Treatment of ALK positive cancers

Abstract

The disclosure provides methods and compositions for treating cancer, e.g., lung cancer including NSCLC that are ALK positive. The compositions for treating ALK positive cancers comprise a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide.

Description

TREATMENT OF ALK POSITIVE CANCERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 550,508 filed February 6, 2024, the entire contents of which is incorporated herein by reference.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on February 6, 2025, is named SeqList- 198628-46576. xml and is 3039 bytes in size.FIELD

[0003] This disclosure provides compositions and methods of use for the treatment of anaplastic lymphoma kinase (ALK) positive cancers.BACKGROUND

[0004] Lung cancer is the leading cause of cancer-related death worldwide (Torre et al. (2015) Global cancer statistics, 2012. CA Cancer J Clin 65: 87-108), and non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases (Inamura K ( 2017) Lung cancer: understanding its molecular pathology and the 2015 WHO classification. Front Oncol 7: 193). Lung adenocarcinoma is the most common subtype of lung cancer. Many oncogenic drivers of adenocarcinoma have been found in the clinic, such as the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) (Bayliss R. et al. (2016) Molecular mechanisms that underpin EML4-ALK driven cancers and their response to targeted drugs. Cell Mol Life Sci 73: 1209-1224). ALK was originally discovered in an anaplastic large cell lymphoma (ALCL) cell line, in which the ALK gene was fused with the nucleophosmin 1 (NPM1) gene, resulting in a new fusion protein, NPM-ALK (Moms SW, et al. (1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263: 1281-1284).

[0005] Tumors bearing the EML4-ALK fusion are sensitive to ALK Tyrosine Kinase Inhibitors (TKIs), that form the first and second lines of treatment for these patients. However, ALK+ lung cancers develop resistance to ALK inhibitors, creating the need for newer treatment strategies in ALK+ NSCLC.SUMMARY

[0006] Embodiments are directed to compositions that encode TUSC2 for use in the treatment of cancers that are ALK positive (e.g., have the EML4-ALK fusion or other fusion with ALK and / or overexpress ALK), including lung cancers, e.g., NSCLC. In embodiments, the disclosure provides a method for sensitizing ALK TKI-resistant tumor cells to ALK TKIs by exposing the tumor cells to a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the disclosure provides a method for inducing apoptosis in cancer cells that are ALK positive by exposing the cancer cells to a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the ALK positive cancer cells are resistant to one or more ALK TKIs. Accordingly, in certain aspects a method of treating cancer in a subject comprises administering to the subject in need thereof a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, wherein the cancer cells are ALK positive (e.g., have the EML4-ALK fusion or other fusion with ALK and / or overexpress ALK) and / or are resistant to one or more ALK inhibitors.. In embodiments, the cancer is lung cancer, in particular, non-small cell lung carcinoma (NSCLC). In certain embodiments, the composition comprises a therapeutically effective amount of a liposome comprising a polynucleotide encoding TUSC2 (e.g., a TUSC2 lipid nanoparticles). In certain embodiments, the subject has previously received treatment comprising administration of ALK inhibitors (also referred to herein as ALK TKI inhibitors). In certain embodiments, the NSCLC is refractory to treatment with ALK inhibitors. In certain embodiments, ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF- 802 / R05424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X-396, CEP-28122 / CEP-37440, 5H- pyrrolo[3,2-t ]pyrimidine or combinations thereof. In certain embodiments, expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLCcells prior to administering the polynucleotide construct. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2. In certain embodiments, the polynucleotide construct comprises non-viral vectors or viral vectors. In certain embodiments, the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors. In certain embodiments, the liposome comprises a DOTAP:cholesterol liposome. In certain embodiments, the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3. In certain embodiments, the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof. In certain embodiments, the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra- cistema magna (i.c.m.), or intrasternally. In certain embodiments, the method further comprises administering to the subject one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, radiation, surgery or combinations thereof.

[0007] In another aspect, the disclosure provides a method of diagnosing and treating an ALK+cancer in a subject, comprising assaying a cancer sample obtained from the subject for cells that are ALK positive, and administering to the subject a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, thereby diagnosing and treating the ALK+ cancer in the subject. In embodiments, the ALK+ cancer is non-small cell lung carcinoma (NSCLC), the method optional comprises assaying for NSCLC cells expressing one or more NSCLC specific biomarkers in a sample obtained from a subject and diagnosing the subject as having NSCLC when one or more NSCLC specific biomarkers are detected.

[0008] In certain embodiments, the composition comprises a therapeutically effective amount of a liposome comprising a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In certain embodiments, the subject has previously received treatment comprising administration of ALK inhibitors. In certain embodiments, the NSCLC is refractory to treatment with ALK inhibitors. In certain embodiments, ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X- 376 / X-396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-t ]pyrimidine or combinations thereof.In certain embodiments, expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide that has at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2. In certain embodiments, non-viral vectors or viral vectors comprise the polynucleotide construct. In certain embodiments, the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors. In certain embodiments, the liposome comprises a DOTAP: cholesterol liposome (for example, Reqorsa, Quaratusugene Ozeplasmid (QO)). In certain embodiments, the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3. In certain embodiments, the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof. In certain embodiments, the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra- cistema magna (i.c.m.), or intrasternally. In certain embodiments, the method further comprises administering to the subject one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, radiation, surgery, or combinations thereof.

[0009] In another aspect, a method of treating lung tumors in a subject comprises administering to the subject a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, or a liposome comprising a TUSC2 polypeptide or a TUSC2 polynucleotide, wherein the lung tumors are resistant to treatment with ALK inhibitors. In certain embodiments, the lung tumors comprise non-small cell lung carcinomas (NSCLCs), thereby treating the lung tumors. In certain embodiments, the NSCLC cells are anaplastic lymphoma kinase (ALK) positive. In certain embodiments, ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF- 802 / R05424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X-396, CEP-28122 / CEP-37440, 5H- pyrrolo[3,2-t ]pyrimidine or combinations thereof. In certain embodiments, expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2. In certainembodiments, the polynucleotide construct comprises non-viral vectors or viral vectors. In certain embodiments, the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors. In certain embodiments, the liposome comprises a DOTAP:cholesterol liposome. In certain embodiments, the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3. In certain embodiments, the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof. In certain embodiments, the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra- cistema magna (i.c.m.), or intrasternally. In certain embodiments, the method further comprises administering to the subject one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, radiation, surgery or combinations thereof.

[0010] In reference to the vector encoding the polypeptide composition of the present disclosure, the functional portion can comprise, for instance, about 90%, 95%, or more, of the TUSC2 polypeptide. The functional portion of the polypeptide composition of the present disclosure can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of either of the wild type TUSC2 polypeptides.

[0011] In another aspect, a method for inducing apoptosis in an ALK positive cancer cell or sensitizing an ALK positive cancer cell to an agent or composition, comprises contacting the cancer cell with a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, wherein the cancer cells are anaplastic lymphoma kinase (ALK) positive, thereby sensitizing or inducing apoptosis of the cell by the composition. In certain embodiments, the cancer cell is non-small cell lung carcinoma (NSCLC) cell. In certain embodiments, the composition comprises a effective amount of a liposome encapsulating the polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide. In certain emodiments, the cancer cell had previously been exposed to an ALK inhibitor. In certain embodiments, the cancer cell is refractory to ALK inhibitors. In certain embodiments, ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR- 011, X-376 / X-396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-d]pyrimidine or combinations thereof. In certain embodiments, expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLC cells prior to administering thepolynucleotide construct. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2. In certain embodiments, the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2. In certain embodiments, the polynucleotide construct comprises non-viral vectors or viral vectors. In certain embodiments, the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors. In certain embodiments, the liposome comprises a DOTAP: cholesterol liposome. In certain embodiments, the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3. In certain embodiments, the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof.

[0012] In embodiments, exposure of the ALK positive cancer cell to an effective amount of a composition comprising a polynucleotide sequence encoding a TUSC2 polypeptide disclosed herein induces apoptosis in the ALK positive cancer cell or sensitized the ALK positive cancer cell to an ALK inhibitor disclosed herein, or other anti-cancer agent disclosed herein.

[0013] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 is a series of brightfield and fluorescence microscopy pictures demonstrating that cells transfected with pcDNA3.1 containing EGFP, show GFP signal 24 hours post transfection, indicating successful transfection.

