six groups and treated with DOTAP- Chol-mFas nanoparticles with various amounts of codon usage optimized mouse Fas cDNA- expressing plasmid as indicated. Tumor size and weight were analyzed at the end of the experiment.

[0024] Figs. 9A-9E show that tumor cells express FasL and kill FAS-expressing tumor cells. Fig. 9A: Jurkat and EL4 cells were stained with IgG isotype control antibody (gray area) or FAS-specific antibody (black line), and analyzed by flow cytometry. Shown are representative histographs of FAS protein staining. Fig. 9B: Jurkat and EL4 cells were cultured in the presence of FasL at the indicated concentrations for 24h. Cells were collected and stained with Annexin V and PI. The stained cells were then analyzed by flow cytometry. Shown are quantification of cell death. Fig. 9C: LS41 IN and CT26 cells were stained for FAS protein as in Fig. 9A. Fig. 9D: LS41 IN and CT26 cells were analyzed by Western blotting and sequentially probed with FasL-specific antibody and P-actin. Fig. 9E: Jurkat and LS41 IN cells were cultured either alone or co-cultured for 24h. EL4 and CT26 cells were cultured either alone or co-cultured for 24h. Cells in the culture supernatants were collected, stained with Annexin V and PI, and analyzed by flow cytometry. Right panel: representative dot plots. % cells death (Annexin V⁺ PI⁺) was quantified and presented on the right.

[0025] Figs. 10A-10C show that mouse tumor cell-expressed FasL induces FAS-mediated apoptosis in human tumor cells. Fig. 10A: Jurkat cells were co-cultured with CT26 and MC38 cells, respectively, for 24h. Cells in the culture supernatant were collected, stained with Annexin V and PI, and analyzed by flow cytometry. Fig. 10B: Jurkat cells were co- cultured with CT26 and MC38 as in Fig. 10A in the presence of mouse FasL neutralization monoclonal antibody for 24h and analyzed by flow cytometry as in Fig. 10 A. Shown are representative dot plots. Fig. 10C: The co-culture supernatant of Jurkat and MC38 as shown in Fig. 10B were quantified. The bottom line is Jurkat cell death without co-cultured tumor cells. The top line is Jurkat cell death in the presence of MC38 cell co-culture and mouse FasL neutralization antibody at the indicated concentrations.

[0026] Fig. 11 shows liver toxicity analysis of cationic lipid-encapsulated mFas DNA nanoparticle therapy. Top panel: study design. Bottom panel: tumor-free mice were treated with control nanoparticles (DOTAP-Chol) or cationic lipid-encapsulated FAS nanoparticles as shown in the top panel. Liver tissues were collected, fixed, embedded, sectioned, and stained with H&E. Shown are liver cells.

[0027] Fig. 12 is a schematic and a graph showing that mouse FAS gene immunotherapy significantly suppressed colon tumor growth in a dose-dependent manner in immune competent mice.

[0028] Fig. 13 is a Kaplan-Meier curve showing the significantly increased survival time (by 10 days) of mice with metastatic CRC treated with mFas monotherapy (DOTAP-mFAS gene therapy).

DETAILED DESCRIPTION

[0029] Provided herein are nucleic acid constructs including a polynucleotide sequence encoding human FAS isoform 1. Also contemplated are nonviral vectors having the nucleic acid constructs disclosed herein and DOTAP:cholesterol liposomes. Also contemplated are viral vectors having the nucleic acid constructs disclosed herein. Further contemplated herein are compositions having the nonviral vectors or viral vectors disclosed herein. Also provided herein are methods of using the compositions disclosed herein for the treatment of cancer in a human subject in need thereof. These methods can further include administering a second anti-cancer therapy to the subjects in need thereof, i.e., combination therapy.

[0030] In contrast to currently available technologies (e.g., FAS agonistic mAbs), which do not increase FAS in cancer cells, the nonviral vectors, viral vectors, compositions and formulations described herein are useful for increasing or restoring FAS to FAS-deficient cancer cells, e.g., restoring epigenetically silenced FAS expression, thus overcoming downregulation or loss of FAS/FasL death signaling in human tumors. In addition to increasing FAS levels, the nonviral and viral vectors, compositions and formulations described herein are useful for overcoming FAS signaling inhibition caused, e.g., by antagonistic intracellular proteins such as PTPN13 that inhibit FAS transport to the cell surface. Expression of FAS from the nucleic acid construct overcomes the antagonistic effect of these inhibitors. When cancer cells take up the vectors described herein, FAS isoform 1 is expressed, and is transported to the cell surface where it interacts with the FasL ligand. By increasing FAS isoform 1 in FAS-deficient cancer cells, FAS/FasL binding is increased and therefore apoptotic signaling (including interactions between apoptotic proteins and cysteine caspases) is increased, resulting in apoptosis of the cancer cells. Additionally, increasing FAS expression in tumor cells increases their sensitivity to immunotherapy and T cells, and overcomes PTPN13 inhibition. The data described below demonstrate that the restoration of FAS expression in colon tumor cells and tumor-selective delivery of FAS cDNA nanoparticles is sufficient to suppress metastatic colon tumor growth in vivo.

I. Nucleic Acid Constructs

[0031] Nucleic acid constructs described herein include (i) a polynucleotide sequence encoding a FAS protein (for example, human FAS isoform 1) flanked by a 5' untranslated region (UTR) and a 3' UTR, and (ii) a promoter (e.g., CMV) operably linked to the polynucleotide sequence encoding the FAS protein. In embodiments, the nucleic acid constructs further include a selectable marker. In a typical embodiment, the nucleic acid construct is used for recombinant production of human FAS isoform 1 in a cancer cell (e.g., in a subject’s cancer cells). Nucleic acid constructs include expression constructs and plasmids. The term “expression construct” refers to a genetic construct that includes a nucleic acid coding for an RNA capable of being transcribed in a cell as well as additional nucleic acid sequences that promote expression of the nucleic acid coding sequence.