[0015] FIGS. 2A-2G are a series of graphs and blots demonstrating that Quaratusugene Ozeplasmid (QO) can overexpress TUSC2 in EML4-ALK+NSCLC and non-ALK cell lines. FIGS. 2A-2B, 2F: TUSC2 is overexpressed in non-ALK lung cancer cell lines, FIGS. 2C- 2F, and is significantly overexpressed in EML4-ALK+NSCLC cell lines (transcriptional and translational levels). FIG. 2G: The protein expressions have been quantified and normalized to the housekeeping control. For all qPCR (2A-2G), data have been analyzed using t-test. *** represents 0.0001 < p < 0.001, **** represents p < 0.0001.

[0016] FIG. 3 is a series of colony formation assays and graphs on the A549 (top panels) and DFCL032 (bottom panels) cell lines following transfection with TUSC2 lipid nanoparticles (Reqorsa), TUSC2 expression plasmid (TUSC2), and empty plasmid (pcDNA).

[0017] FIG. 4 is a series of graphs and blots demonstrating caspase 3 / 7 activity when cells are treated with Reqorsa and TUSC2 plasmid, the successful overexpression being demonstrated by the Western blot data.

[0018] FIG. 5 is a series of blots demonstrating the change in apoptotic markers when treated with Reqorsa and TUSC2 plasmid.

[0019] FIG. 6 is a series of blots of A549, DFCI-032, NCI-H2170, NCI-H3122 and NCI- H2228 cell lines transiently transfected with 3.5 pg of pcDNA3.1, Reqorsa or TUSC2 plasmid (as a positive control). 48 hours post transfection, the cells were collected, and protein was isolated using RIPA buffer. 50 pg of protein lysate was loaded on a precast 8- 16% gel to detect TUSC2 protein expression.

[0020] FIG. 7 is a series of graphs demonstrating the resistance of the generated resistant cell lines to Alectinib. The parental cell line (NCI-3122) along with its corresponding resistant cell lines (Cmax, Step, High) were seeded in a 96-well plate at a density of 2000 cells / well in adherent condition. Next day, the cells were treated with 8 doses of Alectinib (1 :5 dilution starting from a 5mM stock) and their viability was recorded after 5 days.

[0021] FIG. 8 is a series of graphs demonstrating the effect of TUSC2 lipid nanoparticles (Reqorsa) or TUSC2 expression plasmid on cell viability in ALK+NSCLC.

[0022] FIG. 9 is a series of graphs demonstrating the effect of TUSC2 lipid nanoparticles (Reqorsa) or TUSC2 expression plasmid on cell viability in ALK+NSCLC.

[0023] FIG. 10 is a series of graphs to study the effect of overexpressing TUSC2 on cellular proliferation.

[0024] FIG. 11 is a series of graphs to study the effect of overexpressing TUSC2 on cellular proliferation.

[0025] FIG. 12 is a series of graphs to study the effect of overexpressing TUSC2 on cellular proliferation.

[0026] FIG. 13 is a series of graphs quantifying caspase 3 / 7 activity.

[0027] FIG. 14 is a series of graphs quantifying caspase 3 / 7 activity.

[0028] FIG. 15 is a series of graphs to study the effect of overexpressing TUSC2 in sensitizing the cells to ALK tyrosine kinase inhibitors (TKIs).

[0029] FIGS. 16A-16J are a series of graphs, blots photographs of cultured cells demonstrating that Quaratusugene Ozeplasmid (QO) mediated TUSC2 overexpression can induce apoptosis in EML4-ALK+NSCLC cell lines. FIG. 16A: QO mediated overexpression of TUSC2 increases the caspase 3 / 7 activity in non- ALK NSCLC cell lines FIG. 16B, and in EML4-ALK+NSCLC cell lines. FIG. 16C: QO mediated overexpression of TUSC2increases the expression of cleaved PARP. FIG. 16D: The protein expressions have been quantified and normalized to the housekeeping control. FIGS. 16E-16H: The same combination decreases the colony formation ability of the cells. All data have been analyzed using t-test. ****: p< 0.0001,**:0.001<p<0.01,*:0.01<p< 0.05, ns: p > 0.05.

[0030] FIGS. 17A-17B are a series of graphs demonstrating the generation of ALK inhibitor, Alectinib resistant cell lines. FIG. 17A: NCI-H2228 and FIG. 17B, NCLH3122 cell lines were made resistant to the ALK inhibitor, Alectinib by 3 independent methods. The cells were treated for a prolonged period with their Cmax values (referred to herein as “Cmax” or Cmax method), ICso values (referred to herein as “IC50” of IC50 method) and a range of drug doses starting from a low value and gradually escalating the dose (referred to herein as “Start low” or start low method), to establish these cell lines, and were respectively labelled as Cmax, Start ICso and Start low. Each method was generated in triplicates.

[0031] FIGS. 18A-18L are a series of graphs and blots demonstrating that Quaratusugene Ozeplasmid mediated TUSC2 overexpression can induce apoptosis in Alectinib resistant EML4-ALK+NSCLC cell lines. FIGS. 18A, 18B: QO mediated overexpression of TUSC2 in Alectinib resistant NCI-H228 cells significantly increases the caspase 3 / 7 activity. FIG. 18C: QO mediated overexpression of TUSC2 increases the expression of cleaved caspase 3 in these cells. FIGS. 18D, 18E: The protein expressions have been quantified and normalized to the housekeeping control. All data have been analyzed using t-test. ****:p< 0.0001, *** : 0.0001< p< 0.001 **:0.001<p<0.01. FIGS. 18F-18G: Quaratusugene Ozeplasmid mediated TUSC2 overexpression can induce apoptosis in Alectinib resistant EML4-ALK+NSCLC cell lines. QO mediated overexpression of TUSC2 in Alectinib resistant cells significantly increases the caspase 3 / 7 activity in these cells. All data have been analyzed using t-test.****:p< 0.0001, ***:0.0001< p< 0.001 **:0.001<p<0.01, ns: p > 0.05. FIGS. 18H-18L: Quaratusugene Ozeplasmid mediated TUSC2 overexpression can induce apoptosis in Alectinib resistant EML4-ALK+NSCLC cell lines. FIGS. 18H, 18K: QO mediated overexpression of TUSC2 increases the expression of cleaved caspase 3 and cleaved PARP in the resistant cells. FIGS. 181, 18 K, 18L: The protein expressions have been quantified and normalized to the housekeeping control.DETAILED DESCRIPTION

[0032] ALK Positive Cancer / Tumors

[0033] The disclosure provides methods of treating cancers (including lung cancers, e.g., NSCLC) that are ALK positive (ALK+, e.g., have the EML4-ALK fusion or other fusion with ALK and / or overexpress ALK) by administering a composition to a subject in need thereof comprising a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the disclosure provides a method for sensitizing ALK TKI-resistant tumor cells to ALK TKIs by exposing the tumor cells to a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the disclosure provides a method for inducing apoptosis in cancer cells that are ALK+ by exposing the cancer cells to a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the cancer ALK+ cancer cells are resistant to one or more ALK TKIs.

[0034] In certain cancers, Anaplastic Lymphoma Kinase (ALK) point mutations or chromosomal rearrangements result in aberrant activation of ALK and downstream signaling cascades. Activation of ALK is a potent oncogenic driver in Non-Small Cell Lung Carcinoma (NSCLC), contributing to approximately 5% of NSCLC as a distinct clinicopathological subset. In NSCLC (and other cancer types that include ALCL, diffuse large B cell lymphoma, inflammatory myofibroblastic tumors, glioma and colon cancer) the oncogenic driver is often a structural ALK rearrangement resulting in the kinase domainencoding region of ALK at the 3' end fused to various partner genes at the 5' end. More than 90 different ALK fusion proteins have been found in different cancers.

[0035] Fusion of ALK with echinoderm microtubule-associated protein-like 4 (EML4) is the most common ALK fusion in NSCLC, identified in about 85% of cases. EML4-ALK is produced via fusion of the ALK gene to the gene (Holla VR, et al. (2017) ALK: a tyrosine kinase target for cancer therapy. Cold Spring Harb Mol Case Stud 3 : aOOl 115), resulting in constitutive activation of ALK and, in turn, to dysregulation of signaling pathways in the host cell, leading to tumorigenesis (Golding B, el al. (2018) The function and therapeutic targeting of anaplastic lymphoma kinase ( ALK) in non-smaH cell lung cancer (NSCLC). Mol Cancer 17: 52).