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 (e.g., DNA vaccines, immunotherapy), 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 sequence). Such sequences and elements are disclosed below under Control/Regulatory Sequences. Expression constructs/plasmids for inclusion in the vectors described herein can be produced in suitable host producer cells (e.g., E. colt) using suitable methods, e.g., fed-batch fermentation, batch fermentation, etc. For example, the HyperGRO™ inducible fed-batch fermentation process is used commercially to manufacture research grade plasmid DNA at Nature Technology Corporation (Lincoln, NE). The HyperGRO™ process yields plasmid productivity of up to 2,600 mg/L with low levels of nicking or multimerization. The high yield of plasmid per gram of bacteria improves final product purity since the 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 vectors. Plasmid DNA can be extracted from fermentation cells using alkaline lysis. Commercial plasmid manufacturers can utilize purification processes, such as anion exchange chromatography followed by hydrophobic interaction chromatography, that purify plasmid DNA away from impurities (e.g., endotoxin, bacterial RNA, genomic DNA).

[0032] In some embodiments, a nucleic acid construct as disclosed herein is a plasmid, which contains one or more of the following elements: RNA-OUT, CMV enhancer/promoter, CMV-human T-lymphotropic virus type I (HLTV-I) R Region Exon 1, HTLV-I R element, B globin intron, splicing enhancer, Exon 2, Kozak sequence, FAS isoform 1, polyA signal, trpA terminator, and origin. The plasmid can also include a bacterial backbone for production of the plasmid in bacterial cells. In some tissues, bacterial regions of approximately 1,000 bp or more promote transgene silencing. Thus, in embodiments, the bacterial backbone is about 400 base pairs (bp) to about 500 bp, such as about 450 bp. In embodiments the bacterial backbone is about a 454 bp sequence. 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, 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, a splicing enhancer is included within the intron and/or a flanking exon to increase transgene expression through increased intron splicing. i. FAS Polynucleotide and Amino Acid Sequences

[0033] The nucleic acid constructs described herein include a polynucleotide sequence encoding a FAS protein. In embodiments, the FAS protein is human FAS isoform 1 (SEQ ID NO: 1, also known as variant 1). Previous reports have identified as many as eighteen splice variants of human FAS. Among them, three transcripts, variant 1, variant 2 and variant 3, encode proteins that normally occur in cells. Variant 1 encodes SEQ ID NO: 1, a FAS receptor protein of 335 amino acids including a signal peptide which corresponds to amino acids 1 to 16 of the 335 amino acid sequence. The mature FAS protein, lacking the signal peptide, is a 319 amino acid, transmembrane protein that localizes at the cell surface forming a homotrimer. The first amino acid in the mature protein corresponds to amino acid 17 in SEQ ID NO: 1 (the peptide signal-containing isoform 1 sequence). In some embodiments, the FAS protein lacks the peptide signal, e.g., variant 2. Variant (isoform) 2 encodes a FAS protein lacking the transmembrane region (exon 6) and corresponds to a soluble receptor. Both isoforms 1 and 2 are normal products whose production via alternative splicing is regulated by the cytotoxic RNA binding protein TIA1. Variant 3 encodes a protein of 220 amino acids with a distinct and shorter C-terminus compared to variant 1. Many of the other isoforms are rare haplotypes that are usually associated with a state of disease.

[0034] In embodiments, the polynucleotide sequence encoding human FAS isoform 1 is SEQ ID NO: 2, or is a polynucleotide sequence having greater than 85%, greater than 90%, or greater than 95% sequence identity to SEQ ID NO: 2. In embodiments, the FAS protein encoded by the polynucleotide sequence is human FAS isoform 1 and includes the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having greater than 90%, greater than 95%, or greater than 98% sequence identity to SEQ ID NO: 1. In embodiments, the polynucleotide sequence encodes an amino acid sequence including amino acids 26 to 335 of SEQ ID NO: 1, or an amino acid sequence having greater than 90%, greater than 95%, or greater than 98% sequence identity to amino acids 26 to 335 of SEQ ID NO: 1. 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. SEQ ID NO: 1 (human FAS isoform 1)

MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQ FCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGL EVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRS NLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDL SKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLH GKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV

SEQ ID NO: 2 (h-FAS variant 1)

ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTATC GTCCAAAAGTGTTAATGCCCAAGTGACTGACATCAACTCCAAGGGATTGGAATT GAGGAAGACTGTTACTACAGTTGAGACTCAGAACTTGGAAGGCCTGCATCATGA TGGCCAATTCTGCCATAAGCCCTGTCCTCCAGGTGAAAGGAAAGCTAGGGACTG CACAGTCAATGGGGATGAACCAGACTGCGTGCCCTGCCAAGAAGGGAAGGAGTA CACAGACAAAGCCCATTTTTCTTCCAAATGCAGAAGATGTAGATTGTGTGATGAA GGACATGGCTTAGAAGTGGAAATAAACTGCACCCGGACCCAGAATACCAAGTGC AGATGTAAACCAAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTG CACCAAATGTGAACATGGAATCATCAAGGAATGCACACTCACCAGCAACACCAA GTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTG CCAATTCCACTAATTGTTTGGGTGAAGAGAAAGGAAGTACAGAAAACATGCAGA AAGCACAGAAAGGAAAACCAAGGTTCTCATGAATCTCCAACTTTAAATCCTGAA ACAGTGGCAATAAATTTATCTGATGTTGACTTGAGTAAATATATCACCACTATTG CTGGAGTCATGACACTAAGTCAAGTTAAAGGCTTTGTTCGAAAGAATGGTGTCAA TGAAGCCAAAATAGATGAGATCAAGAATGACAATGTCCAAGACACAGCAGAAC AGAAAGTTCAACTGCTTCGTAATTGGCATCAACTTCATGGAAAGAAAGAAGCGT ATGACACATTGATTAAAGATCTCAAAAAAGCCAATCTTTGTACTCTTGCAGAGAA AATTCAGACTATCATCCTCAAGGACATTACTAGTGACTCAGAAAATTCAAACTTC AGAAATGAAATCCAAAGCTTGGTCTAG ii. Control/Regulatory Sequences

[0035] The nucleic acid constructs disclosed herein include control and regulatory sequences that are operably linked to the polynucleotide sequence encoding a FAS protein. The nucleic acid constructs disclosed herein can include appropriate control sequences for expression of the human FAS isoform 1 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 polynucleotide 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 FAS protein, the relationship is such that the control element modulates expression of the FAS 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), 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 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 FAS 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 FAS coding sequence can include an intron. As another example, a chimeric intron (e.g., from the P-globulin and immunoglobulin heavy chain genes) upstream of the transgene (human FAS variant 1) can be used. Additionally or alternatively, the 5' UTR can include a 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. iii. Selectable Markers

[0036] In embodiments, the nucleic acid constructs as disclosed herein include a 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 antibiotic-resistance markers is undesirable, including in production of nonviral 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.

II. Nonviral Vectors

[0037] The term “vector” as used herein refers to a vehicle for delivering genetic material (e.g., RNA or DNA) into a cell, including for example, viral vectors (such as AAV and lentiviral vectors) and nonviral vectors. The term “nonviral vector” is used herein to refer to a nonviral vehicle for delivering genetic material into a cell. In embodiments, the nonviral 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. The liposome formulations described herein deliver the nucleic acid construct into the target cell; entering target cells via endocytosis pathways to avoid lysosomal degradation. Once a liposome formulation binds to a negatively-charged cancer cell, the nucleic acid construct is transfected into the cell (endocytosis) and expressed FAS is later transported to the surface of the cell. The non-viral vectors described herein result in a high level of transfection efficiency with the lowest level of toxicity. The nonviral vectors display superior specificity and protect against degradation of the nucleic acid construct by the target cell during transfection. The lipid formulations are designed for stability, increased half-life of the formulation and the prevention of aggregation of the lipid particles. In the liposomal nonviral vectors, 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, 50 pg, 75 pg, 100 pg, 110 pg, 120 pg, 125 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 210 pg, 220 pg, 225 ig, 230 pig, 240 pig, 250 pig, 260 pig, 270 pig,

275 pig, 280 pig, 290 pig, 300 pig, 310 pig, 320 pig, 325 pig, 330 pig, 340 pig, 350 pig, 360 pig,

370 pig, 375 pig, 400 pig, 425 pig, 450 pig, 500 pig, 550 pig, 600 pig, 650 pig, 700 pig, 750 pig,

800 pig, 850 pig, 900 pig, 950 pig, or 1000 pig of the nucleic acid construct 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, 2 mM, 3 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.

[0038] In embodiments, the nonviral vector composition comprises a nucleic acid construct (e.g., FAS expression construct) and DOTAP:cholesterol present at about 0.1 mg/ml to about 1 mg/ml of nucleic acid construct (including, e.g., about 0.1 mg/ml to about 0.5 mg/ml, about 0.1 mg/ml to about 0.4 mg/ml, about 0.1 mg/ml to about 0.3 mg/ml, about 0.2 mg/ml to about 0.5 mg/ml, or about 0.2 mg/ml to about 0.4 mg/ml of the nucleic acid construct) and about 0.1 mM to about 6 mM DOTAP: cholesterol (including, e.g., about 0.1 mM to about 1 mM, about 0.5 mM to about 1 mM, about 0.7 mM to about 2 mM, about 1 mM to about 6 mM, and about 3 mM to about 5 mM DOTAP: cholesterol). In embodiments, the nonviral vector composition comprises a nucleic acid construct (e.g., FAS expression construct) and DOTAP:cholesterol present at about 0.1 mg/ml to about 0.5 mg/ml of the nucleic acid construct and about 3 mM to about 5 mM DOTAP: cholesterol.

[0039] The nonviral 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 nonviral vector constructs is between 300 and 325 nm. i. DOTAP:Cholesterol Liposomes

[0040] DOTAP:cholesterol liposomes are nanoparticle liposomal formulations composed of 1-2-di oleoyl -3 trimethyl ammonium propane (also known as l,2-bis(oleoyloxy)-3- (trimethyl ammonio) propane or DOTAP) and cholesterol (Templeton et al., Nat. Biotechnol., 1997 15:647-652). 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 0.1 to 8 millimolar (mM) (e.g., 0.1 mM, 0.5 mM, 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 0.1 to 8 mM (e.g., 0.1, 0.5 mM, 1 mM, 2 to 7 mM, 3 to 6 mM, 4 to 5 mM, or 8 mM). In some embodiments of a nonviral 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. ii. Extrusion Techniques

[0041] 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 375°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 under argon gas. iii. Microfluidization Techniques

[0042] In other embodiments, the DOTAP:cholesterol liposomes are produced using a microfluidization method. Microfluidization can be used when consistently small (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.

III. Methods of Making Nonviral Vectors

[0043] Once manufactured, DOTAP: cholesterol liposomes can be used to encapsulate nucleic acids (e.g., a nucleic acid construct as described herein) resulting in the nonviral vectors described herein. In some embodiments, a nonviral 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 herein followed by rapid mixing. For example, equal volumes of nucleic acid construct and DOTAP:cholesterol, can be mixed at concentrations to obtain a resulting non-viral vector composition containing the nucleic acid construct and DOTAP:cholesterol (e.g., DOTAP-Chol-FAS) having a desired concentration of nucleic acid construct and a desired concentration of DOTAP: cholesterol. In embodiments, the nucleic acid and DOTAP: cholesterol mixture comprises about 0.1 mg/ml to about 1 mg/ml of nucleic acid construct (including, e.g., about 0.1 mg/ml to about 0.5 mg/ml, about 0.1 mg/ml to about 0.4 mg/ml, about 0.1 mg/ml to about 0.3 mg/ml, about 0.2 mg/ml to about 0.5 mg/ml, or about 0.2 mg/ml to about 0.4 mg/ml of the nucleic acid) and about 0.1 mM to about 6 mM DOTAP:cholesterol (including, e.g., about 0.1 mM to about 1 mM, about 0.5 mM to about 1 mM, about 0.7 mM to about 2 mM, about 1 mM to about 5 mM, about 3 to about 5 DOTAP:cholesterol). In embodiments, the nucleic acid and DOTAP: cholesterol mixture comprises about 0.1 mg/ml to about 0.5 mg/ml of the nucleic acid construct and about 3 mM to about 5 mM DOTAP:cholesterol.