[0036] The presence of the EML4-ALK fusion gene, and other ALK fusion genes, can be detected in a tumor sample using fluorescence in situ hybridization (FISH) and FDA- approved kits are commercially available for this purpose. Alternatively, the fusion gene canbe detected in a tumor sample by immunohistochemistry, e.g., with an antibody that specifically recognizes the ALK fusion protein.

[0037] Tumors bearing the EML4-ALK fusion are sensitive to ALK Tyrosine Kinase Inhibitors (TKIs), that form the first and second line of treatment for these patients. However, ALK+lung cancers develop resistance to ALK inhibitors, creating the need for additional treatment strategies in ALK+NSCLC.

[0038] Crizotinib was approved by the FDA in 2011 as the first ALK inhibitor. Clinical data have demonstrated that crizotinib is superior to cytotoxic chemotherapy in patients with ALK-positive NSCLC (Shaw AT. el al. (2013) Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. A;£ gZ / Wt? 368: 2385 -2394). Although crizotinib initially results in significant clinical benefits to patients, disease inevitably progresses after treatment because of acquired resistance mutations (Tucker ER, et al. Cancer Res 75: 2770-2774). Mechanisms of acquired resistance are classified into two types: ALK-dependent resistance mechanisms, such as ALK amplification and ALK secondary resistance mutations, and ALK- independent resistance mechanisms, including activation of bypass signaling pathways and lineage changes (Romanidou O et al. (2016) Overcoming resistance to first / second generation epidermal growth factor receptor tyrosine kinase inhibitors and ALK inhibitors in oncogene- addicted advanced non-small cell lung cancer. Ther Adv Med Oncol 8(3): 176-187).

[0039] Secondary ALK resistance mutations were identified in 20-30% of post-crizotinib treatment samples (Awad MM, Shaw AT (2014) ALK inhibitors in non-small cell lung cancer: crizotinib and beyond. Clin Adv Hematol Oncol 12: 429-439). Among these mutations, LI 196M and G1269A are the most commonly detected in the clinic (Lin JJ, Riely GJ, Shaw AT (2017) Targeting ALK: precision medicine takes on drug resistance. Cancer Discov 7: 137-155). To overcome crizotinib resistance mutations, second-generation ALK inhibitors such as ceritinib, alectinib, and brigatinib were developed and approved successively; all of these tyrosine kinase inhibitors (TKIs) show potency against LI 196M and G1269A (Yue Lu et al. (2021) A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC. EMBO Mol Med. 14: el4296. doi.org / 10.15252 / emmm.202114296). Unsurprisingly, patients inevitably relapse on these second-generation inhibitors, and approximately 50-70% acquire secondary ALK resistance mutations. G1202R is a solvent front mutation in the kinase domain and impairs the binding of all first- and second-generation ALK inhibitors by introducing steric hindrance, and thus confers strong clinical resistance to ALK inhibitors. Although G1202R is less frequently detected in post-crizotinib treatment samples (2%), it is the predominant resistance mutationin patients treated with second-generation ALK inhibitors (40-65%) (Gainor JF, et al. (2016) Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in ALK- rearranged lung cancer. Cancer Discov 6: 1118-1133; Yue Lu et al. (2021). The third- generation ALK inhibitor lorlatinib was approved by the FDA in 2018 for the treatment of ALK-positive NSCLC (Syed YY (2019) Lorlatinib: first global approval. Drugs 79(1): 93- 98); it showed promising activity against all TKI resistance mutations in ALK in both cellular assays and in vivo mouse models, and most importantly, it could overcome the resistance caused by the G1202R mutation (Gainor et al. 2016). Due to the abovementioned high- frequency ALK TKI resistance mutations in the clinic, there is a need for more effective treatments for cancers which become resistant to treatment by ALK inhibitors.

[0040] Next-generation ALK inhibitors'. Second-generation ALK inhibitors were developed to enhance anti-ALK activity, to overcome crizotinib-resistant mutations and to improve activity in CNS disease. The molecular characteristics of these drugs are listed in Table 1. ALK inhibitors in development are listed in Table 2 (Sullivan I, Planchard D. ALK inhibitors in non-small cell lung cancer: the latest evidence and developments. Ther Adv Med Oncol. 2016 Jan;8(l):32-47. doi: 10.1177 / 1758834015617355. PMID: 26753004; PMCID: PMC4699265).Table 1Molecular characteristics of second-generation ALK inhibitors.Drugs Targets Activity Activity Activity Activity against other Lack of other than against against against crizotinib- resistant activityALK L1196M C1156Y G1202R mutations against resistance resistance resistance resistance mutation mutation mutation mutationsCeritinib IGF-R1, Yes No No G1269A, 11171T, G1202R,InsR, S1206Y, L1152R, F1174CROS1 F1174L, V1180LAlectinib LTK, GAK Yes Yes No G1269A, S1206Y, G1202R,L1152R, F1174L, V1180L,1151T-ins 11171TBrigatinib ROSt, Yes Yes Yes G1269A, S1206Y, NAEGFR 1151T-ins, F1174C,11171T, D1203N, E1210K, F1245CEntrectinib TrkA, Yes Yes NA NA NATrkB,TrkC,ROS1PF- ROS1 Yes NA Yes G1269A NA06463922TSR-011 TrkA, Yes NA NA NA NATrkB,TrkCASP3026 ROS1, Yes NA NA F1174L NAACKX-396 MET Yes Yes NA NA NACEP-37440 FAK NA NA NA NA NAALK, anaplastic lymphoma kinase; NA, not available.

[0041] Table 2.ALK inhibitors in early phase of development.Drug name Study name or Phase Most common grade 1-2 AEsClinicalTrials.gov identifierEntrectinib NCT02097810 1 / 11 Paraesthesia (42%) nausea (37%), myalgia (34%), asthenia (27%), dysgeusia (27%), vomiting (21%), arthralgia (19%), diarrhoea (19%)PF- NCT01970865 1 / 11 Hypercholesterolemia (48%), peripheral oedema (23%)06463922 and peripheral neuropathy (21%)TSR-011 NCT02048488 1 / 11 Fatigue (17.4%), constipation (15.9%), QTc prolongation(15.9%), diarrhoea (14.5%) and headache (13%)ASP3026 NCT01401504 1 Fatigue (44%), vomiting (39%), nausea (37%), and constipation (24%)X-396 NCT01625234 1 / 11 Rash (31%), nausea (31%), vomiting (29%), fatigue(26%), oedema (17%) and pruritus (11%)CEP-37440 NCT01922752 1 NACEP-28122 Preclinical NAAEs, adverse events; ID, identifier; NA, not available.

[0042] Accordingly, this disclosure provides methods of treating a subject having lung tumors that are resistant to treatment with one or more ALK inhibitors by administering to the subject a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, or a liposome comprising a TUSC2 polypeptide or a TUSC2 polynucleotide.

[0043] Non-Small Cell Lung Carcinoma (NSCLC) Biomarkers

[0044] Genomic Biomarkers in NSCLC

[0045] Advances in elucidating the molecular biology of lung cancer have led to the identification of a number of biomarkers that could be relevant in the clinical management of patients with NSCLC. (Villalobos P, Wistuba II. Lung Cancer Biomarkers. Hematol Oncol Clin North Am. 2017 Feb;31(1): 13-29. doi: 10.1016 / j.hoc.2016.08.006. PMID: 27912828; PMCID: PMC5137804).

[0046] Epidermal growth factor receptor (EGFR): The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor member of the ERBB family. The EGFR gene is located on the short arm of chromosome 7 at position 12. When the extracellular ligand binds to EGFR, it generates homo- or heterodimerization of the receptor, leading to phosphorylation of sites in the cytoplasmic tyrosine kinase and activation of various intracellular pathways,including the phosphatidylinositol 3 -kinase (PI3K) / AKT / mammalian target of rapamycin (mTOR) and RAS / RAF / mitogen-activated protein kinase (MAPK) pathways, which lead to cell proliferation, metastasis, and prevention of apoptosis. EGFR is overexpressed in 62% of NSCLCs, and its expression has been associated with poor prognosis. Approximately 10% of patients with adenocarcinoma of the lung in the United States and 30% to 50% in East Asia have lung tumors associated with EGFR mutations. These mutations occur within exons 18- 21, which encode for a portion of the EGFR kinase domain. Approximately 90% of EGFR mutations occur as in-frame deletions in exon 19 or as missense mutations in exon 21 (44% and 41% of all mutations, respectively). Activating mutations in the kinase domain of EGFR trigger ligand-independent tyrosine kinase activation, leading to hyperactivation of downstream antiapoptotic signaling pathways. EGFR mutations are found more often in adenocarcinomas with lepidic features from female never smokers (10). The high response rates (55%-78%) to treatment with tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib and afatinib, in patients with EGFR-mutant tumors, and the significantly greater progression-free survival (PFS) of these patients, have made EGFR TKIs the standard treatment for patients with these mutations. However, most of these patients develop resistance and relapse in a short time, owing to the occurrence of a new mutation (T790M) in exon 20 of the EGFR kinase domain (50%), amplification of the MET oncogene (21%), or mutations of PI3KCA.