[0044] In embodiments, for example, 100 pg of nucleic acid construct can be added to 5 mM lipids (e.g., in 100 pl) followed by rapid mixing.

[0045] In other methods, nonviral vectors can be produced using the heating, sonicating, and sequential extrusion methods described above. In some embodiments, nonviral vectors are produced using the microfluidization methods described above.

[0046] Once nonviral 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). IV. Viral Vectors

[0047] The term “viral vector” is used herein to refer to a recombinant viral vector for delivering genetic material (e.g., a polynucleotide sequence encoding a FAS protein such as human FAS isoform 1) 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 FAS protein such as human FAS isoform 1 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.

[0048] A “recombinant AAV vector” or “rAAV vector” comprises a rAAV genome derived from the wild type 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 an rAAV genome is encapsidated or packaged into an AAV particle, the particle can be referred to as an “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 sequence 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. [0049] 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 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 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.

V. Compositions / Pharmaceutical Formulations

[0050] Compositions including the nucleic acid constructs, nonviral vectors, and viral vectors are described herein. In embodiments, the composition includes a nonviral vector as described herein and a pharmaceutically acceptable excipient. In embodiments, the composition includes a nonviral vector as described herein and dextrose, e.g., about 5% dextrose in water or about 5% dextrose in saline. In other embodiments, the composition includes a nonviral vector as described herein and about 0.9% (e.g., 0.8%, 0.9%, 1.0%, etc.) sodium chloride. In additional embodiments, the composition includes a nonviral vector comprising a nucleic acid construct described herein and a combination of about 5% dextrose and about 0.9% sodium chloride.

[0051] The compositions, nucleic acid constructs, nonviral 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 exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington. Other substances may be added to the compositions to stabilize and/or preserve the compositions. As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” mean 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 or physiologically 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.

[0052] The compositions described herein may be in a form suitable for sterile injection. To prepare such a composition, the active therapeutic(s) (e.g., nonviral vector) 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 0.5% NaCl or physiological saline (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. [0053] In other embodiments, the 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, nonviral vector or 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 release of a nonviral vector as described herein. In some embodiments, intranasal delivery is used, for example, if the disease is related to the lungs, such as metastatic disease that spreads to the lungs, e.g., osteosarcoma.

[0054] The nucleic acid constructs, nonviral vectors, viral vectors and 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 FAS express! on/signaling to FAS-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, 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, nonviral vector, viral vector or composition as described herein is determined based on preclinical efficacy and safety. In some embodiments, a therapeutically effective amount of nonviral vector as described herein or a composition containing a therapeutically effective amount of the nonviral vector is injected intravenously. In other embodiments, a therapeutically effective amount of nonviral vector as described herein or a composition containing a therapeutically effective amount of the nonviral vector is administered intranasally. The nonviral vectors, viral vectors and 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.

VI. Methods of Treatment

[0055] Methods of treating cancer in a human subject are described herein. As used herein, the term “treating cancer” means administration of a therapeutic agent (e.g., nonviral vectors 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 FAS to FAS-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 FAS signaling/expression to cancer cells that have reduced FAS levels or inhibition of FAS signaling (e.g., colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, leukemia, neuroblastoma, lung cancer, or rectal cancer cells in a human subject). In one embodiment of a method of treating cancer, a 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 composition including a vector as described herein is administered to a human subject in need thereof. In a further embodiment of a method of treating cancer, a composition comprising a nonviral vector described herein is administered to a human subject in need thereof.

[0056] Any suitable methods of administering nucleic acid constructs, nonviral vectors, viral vectors, and compositions to a subject in need thereof may be used. In these methods, the nucleic acid constructs, nonviral vectors, viral vectors and 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, nonviral vectors, and 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, nonviral vectors, and 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.), i.e., at multiple time points. When the compositions are administered multiple times, the administrations may be separated by one day, three days, one week, two weeks, three weeks, 1 month, two months, or six months. [0057] Some methods of treatment described herein are combination therapies that include administering to the human subject a nucleic acid construct, a nonviral vector, a viral vector, or a composition as described herein, and a second anti-cancer therapy. In embodiments the second anti -cancer therapy is radiation therapy. In embodiments the second 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 a second anti-cancer therapy is a checkpoint inhibitor. Use of checkpoint inhibitors as immunotherapy for treating cancer is known in the art ( ee 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). A further example of a second anti-cancer therapy is a BRAF inhibitor such as encorafenib. Another example of a second anti -cancer therapy is an EGFR inhibitor. Examples of EGFR inhibitors include cetuximab, osimertinib, Tarceva® (erlotinib), and nivolumab. In embodiments of a combination therapy as described herein, the nucleic acid constructs, nonviral vectors, viral vectors or compositions are administered to the human subject before the second anti-cancer therapy is administered to the human subject (i.e., at two different time points). In another embodiment, the nucleic acid constructs, nonviral vectors, viral vectors and compositions are administered to the human subject at the same time that (concurrently with) the second anticancer therapy is administered. In another embodiment, the nucleic acid constructs, nonviral vectors, viral vectors and compositions are administered to the human subject after the second anti -cancer therapy is administered to the human subject (i.e., at two different time points). In some embodiments, a composition as described herein can include a nucleic acid construct, nonviral vector, or viral vector as described herein and a second 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. VII. Human Subjects

[0058] The terms “patient,” “subject,” and “individual” are used interchangeably herein, and mean a mammalian (e.g., human) subject in need of treatment for 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 non-small 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. i. Molecular Markers

[0059] 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 a composition containing a nonviral vector 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 CRC. 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.

[0060] 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 ROS1. A human subject having one or more of any of these mutations can be selected for treatment with the compositions, nucleic acid constructs, nonviral vectors, and methods described herein.

[0061] 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 (MSLL). 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 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 composition, nucleic acid construct, or nonviral 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.).

[0062] 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 CRC, other types of gastrointestinal cancer, and endometrial cancer, but may also be found in cancers of the breast, prostate, bladder, and thyroid. [0063] Human subjects having cancer cells with a particular mutation (e.g., BRAF V600E) and/or having MSI and/or dMMR can be treated with a monotherapy or a combination therapy as described herein. In one embodiment of a combination therapy as described herein, the second anti-cancer therapy is specific for a cancer associated with a particular mutation. For example, if a human subject has a BRAF V600E mutation, the second 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 second anti-cancer therapy is specific for a cancer with MSI. For example, if a human subject has an MSI tumor, the second anti -cancer therapy may be a checkpoint inhibitor such as pembrolizumab or nivolumab. In yet another embodiment of a combination therapy as described herein, the second anti-cancer therapy is specific for colon cancer. For example, if a human subject has colon cancer, the second anti-cancer therapy may be a cyclin dependent kinase (CDK) inhibitor.

EXAMPLES

[0064] Example 1. Restoring epigenetically silenced FAS expression via cationic lipid-encapsulated FAS DNA nanoparticle is sufficient to suppress metastatic colon tumor growth in vivo

[0065] It has been shown that tumor cells use an epigenetic mechanism, such as histone deacetylation, to silence protein expression to evade host immune surveillance. Multiple epigenetic mechanisms may be involved in silencing FAS expression in tumor cells. These epigenetic mechanisms may compensate for each other in suppression of FAS expression, which may limit the efficacy of epigenetic agent monotherapy. Experiments described herein demonstrate that restoring FAS expression is sufficient to suppress metastatic colon tumor growth. To this end, codon usage-optimized FAS cDNA was designed and synthesized and cationic lipid-encapsulated FAS cDNA nanoparticles were formulated. It was determined that codon usage optimization results in high FAS expression in colon tumor cells and tumor- selective delivery of a FAS cDNA nanoparticle is sufficient to suppress metastatic colon tumor growth in vivo.

[0066] Materials and Methods

[0067] Cell lines'. Murine colon carcinoma CT26 cell line, murine leukemia cell line EL4, human leukemia cell line Jurkat, and human colon carcinoma cell lines SW480, SW620, and LS41 IN were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Murine colon carcinoma cell line MC38 was provided by Dr. Jeffrey Schlom (National Cancer Institute, Bethesda, MD) and was characterized. Cell lines were tested bi-monthly for mycoplasma and were mycoplasma-free at time of experiments.

[0068] In vivo tumor models'. CT26 subcutaneous tumors were established by injecting CT26 cells (2xl0⁵ cells/mouse) into the right flank of BALB/c mice. SW620 subcutaneous xenografts were established by injecting SW620 cells (2.5xl0⁶ cells/mouse) into the right flank of athymic mice.

[0069] Cationic lipid nanoparticle '. Cationic lipid DOTAP-Cholesterol (1: 1) manufactured in T & T Scientific Corp (Knoxville, TN). Nanoparticle and DNA formulation is carried out in the Pi’s lab.

[0070] Single cell RNA sequencing dataset analysis'. Single cell RNA sequencing datasets of human colorectal cancer patients were extracted from GEO database (GSE146771). Cells are annotated according to dataset designation. Cells were subsetted by tissue of origin (normal colon, peripheral blood, and colorectal tumor and FAS expression level in the indicated cell types were determined using R package.

[0071] DNA methylation analysis'. Colorectal cancer TCGA 450K methylation array and RNA-seq data of FAS were downloaded from UCSC cancer genome browser (Xena). The normalized beta values and FPKM were used to generate the heatmap and correlation plots using Complex Heatmap and ggpubr packages in R3.6.3. The promoter diagram was generated using karyoploteR. Statistically analyses were performed in R package.

[0072] Cell death analysis'. Floating cells were collected from culture supernatant. Adherent cells were detached using trypsin and mixed with floating cells. The cells were suspended in annexin V-binding buffer (lOmM HEPES, pH 7.4, 140mM NaCl, 2.5mM CaC12) and stained with APC-annexin V (Biolegend) and propidium iodide. The stained cells were analyzed by flow cytometry.

[0073] Patient Dataset Analysis'. FAS mRNA datasets and survival datasets were extracted from TCGA database. Survival analyses were performed using the R survival and survminer packages.

[0074] Flow cytometry. Samples were stained with fluorescent dye-conjugated antimouse FAS, FasL, CD8, and anti-human FAS antibodies (Biolegend, San Diego, CA). Samples were acquired on an FACSCalibur with CellQuestPro or LSRFortessa with BD Diva 8.01 (BD Biosciences). All flow cytometry data analysis was conducted with FlowJo vlO.6.0 (BD Biosciences). [0075] Western blotting-. Tumor cells were lysed in total protein lysis buffer and separated in 4-20% SDS-polyacrylamide gels. The blots were probed with anti-human cleaved caspase 3, cleaved PARP (Cell Signaling Tech), FasL (BD Bioscience, San Diego, CA) and P-actin (Sigma-Aldrich, St Louis, MO).

[0076] Statistical analysis'. Statistical analysis was conducted using Prism8 (Graphpad) and p-values were calculated by a two-tailed Student’s t-test. Significance between survival groups were computed by two-sided log-rank test (Survival package, R).