[0047] Anaplastic lymphoma kinase (ALK): Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor member of the insulin receptor superfamily. The ALK gene is located on the short arm of chromosome 2 at position 23. ALK gene rearrangement was originally identified in anaplastic large cell lymphoma and was subsequently described in a subset of NSCLC tumors harboring a fusion of ALK and EML4 genes. This rearrangement encodes a chimeric protein with constitutive kinase activity, which promotes malignant growth and proliferation. The EML4-ALK fusion has been detected in about 3.7% to 7% of NSCLCs, usually in adenocarcinomas with signet-ring cells or cribriform histology features and is more common in young patients who have never smoked. There are several EML4- ALK rearrangement variants and also ALK fusion with other less frequent partners, such as kinesin family member 5B (KIF5B), TRK-fused gene (TFG), kinesin light chain 1 (KLC1), and huntingtin-interacting protein 1 (HIP1) genes, resulting in oncogenic transformation. It has been shown that EGFR, Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and ALK molecular alterations are generally mutually exclusive events; nevertheless, they have been described in up to 2.7% of lung adenocarcinoma cases with concurrent molecularalterations. The ALK fusion defines a distinct subpopulation of patients with lung adenocarcinoma who are highly responsive (57%-74%) to ALK inhibitors such as crizotinib. Patients treated with crizotinib demonstrated significantly better median PFS and response rate compared to patients who received chemotherapy. As a result, testing for ALK rearrangements in patients with advanced lung adenocarcinoma is recommended in current clinical practice guidelines. However, despite initial responses, patients develop acquired resistance to ALK inhibitors such as crizotinib, owing to secondary mutations within the kinase domain of ALK, for example in EML4-ALK; these include LI 196M, Cl 156Y, and Fl 174L, among others. Several second-generation ALK inhibitors that target ALK -positive NSCLC, such as alectinib, ceritinib, and AP26133, have been developed.

[0048] Diagnostic approaches to detect ALK fusion genes and their results include break- apart fluorescence in situ hybridization (FISH), IHC, and reverse-transcription PCR (RT- PCR). Break-apart FISH has been established in clinical trials as the standard method for confirmation of ALK status (Cabillic F, et al. Parallel FISH and immunohistochemical studies of ALK status in 3244 non-small-cell lung cancers reveal major discordances. J Thorac Oncol. 2014;9(3):295-306.22). FISH and IHC have shown high concordance in several reports, especially with the development of IHC antibodies (clones 5A4 and D5F3) with better sensitivities and specificities (83%- 100%) for the detection of ALK rearrangement. As a result, IHC detection of the ALK protein may be used as a screening tool to test samples for ALK rearrangements or as a tool to evaluate cases that are not interpretable by FISH.

[0049] Kirsten rat sarcoma viral oncogene homolog (KRAS): KRAS is an oncogene located on the long arm of chromosome 12 at position 12.1. It is a member of the RAS family of membrane-associated G proteins and encodes for a protein with intrinsic GTPase activity, which is involved in a variety of cellular responses including proliferation, cytoskeletal reorganization, and survival. KRAS acts downstream of a number of tyrosine kinases receptors, including EGFR, and is associated with activation of the RAS / RAF / MAP kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) and RAS / MAPK signaling pathways. KRAS mutations occur in 25% to 35% of patients with NSCLC, principally adenocarcinomas with a solid pattern, and are found more often in white patients compared to Asians, in former or current smokers, but without sex predilection. Mutations in the form of single-nucleotide missense variants are found in codons 12 and 13 in approximately 95% of cases. In never smokers, the most common KRAS mutations are G12D and G12V, whereas G12C is the most common mutation associated with smoking. The presence of KRASmutation may be associated with unfavorable outcome and could be a negative predictor of responsiveness to chemotherapy. In addition, it is associated with an increased likelihood of having a second primary tumor and is a predictor of resistance to targeted therapy with EGFR-TKIs, such as gefitinib or erlotinib, in patients with NSCLC.

[0050] ROS proto-oncogene 1, receptor tyrosine kinase (ROS1)'. ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) is a tyrosine kinase receptor member of the insulin receptor family and is located on the long arm of chromosome 6 at position 22. ROS1 plays a role in epithelial cell differentiation during the development of a variety of organs, but no ligand for this receptor has been identified. ROS1 rearrangements were originally described in glioblastoma and have also been reported in cholangiocarcinoma and ovarian cancer. Approximately 1% to 2% ofNSCLCs harbor ROS 1 rearrangements, and several fusion partners, including CD74, solute carrier family 34, member 2 (SLC34A2), leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3), ezrin (EZR), syndecan 4 (SDC4), tropomyosin 3 (TPM3), and FIG, have been reported in these tumors. All of these fusions result in a chimeric protein that has been reported to be oncogenic. RO SI -rearranged NSCLC typically occurs in young, female, never smokers with a histologic diagnosis of adenocarcinoma and is usually mutually exclusive with other oncogenic drivers (EGFR, KRAS, ALK). Clinical trials have reported that patients with advanced NSCLC harboring ROS1 rearrangement have benefited from crizotinib treatment, showing response rates up to 80%. Ongoing phase 1 and 2 studies are investigating the activity of crizotinib and ceritinib (ALK inhibitor) in RO SI -rearranged NSCLC.

[0051] Human epidermal growth factor receptor 2 (HER2): The human epidermal growth factor receptor 2 gene HER2 (ERBB2) is a proto-oncogene located on chromosome 17 at position 12. It encodes for a tyrosine kinase receptor member of the ERBB receptor family. HER2 lacks a specific ligand. Nevertheless, it can be combined with other ERBB receptors to form a heterodimer. This allows for the activation of important signal transduction pathways, including the MAPK and PI3K pathways, involved in cell proliferation, differentiation, and migration. HER2 expression and / or amplification is found in many cancers including breast and gastric cancer. Overexpression of HER2 has been reported in 7% to 34.9% ofNSCLCs and has been associated with poor prognosis in patients with these tumors. Activating mutations of HER2 have been found in 1.6% to 4% of lung cancers. These mutations occur in the 4 exons of the tyrosine kinase domain (exons 18-21) and are found more often in adenocarcinomas in female, Asian, never or light smokers. HER2 mutations are almost always mutually exclusive with other driver oncogene alterations in lung cancer describedabove. Different studies reinforce the importance of screening lung adenocarcinomas for HER2 mutation as a method to select patients who could benefit from HER2 -targeted therapies (afatinib and trastuzumab), which have shown response rates of approximately 50%. Several clinical trials of targeted agents, such as trastuzumab, neratinib and pyrotinib, among others, are being conducted in patients with HER2 mutation. HER2 mutations are usually assessed via sequencing approaches.

[0052] RET proto-oncogene: The RET proto-oncogene is located on the long arm of chromosome 10 at position 11.2. It encodes for a tyrosine kinase receptor for the glial cell line-derived neurotrophic factor family of ligands and is involved in cell proliferation, migration, and differentiation, as well as neuronal navigation. RET chromosomal rearrangements were originally described in papillary thyroid carcinoma. Approximately 1% to 2% of NSCLCs harbor RET fusions, and several fusion partners, including kinesin family member 5B (KIF5B) (90%), coiled-coil domain containing 6 (CCDC6), nuclear receptor coactivator 4 (NCOA4), and tripartite motif-containing 33 (TRIM33), have been described. RET-rearranged NSCLC typically occurs in adenocarcinomas with more poorly differentiated solid features in young never smokers, and it is mutually exclusive with known driver oncogenes. In vitro studies showed that RET fusions lead to oncogenic transformation, which can be inhibited by multitargeted kinase inhibitors such as vandetanib, sorafenib, and sunitinib.

[0053] MET proto-oncogene: The MET gene is located on the long arm of chromosome 7 at position 31. This oncogene encodes for a tyrosine kinase receptor (hepatocyte growth factor receptor), which activates multiple signaling pathways that play fundamental roles in cell proliferation, survival, motility, and invasion. Pathologic activation of MET includes mutation, gene amplification, and protein overexpression. MET alterations were first reported in patients with renal papillary carcinoma and mutations in the MET kinase domain leading to constitutive activation of the receptor. In lung cancer, MET mutations are found in the extracellular semaphorin and juxtamembrane domains, occurring in 3% of squamous cell lung cancers and 8% of lung adenocarcinomas. MET amplifications are found in 4% of lung adenocarcinomas and 1% of squamous cell lung cancers and are associated with sensitivity to MET inhibitors. In NSCLC, MET and hepatocyte growth factor protein expression, along with high MET gene copy number, have been described as poor prognosis factors. Activating point mutations affecting splice sites of exon 14 of the MET gene (METexl4), which occur in 4% of lung adenocarcinomas, represent a possible oncogenic driver and identify a subset of patients who may benefit from MET inhibitors such as capmatinib and crizotinib.

[0054] B-RAF proto-oncogene, serine / threonine kinase (BRAF): The B-RAF protooncogene, serine / threonine kinase (BRAF) oncogene is located on the long arm of chromosome 7 at position 34. It encodes for a serine / threonine kinase, which is involved in the RAS / RAF / MEK / ERK signaling pathway. When activated by oncogenic mutations, BRAF phosphorylates MEK and promotes cell growth, proliferation, and survival. The highest incidence of BRAF mutation is in malignant melanoma (27%-70%), followed by papillary thyroid cancer, colorectal cancer, and serous ovarian cancer. BRAF mutations have also been reported in 1% to 3% of NSCLCs). In contrast to melanoma, only half of BRAF mutations in NSCLC are V600E mutations. Other non-V600E mutations reported in NSCLC include G469A (-35%) and D594G (-10%). All BRAF mutations are mutually exclusive with other driver alterations such as those of EGFR, KRAS, and ALK. BRAF-mutated NSCLC has been reported to be mostly adenocarcinoma, and in contrast to patients with EGFR mutations or ALK rearrangements who are mostly never smokers, patients with BRAF mutations are mostly current or former smokers. Nevertheless, patients with NSCLC and BRAF V600E mutations have a worse prognosis and lower response to platinum-based chemotherapy than patients with wild-type BRAF. BRAF inhibitors, such as vemurafenib and dabrafenib, have high and selective activity against the V600E-mutant BRAF kinase, with overall responses rates from 33% to 42%. BRAF and MEK inhibitors targeting BRAF mutation-positive NSCLC, such as trametinib, selumetinib, and dasatinib, among others, are currently under evaluation in clinical trials.

[0055] Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA): PI3Ks are heterodimeric lipid kinases composed of catalytic and regulatory subunits and are part of several downstream pathways involved in cell growth, transformation, adhesion, apoptosis, survival, and motility. The PIK3CA gene is located on the long arm of chromosome 3 at position 26.3. It encodes for the catalytic subunit pl 10 alpha of P13Ks. PKI3CA amplifications, deletions, and somatic missense mutations have been reported in many tumors including lung cancers. Mutations are found in 1% to 4% of patients with NSCLC, usually affecting exons 9 and 20 (80%). These mutations are not mutually exclusive with other driver alterations and have been reported more frequently in lung squamous cell carcinoma compared to adenocarcinoma (6.5% vs 1.5%). Studies have shown that PIK3CA mutations in EGFR-mutated lung cancer confer resistance to EGFRTKIs and are a negative prognostic predictor in patients with NSCLC treated with EGFR-TKIs.

[0056] Neurotrophic receptor tyrosine kinase 1(NTRK1): The neurotrophic receptor tyrosine kinase 1 (NTRK1) proto-oncogene is located on chromosome lq21-22 and encodesfor a receptor tyrosine kinase, also known as tropomyosin-related kinase (TRK) A, belonging to the TRK superfamily of receptor tyrosine kinases. NTRK1 is involved in the regulation of cell growth and differentiation via activation of several signal transduction pathways including MAPK, PI3K, and phospholipase C-gamma. In lung cancer, approximately 3% of adenocarcinomas harbor NTRK1 fusions, and some fusion partners, including myosin phosphatase RHO-interacting protein (MPRIP)-NTRKl and CD74-NTRK1, have been reported.

[0057] Fibroblast growth factor receptor (FGFR): The fibroblast growth factor receptor(FGFR) gene is located on chromosome 8 at position 12 and encodes for a tyrosine kinase receptor belonging to the FGFR family. The FGFR family includes 4 receptor tyrosine kinases (FGFRs 1-4). When ligand-receptor binding occurs, FGFR dimerizes and phosphorylates FGFR substrate 2-alpha (FRS2a), leading to activation of different pathways, including the RAS / MAPK and PI3K / AKT / mT0R pathways, promoting cell survival, motility, invasiveness, and proliferation. FGFR has been identified as an oncogenic driver in breast, gastric, endometrial, urothelial, and brain tumors, among others. In lung cancer, the incidence of FGFR1 amplification is significantly higher in squamous cell carcinoma (20%) compared to adenocarcinoma (3%) and is more frequent in current smokers compared to former and never smokers. Some studies have recognized FGFR amplification as an independent negative prognostic factor in patients with NSCLC. Phase 1 and 2 clinical trials of FGFR inhibitors (dovitinib, nintedanib, ponatinib, and AZD4547, among others) are ongoing in patients with NSCLC.

[0058] Discoidin domain receptor tyrosine kinase 2 (DDR2): The discoidin domain receptor tyrosine kinase 2 gene (DDR2) is located on the long arm of chromosome 1 at position 23.3 and encodes for a tyrosine kinase receptor that is expressed in mesenchymal tissues and which binds fibrillar collagen as ligand. DDR2 activates important signaling pathways including SRC, SRC homology domain-containing (SHC), Janus kinase (JAK), ERK1 / 2, and PI3K and promotes cell migration, proliferation, and survival. In lung cancer, DDR2 mutations occur in 3% to 4% of lung squamous cell carcinomas compared to 0.5% of adenocarcinomas and are only present in smokers

[0059] Immunotherapy Markers in Lung Cancer: Immunotherapy has re-emerged strongly with the development of checkpoint inhibitors as treatments for NSCLC. Immune checkpoints are inhibitory pathways with the functions of maintaining self-tolerance and modulating immune responses. Immune checkpoint proteins that have been studied more comprehensively in many types of cancer, including lung cancer, are cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed death-ligand 1 receptor (PD-1), which are expressed mainly on T cells, and programmed death-ligand 1 (PD-L1), which is expressed on tumor cells and tumor inflammatory infiltrate including macrophages, dendritic cells, and T cells. Many other checkpoint molecules, such as T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), B- and T-lymphocyte-associated protein (BTLA), V-domain Ig suppressor of T-cell activation (VISTA), and lymphocyte-activation gene 3 (LAG3), have been identified.

[0060] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4): Monoclonal antibodies that inhibit CTLA-4, such as ipilimumab, are available to prevent the binding of CTLA-4 with its ligands (CD80 / CD86), leading to reactivation of the antitumor immune response mediated by specific T cells. A phase 2 study of ipilimumab in combination with chemotherapy in patients with advanced NSCLC showed very promising results, with a significant improvement in PFS versus a control group treated with chemotherapy alone. A phase 3 trial of ipilimumab in combination with chemotherapy in patients with squamous histology NSCLC has been carried out (Carrizosa DR, Gold KA. New strategies in immunotherapy for non-small cell lung cancer. Transl Lung Cancer Res. 2015;4(5):553— 559).