[0077] Results

[0078] FAS is a suppressor of human CRC metastasis'. Analysis of human cancer genomics database revealed that the FAS locus is not focally amplified across a dataset of 3,131 tumors, suggesting that FAS is unlikely an oncogene. The FAS locus is significantly focally deleted across the entire dataset of these same 3,131 tumors, including human CRC (Broad Institute tumorscape database), suggesting that FAS is likely a colorectal tumor suppressor in humans. To functionally validate this notion, FAS mRNA expression datasets were extracted from the TCGA database and compared FAS expression level between normal colon tissues and colorectal tumors. FAS expression is significantly down-regulated in colorectal tumors as compared to normal tissue (Fig. 1A). Analysis of FAS expression level in different stages of human colorectal tumors indicated that FAS expression decreases with tumor progression and the lowest FAS expression is in stage IV colorectal tumors (Fig. IB & C). Kaplan-Meier survival analysis of FAS expression levels in tumors with patient survival time indicates that FAS expression level is positively correlated with CRC patient survival (Fig. ID). These findings indicate that FAS functions as a suppressor of human CRC.

[0079] FAS expression is repressed by its promoter DNA hypermethylation in tumor cells of human CRC patients. FAS expression can be repressed by its promoter DNA hypermethylation in human colon tumor cell lines (Petak et al. Cell Death Differ 2003;

10:211-7). Analysis of the human genomic DNA sequence identified a CpG island surrounding the FAS transcription start site (Fig. 2A). To determine the FAS promoter DNA methylation status in human CRC patients, DNA methylation datasets were extracted from TCGA database and analyzed for the FAS promoter DNA methylation. DNA methylation peaks were detected in three CpG sites including the CpG island at the FAS promoter (Fig. 2B). The FAS promoter DNA is hypermethylated in these three CpG sites in human colorectal tumors as compared to normal colon tissue (Fig. 2B). Correlation of FAS mRNA expression level with the FAS promoter DNA hypermethylation level revealed that the FAS promoter DNA hypermethylation is inversely correlated with FAS expression level in tumors from human CRC patients (Fig. 2C). These findings indicate that FAS expression is repressed by its promoter DNA hypermethylation in human CRC patients.

[0080] Single cell RNA sequencing indicates that FAS is highly expressed in immune cells and normal epithelial cells in human CRC patients. To determine the cellular FAS expression profiles in human CRC patients, single cell RNA-seq datasets were extracted from the raw datasets in the GEO database (GSE146771) and analyzed for FAS expression at the single cell level. Cell types that express FAS were annotated (Fig. 3A). Normal colon tissues share similar FAS expression cellular patterns as colorectal tumor cells (Fig. 4B). FAS expression was detected in all subsets of lymphocytes and epithelial cells. FAS expression level is lower in colorectal tumor cells (Fig. 3C).

[0081] Development of cationic lipid encapsulated codon-optimized FAS cDNA- expressing plasmid DNA nanoparticle. Cationic lipid-based nanoparticles are an efficient nucleic acid delivery system that selectively deliver DNA to tumors in vivo. To restore FAS expression, Fas cDNA-expressing plasmids were encapsulated. Because FasL is primarily expressed on T cells, an immune competent syngeneic colon tumor mouse model was first used. A codon usage optimization strategy was taken to synthesize mouse FAS cDNA with optimized coding sequences to maximize protein expression. The synthesized gene was cloned to a bacterial expression plasmid. The plasmid DNA was then encapsulated into cationic lipid (DOTAP-Chol, 1 : 1) to formulate nonviral vector nanoparticles (DOTAP-Chol- mFas)(Fig. 4A).

[0082] Restoring FAS expression overcomes colon tumor resistance to FasL-induced apoptosis in vitro. The mouse colon tumor CT26 cell line has an MSS genotype and is highly metastatic. CT26 cells express lower level of FAS than the MSI subtype of MC38 tumor cells and are resistant to FasL-induced apoptosis. Therefore, to restore FAS expression in CT26 cells, codon usage-optimized mFas was used. Titration of DNA and DOTAP-Chol formulation achieved as high as 50% FAS⁺ cells (Fig. 4B & C). Exogenous restoration of FAS expression in CT26 cells rendered tumor cells sensitive to FasL-induced apoptosis (Fig. 4C). All FAS⁺ tumor cells were effectively eliminated by FasL in vitro (Fig. 4C & D). Taken together, it was determined that restoring FAS expression in mouse colon tumor cells is sufficient to render FasL-resistant metastatic mouse colon tumor cells susceptible to FasL- induced apoptosis in vitro.

[0083] FAS DNA nanoparticle therapy is sufficient to suppress mouse colon tumor growth in immune competent mice. To determine whether the above finding can be translated to colon tumor growth suppression in vivo, CT26 tumor-bearing mice were treated with various doses of DOTAP-Chol-mFas. A mFas dose-dependent suppression of tumor growth was observed, and a dose of 25 pg mFas DNA encapsulated in 4 mM DOTAP-Chol achieved the highest tumor suppression efficacy (Fig. 8). DOTAP-Chol was used as control in this study. Next, tumor-bearing mice were treated at an early stage (Fig. 5 A). One dose of DOTAP- Chol-mFas treatment significantly suppressed tumor growth (Fig. 5B). The tumor was then allowed to grow to a larger size before the treatment and the tumor-bearing mice were treated three times with DOTAP-Chol-mFas (Fig. 5C). DOTAP-Chol-mFas also significantly suppressed growth of the established large tumors (Fig. 5D). Tumor tissues were then collected and analyzed for CD8⁺ T cell tumor infiltration and FasL expression. No significant change in tumor-infiltration CTL level was observed, and approximately 50% of the colon tumor-infiltrating CTLs are FasL⁺ (Fig. 5E). Taken together, it was determined that DOTAP- Chol-Fas therapy is sufficient to suppress colon tumor growth in vivo.