[0061] Programmed death-ligand 1 receptor (PD-1): Several monoclonal antibodies targeting the interaction between PD-1 and its ligands PD-L1 and PD-L2 are available. There are different ways to block the PD-1 pathway; one is to use antibodies directed against PD-1 or by blocking its ligand PD-L1. Clinical trials in NSCLC have shown sustained responses in approximately 20% of unselected patients to treatment with monoclonal antibodies against PD-1, such as nivolumab and pembrolizumab, and with antibodies against PD-L1 such as MPDL3280A. The FDA has approved the use of nivolumab in advanced NSCLC on or after platinum -based chemotherapy and pembrolizumab as second-line treatment for NSCLC after chemotherapy. A study has reported that greater nonsynonymous mutation burden is associated with improved objective response, durable clinical benefit, and PFS in patients with NSCLC treated with pembrolizumab (Rizvi NA, Hellmann MD, Snyder A, el al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348(6230): 124-128). Furthermore, IHC PD-L1 positivity in NSCLC has been identified as a potential predictor of response to anti- PD-1 and anti-PD-Ll monoclonal antibody therapy and also as a prognostic biomarker (Sun JM, et al. Prognostic significance of programmed cell death ligand 1 in patients with non-small-cell lung cancer: a large cohort study of surgically resected cases. J Thorac Oncol. 2016 Apr 18).

[0062] TUmor Suppressor Candidate 2 (TUSC2)

[0063] TUSC2 (also known as FUS1) is a tumor suppressor gene originally described as a member of the tumor suppressor gene cluster from human 3p21.3 chromosomal region that is frequently deleted in lung cancer. Human TUSC2 is a small protein (110 amino acids) with an estimated MW of 12 kD. According to computer modeling, TUSC2 lacks transmembrane domains, is highly hydrophobic and contains helix-coil domain secondary structures. At the N-terminus, TUSC2 contains a myristoylation signal (Met-Gly-X-X-X-Ser / Thr), and experiments confirm that TUSC2 is myristoyl ated. In normal tissues, TUSC2 is ubiquitously expressed. TUSC2 has been demonstrated to act as tumor suppressor gene in lung, breast, bone, and other cancers. TUSC2 acts as a tumor suppressor in both in vitro and in vivo experiments. When transfected into cancer cell lines that lack TUSC2, TUSC2 decreases growth of the cells. When TUSC2 is expressed in mice carrying human tumor xenografts, it leads to decreased growth of the tumors.

[0064] The TUSC2 gene is often deleted or inactivated in cancers. The TUSC2 gene is located on the short arm of chromosome 3, specifically in the region termed 3p21.3, and this region is commonly deleted in a number of cancer types, including non-small cell lung cancer, small cell lung cancer, mesothelioma, breast cancer, and others. Most commonly, this occurs as haplo-deletion (deletion of one of the two TUSC2 gene copies), but in some cases both copies of the TUSC2 gene are deleted.

[0065] The TUSC2 protein is an example of a group of proteins termed calcium-myristoyl switches. The TUSC2 protein requires myristylation in order to be active, and TUSC2 protein that is not myristoylated is quickly degraded. The myristoylated amino terminal end of the protein is hydrophobic and is normally located inside the folded protein. However, in the presence of elevated levels of calcium, structural changes occur so that the myristoylated end of the protein is released and can then bind to the inner membrane of the mitochondria. This moves the TUSC2 protein from the free space of the mitochondrion to association with the inner membrane of the mitochondria. One way that TUSC2 protein can be inactivated in cancer is for protein myristylation to be blocked, leading to TUSC2 protein levels in those cancers to be decreased or absent. This phenomenon explains TUSC2 protein absence in cases where the gene is haplo-deleted.

[0066] TUSC2 is a master regulator gene, with multiple effects. TUSC2 expression inhibits a wide variety of cellular processes, including inhibiting a wide range of tyrosine kinases that stimulate cellular proliferation, activate apoptotic proteins that lead to cell death, and increase immune cells that attack the tumor.

[0067] Nucleic Acid Constructs

[0068] Nucleic acid constructs described herein include a polynucleotide sequence encoding a TUSC2 protein (for example, human TUSC2 protein). The TUSC2 coding sequence is flanked by a 5' untranslated region (UTR) and a 3' UTR, and is operably linked to a promoter (e.g., CMV). 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 TUSC2 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 a RNA capable of being transcribed and translated in a cell. Methods for constructing expression constructs and plasmids through standard recombinant techniques are known in the art. Methods for designing expression constructs / plasmids for gene therapy applications (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 consensus). Such sequences and elements are disclosed below under Control / Regulatory Sequences.

[0069] Expression constructs / plasmids for inclusion in the vectors described herein can be produced in suitable host producer cells (e.g., E. coif) using suitable methods, e.g., fed-batch fermentation, batch fermentation, etc. For example, the HyperGRO™ inducible fed-batch fermentation process 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. High yield of plasmid per gram of bacteria improves final product purity since plasmid is enriched relative to host cell impurities. Boehringer Ingelheim (Vienna, Austria) has developed an alternative high yield fermentation process which is commercially available for cGMP production of plasmid DNA vectors. Plasmid DNA can be extracted from producer cells using alkaline lysis. Commercial plasmid manufacture 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).

[0070] In embodiments, the expression construct comprising the polynucleotide sequence encoding a TUSC2 protein is a covalently closed linear DNA (“doggybone DNA” or dbDNA”). dbDNA may be created starting with a circular double-stranded DNA molecule (e.g., a plasmid) containing a TUSC2 encoding sequence flanked on each side by 56 bppalindromic protelomerase recognition sequences. The DNA starting material is then denatured and Phi29 DNA polymerase is primed. Phi29 initiates rolling circle amplification of the template, creating double-stranded concatameric repeats of the original construct. Protelomerase is added, which binds to the recognition sites flanking the TUSC2 encoding sequence and performs a cleavage-joining reaction that results in monomeric doublestranded, linear, covalently closed DNA constructs. One of a panel of common restriction enzymes is added to cut undesired backbone DNA sequences, exposing open ended DNA that can be removed through digestion with exonuclease. dbDNA is purified from small fragments and reaction components with size separation to leave only the dbDNA construct comprising the TUSC2 encoding sequence. The resulting dbDNA construct can be used as a starting material for further amplification reactions. dbDNA constructs and methods of making them are disclosed in W02010086626 (PCT / GB2010 / 000165), incorporated herein by reference.

[0071] TUSC2 Polynucleotide and Amino Acid Sequences

[0072] The nucleic acid constructs described herein include a polynucleotide sequence encoding a TUSC2 protein. The TUSC2 protein encoded by the TUSC2 polynucleotide sequence and expressed from the expression constructs described herein may be a truncated TUSC2 or may have one or more substitutions that retain a tumor suppressor function of the expressed protein. In embodiments, the TUSC2 protein comprises amino acids 2-110 of SEQ ID NO:2. In embodiments, the TUSC2 protein has greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99% sequence identity with sequence SEQ ID NO:2.

[0073] In embodiments, the polynucleotide sequence encoding human TUSC2 is SEQ ID NO: 1, or is a polynucleotide sequence having greater than 85%, greater than 90%, or greater than 95% sequence identity to SEQ ID NO: 1. The human TUSC2 DNA coding sequence is provided as SEQ ID NO: 1 : atgggcgccagcgggtccaaagctcggggcctgtggcccttcgcctcggcggccggaggcggcggctcagaggcagcaggagc tgagcaagctttggtgcggcctcggggccgagctgtgccccccttcgtattcacgcgccgcggctctatgttctatgatgaggatgggg atctggctcacgagttctatgaggagacaatcgtcaccaagaacgggcagaagcgggccaagctgaggcgagtgcataagaatctg attcctcagggcatcgtgaagctggatcacccccgcatccacgtggatttccctgtgatcctctatgaggtgtga

[0074] In certain embodiments, the TUSC2 protein is human TUSC2. The human TUSC2 amino acid sequence is provided as SEQ ID NO:2: MGASGSKARGLWPFASAAGGGGSEAAGAEQALVRPRGRAVPPFVFTRRGSMFYDE DGDLAHEFYEETIVTKNGQKRAKLRRVHKNLIPQGIVKLDHPRIHVDFPVILYEV

[0075] The nucleic acids of the disclosure can, for example, encode the amino acid sequence of the TUSC2 polypeptide with at least one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Vai, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.

[0076] The nucleic acids of the disclosure can, for example, encode functional variants which also include extensions of the TUSC2 polypeptide. For example, a functional variant of the TUSC2 polypeptide can include 1, 2, 3, 4 and 5 additional amino acids from either the N-terminal or C-terminal end of the TUSC2 protein.

[0077] Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the TUSC2 polypeptide with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the wild type TUSC2 polypeptide e.g. SEQ ID NO: 2. The TUSC2 polypeptide can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the functional variant, e.g., other amino acids, do not materially change the biological activity of the functional variant.

[0078] In certain embodiments, the nucleic acid sequence encodes a polypeptide comprising a sequence of at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2.

[0079] In certain embodiments, the polynucleotide sequence encoding the TUSC2 protein has been codon optimized. In embodiments, the TUSC2 protein encoded by the polynucleotide sequence is human TUSC2 and includes the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having greater than 90%, greater than 95%, or greater than 98%, or greater than 99% sequence identity to SEQ ID NO:2. 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.

[0080] In some embodiments, the nucleic acids encode a mammalian TUSC2 protein. In certain embodiments, the TUSC2 protein can be murine, porcine, ovine, bovine, human, or combinations thereof. In preferred embodiments, the TUSC2 protein is, or comprises, human TUSC2.

[0081] In accordance with an embodiment, the present disclosure provides a composition comprising a nucleic acid encoding a TUSC2 protein, or a functional portion or fragment, such as, for example, SEQ ID NO: 2.

[0082] In certain embodiments, the nucleic acid sequence comprises a sequence of at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1.

[0083] In some embodiments, the polynucleotides provided herein encoding TUSC2 proteins include codon-optimized sequences. As used herein, the term “codon-optimized” means a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned as compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by choosing different codons without altering the amino acid sequence of the encoded protein. Accordingly, codon-optimization generally refers to replacement of codons with synonymous codons to optimize expression of a protein while keeping the amino acid sequence of the translated protein the same. Codon optimization of a sequence can increase protein expression levels (Gustafsson etal., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22: 346-53) of the encoded proteins, for example, and provide other advantages. Variables such as codon usage preference as measured by codon adaptation index (CAI), for example, the presence or frequency of A, G, C, U nucleotides, mRNA secondary structures, cis-regulatory sequences, GC content, and other variables may correlate with protein expression levels (Villalobos etal., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285).

[0084] Any method of codon optimization can be used to codon optimize polynucleotides and nucleic acid molecules provided herein, and any variable can be altered by codon optimization. Accordingly, any combination of codon optimization methods can be used. Exemplary methods include the high codon adaptation index (CAI) method and others. The CAI method chooses a most frequently used synonymous codon for an entire protein coding sequence. As an example, the most frequently used codon for each amino acid can bededuced from 74,218 protein-coding genes from a human genome. Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon optimized.

[0085] In some embodiments, the nucleotide sequence of any region of an RNA or DNA sequence embodied herein may be codon optimized. In certain embodiments, the primary cDNA template may include reducing the occurrence or frequency of appearance of certain nucleotides in the template strand. For example, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 25% of said nucleotides in the template. In further examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 20% of said nucleotides in the template. In some examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 16% of said nucleotides in the template. The occurrence of a nucleotide in a template may be increased or reduced to a level above or below 15% and may be increased or reduced to a level above or below 12% of said nucleotides in the template.

[0086] In certain embodiments, the polynucleotides of the disclosure can comprise one or more chemically modified nucleotides. Examples of nucleic acid monomers include nonnatural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the base portion or the sugar portion. In nature, most polynucleotides comprise nucleotides that are “unmodified” or “natural” nucleotides, which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). These bases are typically fixed to a ribose or deoxy ribose at the 1' position. The use of RNA polynucleotides comprising chemically modified nucleotides have been shown to improve RNA expression, expression rates, half-life and / or expressed protein concentrations. RNA polynucleotides comprising chemically modified nucleotides have also been useful in optimizing protein localization thereby avoiding deleterious bio-responses such as immune responses and / or degradation pathways.

[0087] Examples of modified or chemically modified nucleotides include 5- hydroxycytidines, 5-alkylcytidines, 5-hydroxyalkylcytidines, 5-carboxycytidines, 5- formylcytidines, 5-alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N4- alkylcytidines, N4-aminocytidines, N4-acetylcytidines, and N4, N4-dialkylcytidines.

[0088] Examples of modified or chemically modified nucleotides include 5- hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5- formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine; N4-methylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4, N4- dimethylcytidine.

[0089] Examples of modified or chemically modified nucleotides include 5- hydroxyuridines, 5-alkyluridines, 5-hydroxyalkyluridines, 5-carboxyuridines, 5- carboxyalkylesteruridines, 5-formyluridines, 5 -alkoxyuridines, 5-alkynyluridines, 5- halouridines, 2-thiouridines, and 6-alkyluridines.

[0090] Examples of modified or chemically modified nucleotides include 5- hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as “SMeOU”), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2- thiouridine, and 6-methyluridine.

[0091] Examples of modified or chemically-modified nucleotides include 5- methoxycarbonylmethyl-2 -thiouridine, 5-methylaminomethyl-2-thiouridine, 5- carbamoylmethyluridine, 5 -carbarn oylmethyl-2'-O-methyluri dine, l-methyl-3-(3-amino-3- carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5- carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2- thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O- methyluridine, and 3,2'-O-dimethyluridine.

[0092] Examples of modified or chemically-modified nucleotides include N6- methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N6-methyladenosine, N6- isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cv.s-hydroxyisopentenyl)adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine, N6-acetyl -adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-0-methyl-adenosine, N6,2'-O-dimethyl- adenosine, N6,N6,2'-O-trimethyl-adenosine, l,2'-O-dimethyl-adenosine, 2'-O- ribosyladenosine, 2-amino-N6-methyl-purine, 1 -thio-adenosine, 2'-F-ara-adenosine, 2'-F- adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0093] Examples of modified or chemically modified nucleotides include N1- alkylguanosines, N2-alkylguanosines, thienoguanosines, 7-deazaguanosines, 8-oxoguanosines, 8-bromoguanosines, O6-alkylguanosines, xanthosines, inosines, and N1- alkylinosines.

[0094] Examples of modified or chemically modified nucleotides include N1- methylguanosine, N2-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-methylguanosine, xanthosine, inosine, and Nkmethylinosine.

[0095] Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

[0096] Examples of modified and chemically modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted deoxyabasic monomer residues.

[0097] Examples of modified and chemically modified nucleotide monomers include 3'- end stabilized nucleotides, 3 '-glyceryl nucleotides, 3 '-inverted abasic nucleotides, and 3'- inverted thymidine.

[0098] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'-O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0099] Examples of modified and chemically modified nucleotide monomers include 2', d'constrained 2'-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNAs.

[0100] Examples of modified and chemically modified nucleotide monomers include 2'- amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.

[0101] Examples of modified and chemically modified nucleotide monomers include N6- methyladenosine nucleotides.

[0102] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2- mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.

[0103] Examples of modified and chemically modified nucleotide monomers include 2'- O-aminopropyl substituted nucleotides.

[0104] Examples of modified and chemically modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'-halogen, a 2'-SR, or a 2'- amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0105] Example of base modifications described above can be combined with additional modifications of nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers may be found in nature.

[0106] In certain embodiments, when the nucleic acid is an RNA, the RNA molecules can be engineered to comprise one or more modified nucleobases. For example, known modifications of RNA molecules can be found, for example, in Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington DC). Modified RNA components include the following: 2'-O-methylcytidine; N4-methylcytidine; N4-2'-O-dimethylcytidine; N4-acetylcytidine; 5-methylcytidine; 5,2'-O- dimethylcytidine; 5-hydroxymethylcytidine; 5-formylcytidine; 2'-3-methylcytidine; 2- thiocytidine; lysidine; 2'-O-methyluridine; 2-thiouridine; 2-thio-2'-O-methyluridine; 3,2'-O- dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2'-O- dimethyluridine; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; uridine 5- oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5- methoxy carbonylmethyluridine; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5- methoxycarbonylmethyl-2'-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2'-O- methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl) uridinemethyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2- thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine; 5- carboxymethylaminomethyl-2'-O-methyl-uridine; 5-carboxymethylaminomethyl-2- thiouridine; dihydrouridine; dihydroribosylthymine; 2'-methyladenosine; 2-methyladenosine; N6Nmethyladenosine; N6,N6-dimethyladenosine; N6,2'-O-trimethyladenosine; 2 methylthio- N6Nisopentenyladenosine; N6-(cis-hydroxyisopentenyl)-adenosine; 2-methylthio-N6-(cis- hydroxyisopentenyl)-adenosine; N6-glycinylcarbamoyl)adenosine; N6threonylcarbamoyl adenosine; N6-methyl-N6-threonylcarbamoyl adenosine; 2-methylthio-N6-methyl-N6- threonylcarbamoyl adenosine; N6-hydroxynorvalylcarbamoyl adenosine; 2-methylthio-N6- hydroxnorvalylcarbamoyl adenosine; 2'-O-ribosyladenosine (phosphate); inosine; 2'O-methyl inosine; 1 -methyl inosine; 1; 2'-O-dimethyl inosine; 2'-O-methyl guanosine; 1 -methyl guanosine; N2-methyl guanosine; N2,N2-dimethyl guanosine; N2,2'-O-dimethyl guanosine; N2,N2,2'-O-trimethyl guanosine; 2'-O-ribosyl guanosine (phosphate); 7-methyl guanosine; N2; 7-dimethyl guanosine; N2; N2; 7-trimethyl guanosine; wyosine; methylwyosine; undermodified hydroxywybutosine; wybutosine; hydroxywybutosine; peroxywybutosine;queuosine; epoxyqueuosine; galactosyl-queuosine; mannosyl-queuosine; 7-cyano-7- deazaguanosine; archaeosine [also called 7-formamido-7-deazaguanosine]; and 7- aminomethyl-7-deazaguanosine.