[0084] Exogenous expression of codon usage optimized FAS induces metastatic human colon tumor cell FAS receptor auto-oligomerization and tumor cell auto-apoptosis in vitro. SW480 and SW620 cell lines are a matched pair of human primary and metastatic colon tumor cell lines established from the same patient (Hewitt et al. J Pathol 2000;192:446-54). FAS expression is lower in SW620 cells than in SW480 cells (Fig. 6A). Human colon tumor cell line LS41 IN is a high-grade primary colon tumor cell line that also has lost FAS expression (Fig. 6A). Both SW620 and LS41 IN are resistant to FasL-induced apoptosis. To restore FAS expression in human colon tumor cells, human FAS cDNA with optimized codon use was synthesized and cloned into a bacterial expression plasmid. The plasmid DNA was then used to transfect (Lipofectamine with FAS plasmid DNA) SW620 and LS41 IN cells, resulting in FAS expression in over 60% of tumor cells (Fig. 6B). Analysis of tumor cell death revealed that the transfected cells die in the absence of FasL and cell death is further increased by FasL in vitro (Fig. 6C & D). In the transfected cell line LS41 IN (and to a lesser degree, cell line SW620), a significant amount of apoptosis (71.5%) was seen in the cell cultures with no added FasL. Almost all FAS⁺ cells are apoptotic (Fig. 7C & D). FasL induced rapid caspase 3 activation and PARP cleavage in SW480 cells (Fig. 6E), but not in SW620 cells (Fig. 6F). Exogenous expression of the codon usage-optimized FAS cDNA resulted in caspase 3 activation and PARP cleavage in the absence of exogenous FasL (Fig. 6G). One possible explanation for the above phenomenon is tumor cell produced FasL engages FAS to kill these tumor cells. To test this hypothesis, human FASL-specific monoclonal antibody was added to the cell culture. Repeated attempts with this FasL blockade approach failed to block tumor cell apoptosis of the tumor cells that express codon usage-optimized FAS. This phenomenon was therefore termed “auto-apoptosis”. FasL enhances this process (Fig. 6G). FasL binding to FAS receptor induces FasL monomer trimerization, followed by trimer oligomerization to initiate the death signal. Analysis of FAS protein distribution indicates that exogenous expression of codon usage-optimized FAS results in FAS oligomerization in the absence of FasL (Fig. 6H), a phenomenon thus termed “auto-oligomerization”. This auto-oligomerization was shown to lead to auto-apoptosis of the tumor cells expressing the transfected hFAS. The above finding suggests that the presence of tumor-infiltrating lymphocytes (TILs) may not be a requirement for DOTAP- hFAS therapy to restore apoptosis in vivo and that DOTAP-hFAS may be effective even in “cold” (few infiltrating immune cells) tumors, such as treatment-resistant MSS CRC tumors. [0085] The above results indicate that the FAS+ cells (transfected cells) are killed by added FasL as well as by auto-oligomerization of the restored FAS protein leading to autoapoptosis. The results also indicate that the FAS signaling pathway in CRC tumor cells is functional indicating that the limiting factor for apoptosis in human metastatic colon tumor cells is the silencing of FAS expression by epigenetic mechanisms. The results further indicate that restoring FAS expression in human colon tumor cells should be an effective way to suppress metastatic human colon tumor growth in vivo.

[0086] Restoring FAS expression is sufficient to suppress metastatic human colon tumor xenograft growth in vivo. To determine whether the above finding can be translated to human colon tumor growth suppression in vivo, a cationic lipid encapsulated codon usage- optimized human FAS cDNA nanoparticle (DOTAP-Chol-hFAS) was produced. SW620 cells were injected into athymic nude mice to established tumor xenografts. Human colon tumor xenografts were then established, and the tumor-bearing mice were treated with DOTAP-Chol-hFAS as shown (Fig. 7D). DOTAP-Chol-hFAS therapy is sufficient to suppress the xenograft tumor growth in vivo (Fig. 7E). To determine whether mouse FasL is capable of inducing human tumor cell FAS-mediated apoptosis, the FasL-sensitive human Jurkat cells and mouse EL4 cells were used (Fig. 9A & B). Human LS41 IN cells and mouse CT26 cells have no cell surface FAS protein (Fig. 9C) but have abundant cellular FasL protein (Fig. 9D). Co-culturing Jurkat cells with LS41 IN resulted in Jurkat cell apoptosis and co-culturing EL4 cells with CT26 cells resulted in EL4 cells apoptosis (Fig. 9E). Mouse MC38 cells induced human Jurkat cell apoptosis when co-cultured together (Fig. 10A). Blocking FasL with a mouse FasL neutralizing monoclonal antibody decreased Jurkat cell apoptosis (Fig. 10C).

[0087] The experimental results described above show that DOTAP-FAS gene immunotherapy effectively restores the expression and apoptotic function of FAS protein that has been epigenetically silenced in CRC tumor cells, both in vitro and in vivo, using both mouse and human FAS constructs. The experimental results also show that DOTAP-FAS gene monotherapy significantly ablates CRC tumors at both early and late stages, notably with a single treatment (single IV dose of only 25 pg), as demonstrated in the early-stage model.

[0088] To determine delivery efficiency of hFAS cDNA to the tumor site, human xenograft-bearing mice were treated with DOTAP-Chol-hFAS nanoparticles, tumor tissues were collected one day later, genomic DNA was isolated, and PCR was performed using a pair of PCR primers that are specific for the codon-optimized human FAS cDNA sequence (Fig. 7A). This PCR specifically detects the codon sequence optimized hFAS cDNA, not the endogenous FAS gene (Fig. 7B). High level of exogenous FAS was detected in the xenografts from mice treated with DOTAP-Chol-hFAS (Fig. 7C), indicating efficient delivery of the codon usage optimized FAS cDNA to the tumor site.

[0089] FAS DNA nanoparticle therapy has no significant toxicity. Conversely, FasL therapy causes lethal liver toxicity (Ogasawara et al., Nature 1993;364:806-9). Therefore, a preliminary toxicity study was performed to determine whether DOTAP-Chol-Fas gene therapy has liver toxicity. Mice were treated with DOTAP-Chol-mFas every three days for 3 times. Serum was collected for liver enzyme profiles to detect liver tissue damage. No significant increase in liver enzymes were observed in the serum (Table 2), even with IV doses as high as 100 pg DNA per mouse. Liver tissues were analyzed by histological means and examined by a board-certified pathologist. No microscopic tissue injury was observed in the treated mice (Fig. 11). Therefore, it was concluded that DOTAP-Chol-mFas therapy is not hepatotoxic.

[0090] Table 2 Mouse liver enzyme profile

[0091] In conclusion, using this nanoparticle system in combination with codon usage- optimized FAS cDNA, it was determined that tumor-selective lipid nanoparticle FAS cDNA delivery is sufficient to restore FAS expression (on human colon tumor cell surfaces) to suppress metastatic human colon tumor xenograft growth in vivo in the absence of immune cells, thereby in the absence of membrane-bound FasL. FasL is expressed on activated T cells and NK cells under physiopathological conditions. It is known that T cells and NK cells are often suppressed in the tumor microenvironment. The findings described herein indicate that DOTAP-Chol-hFAS therapy is sufficient to suppress human colon tumor growth in the immune suppressive tumor microenvironment. DOTAP-Chol-hFAS is therefore an effective agent to restore FAS expression in metastatic human colorectal carcinoma to suppress metastasis.

[0092] Example 2. In vivo Experiments Demonstrating Suppression of Tumor Growth and Survival

[0093] Mouse CRC CT26 cells were implanted subcutaneously (SQ) in immune competent mice on Day 1. DOTAP-Chol-mFAS or the DOTAP-Chol only (control group) was administered intravenously (IV) on Days 5, 8 and 11. Two hundred pl containing 5, 10, 25, 50 or 100 micrograms (pg) of mFas was injected. The mice were sacrificed and the tumors analyzed 7 days later (Fig. 12). In this experiment, mouse deaths were observed in the control group and the 50 pg dose group that were attributed to the 200 pl injection volumes used. The total volume of blood in a 20-gram mouse is only 1 ml. Twenty percent of their blood volume administered quickly via IV proved to be toxic. All further injection volumes were limited to 100 pl and no further deaths were seen. Additionally, solubility issues were encountered in the 100 pg dose group and as a result, this group received only a single injection. Nevertheless, this experiment showed a dose response that became significant at the 50 pg dose level (p=0.0302) as shown in Figure 12. There was no significant difference between the 50 and 100 pg groups. [0094] A survival study using CT26 cells to cause metastatic lung disease in mice was conducted. 2xl0⁵ CT26 cells were injected IV on Day 1. Twenty-five pg of DOTAP-Chol- mFas nanoparticle was administered IV on Days 5, 8 and 11 to the treatment group (N=15) and the control group (N=15) was given empty nanoparticles only. Mice in the control group had a median survival of 23 days. Mice in the treatment group had a median survival of 33 days (pO.OOOl) (Fig. 13).

[0095] 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.

Claims

We Claim:

1. A nucleic acid construct comprising (i) a polynucleotide sequence encoding human FAS isoform 1 flanked by a 5' untranslated region (UTR) and a 3' UTR; (ii) a CMV promoter operably linked to the polynucleotide sequence encoding human FAS isoform 1; and (iii) a selectable marker.

2. The nucleic acid construct of claim 1, wherein the human FAS isoform 1 comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having greater than 95% sequence identity to SEQ ID NO: 1.

3. The nucleic acid construct of claim 1, wherein the polynucleotide sequence encoding human FAS isoform 1 is SEQ ID NO: 2, or is a polynucleotide sequence having greater than 90% sequence identity to SEQ ID NO: 2.

4. The nucleic acid construct according to any one of claims 1 to 3, wherein the 5' UTR comprises at least one intron.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the 5' UTR comprises a HTLV-I R element.

6. A nonviral vector comprising a nucleic acid construct according to any one of claims 1 to 5 and a DOTAP: cholesterol liposome.

7. The nonviral vector of claim 6, wherein the DOTAP: cholesterol ratio is between about 3 : 1 and about 1 :3.

8. A composition comprising the nonviral vector according to claim 6 or claim 7 and a pharmaceutically acceptable excipient.

35

9. The composition of claim 8, wherein the nucleic acid construct concentration is in a range of about 0.1 mg/ml to about 0.5 mg/ml and the DOTAP:cholesterol concentration is in a range of about 3 mM to about 5 mM.

10. The composition according to claim 8 or claim 9, comprising about 5% dextrose, about 0.9% sodium chloride or a combination of both agents.

11. The composition according to any one of claims 8 to 10, wherein the DOTAP:cholesterol liposome has a particle size range of about 40 to 250 nanometers.

12. A method for treating cancer in a human subject comprising administering to the human subject in need thereof a composition comprising a nucleic acid construct according to any one of claims 1 to 5.

13. A method for treating cancer in a human subject comprising administering to the human subject in need thereof a composition comprising the nonviral vector according to claim 6 or claim 7.

14. A method for treating cancer in a human subject comprising administering to the human subject in need thereof a composition according to any one of claims 8 to 11.

15. The method according to any one of claims 12 to 14, wherein the cancer is selected from the group consisting of: colon cancer, pancreatic cancer, breast cancer, melanoma, osteosarcoma, neuroblastoma, leukemia, lung cancer, and rectal cancer.

16. The method according to any one of claims 12 to 15, wherein the composition is administered intravenously or intranasally.

17. The method according to any one of claims 12 to 16, further comprising administering a second anti-cancer therapy to the subject.

36

18. The method of claim 17, wherein the second anti-cancer therapy comprises at least one of: chemotherapy, radiation treatment, and surgery.

19. The method of claim 17, wherein the second anti-cancer therapy is a checkpoint inhibitor or a BRAF inhibitor.

20. The method of claim 17, wherein the second anti-cancer therapy is an EGFR inhibitor.

21. The method of claim 19, wherein the checkpoint inhibitor is pembrolizumab and the BRAF inhibitor is encorafenib.

22. The method of claim 20, wherein the EGFR inhibitor is cetuximab or nivolumab.

23. The method of claim 17, wherein the second anti-cancer therapy is CAR-T therapy.

24. A viral vector comprising a polynucleotide sequence encoding human FAS isoform 1 operably linked to a promoter.

25. The viral vector of claim 21, wherein the viral vector is an Adeno- Associated Virus (AAV) viral vector.

26. A method for treating cancer in a human subject comprising administering to the human subject in need thereof a viral vector according to claim 24 or claim 25.