[0107] Isolated nucleic acid molecules can be produced by standard techniques. For example, PCR techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.

[0108] Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector, e.g. a plasmid. Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion of TUSC2 DNA.

[0109] In some embodiments, the polynucleotide is a synthetic polynucleotide. In some embodiments, the synthetic nucleic acid comprises a modified nucleotide. Modification of the inter-nucleoside linker (i.e., backbone) can be utilized to increase stability or pharmacodynamic properties. For example, inter-nucleoside linker modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the nucleic acid. Generally, a modified inter-nucleoside linker includes any linker other than other than phosphodiester (PO) liners, that covalently couples two nucleosides together. In some embodiments, the modified inter-nucleoside linker increases the nuclease resistance of the nucleic acid compared to a phosphodiester linker. For naturally occurring oligonucleotides, the inter-nucleoside linker includes phosphate groups creating aphosphodiester bond between adjacent nucleosides. In some embodiments, the nucleic acid comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments all of the inter-nucleoside linkers of the nucleic acid or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments the inter- nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.

[0110] Modifications to the ribose sugar or nucleobase can also be utilized herein. Generally, a modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nucleobase moiety. In some embodiments, the nucleic acids, as described, comprise one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA. Numerous nucleosides with modification of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or stability. Such modifications include those where the ribose ring structure is modified. These modifications include replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g. locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids. Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

[0111] Sugar modifications also include modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2', 3', 4' or 5' positions. Nucleosides with modified sugar moi eties also include 2' modified nucleosides, such as 2' substituted nucleosides. Indeed, much focus has been spent on developing 2' substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity. A 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or — OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradicle and includes 2' substituted nucleosides and LNA (2'-4' biradicle bridged) nucleosides. Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O- methyl-RNA, 2 '-alkoxy -RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, and 2'-F-ANA nucleoside. By way of further example, in some embodiments, the modification in the ribose group comprises a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2- methoxy ethyl).

[0112] In some embodiments, the nucleic acid comprises one or more modified sugars. In some embodiments, the nucleic acid comprises only modified sugars. In certain embodiments, the nucleic acid comprises greater than 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the nucleic acid comprises both inter-nucleoside linker modifications and nucleoside modifications.

[0113] The nucleic acids of the disclosure, including DNA, RNA or nucleic acids encoding the fusion polypeptide, may be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, 2 nd edition, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 2003. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.

[0114] The nucleic acids also can be chemically synthesized, either as a single nucleic acid (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion RNA, DNA, of TUSC2 encoding DNA.

[0115] In certain embodiments, the fusion polypeptides are synthesized from an expression vector encoding the DNA molecule, as described in detail elsewhere herein.

[0116] Control / Regulatory Sequences

[0117] The nucleic acid constructs disclosed herein include control and regulatory sequences that are operably linked to the polynucleotide sequence encoding a TUSC2 protein. The nucleic acid constructs disclosed herein can include appropriate control sequences for expression of the human TUSC2 in human cancer cells. “Control sequences” include nucleic acid sequences necessary for replication of a vector in a producer cell (e.g., E. coli cell), as well as nucleic acid sequences necessary for, or involved in, transcription and / or translation of an operably linked 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 TUSC2 protein, the relationship is such that the control element modulates expression of the TUSC2 protein encoding sequence. Examples of control / regulatory sequences include promoters, enhancers, translation initiation signals, termination signals, polyadenylation sequences (e.g., polyA signals derived from bovine growth hormone, SV40, rabbit P-globin), Kozak sequences (e.g., GCCACCATG), posttranslational regulatory elements, introns, splicing enhancers, nuclear targeting sequences, etc.

[0118] To facilitate expression of TUSC2 in the cancer cells, a suitable promoter may be used in the nucleic acid constructs described herein. In embodiments, the CMV promoter or a modified CMV promoter is used. Certain CMV promoters serve dual roles as a promoter and an enhancer. In embodiments a mini-CMV promoter is used. In other embodiments, chimeric promoters that are a fusion of two different promoter sequences or a fusion of a promoter sequence and an inducible element can be used. For example, a chicken P- actin / CMV enhancer combination can be used. Promoters, in addition to the CMV promoter, that can be used to promote transcription of the TUSC2 transgene include simian virus 40 (SV40) early promoter, elongation factor- la, (EFla), phosphoglycerate kinase (PGK), and human P-actin promoter (ACTB). In some embodiments, a tissue-specific promoter can be used. In some embodiments, a nucleic acid construct as described herein includes one or more (e.g., 1, 2, 3, 4, 5, etc.) introns. For example, in a nucleic acid construct as disclosed herein, the 5' UTR, 3' UTR, and / or the TUSC2 coding sequence can include an intron (e.g., intron 2 of the human P globin gene). As another example, a chimeric intron (e.g., from the - globulin and immunoglobulin heavy chain genes) upstream of the transgene can be used. Additionally or alternatively, the 5' UTR can include a HTLV-I R element for enhancementof mRNA translation efficiency and increasing transgene expression. Nuclear targeting sequences, which promote shuttling of the nucleic acid construct into the nucleus, can be included in 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.

[0119] Selectable Markers

[0120] 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.

[0121] Nonviral Vectors

[0122] The term “vector” as used herein refers to a vehicle for delivering genetic material (e.g., RNA or DNA) to a cell, including for example, viral vectors (such as AAV and lentiviral vectors) and nonviral vectors. The term “nonviral vector” is used herein to refer to a nonviral vehicle for delivering genetic material to 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 TUSC2 is expressed. The non-viral vectors described herein result in a high level of transfection efficiency with a low level of toxicity. The nonviralvectors display a high degree of 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 pg, 230 pg, 240 pg, 250 pg, 260 pg, 270 pg, 275 pg, 280 pg, 290 pg,300 pg, 310 pg, 320 pg, 325 pg, 330 pg, 340 pg, 350 pg, 360 pg, 370 pg, 375 pg, 400 pg,425 pg, 450 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850 pg, 900 pg,950 pg, or 1000 pg of nucleic acid per 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.

[0123] 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 250 nm and 325 nm.

[0124] Alternative Delivery Vehicles

[0125] Lipid Formulations / LNPs: Therapies based on the intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. Indeed, naked nucleicacid materials cannot be easily systemically administered due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocyte uptake and their ability in activating the immune response, all features that preclude their clinical development. When exogenous nucleic acid material (e.g., mRNA) enters the human biological system, it is recognized by the reticuloendothelial system (RES) as foreign pathogens and cleared from blood circulation before having the chance to encounter target cells within or outside the vascular system. It has been reported that the half-life of naked nucleic acid in the blood stream is around several minutes (Kawabata K, Takakura Y, Hashida M Pharm Res. 1995 June; 12(6):825-30). Chemical modification and a proper delivery method can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, which increase stability and efficacy of nucleic acid-based therapies. In addition, RNAs or DNAs are anionic hydrophilic polymers that are not favorable for uptake by cells, which are also anionic at the surface. The success of nucleic acid-based therapies thus depends largely on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0126] Moreover, upon internalization into a target cell, nucleic acid delivery vectors are challenged by intracellular barriers, including endosome entrapment, lysosomal degradation, nucleic acid unpacking from vectors, translocation across the nuclear membrane (for DNA), release at the cytoplasm (for RNA), and so on. Successful nucleic acid-based therapy thus depends upon the ability of the vector to deliver the nucleic acids to the target sites inside of the cells in order to obtain sufficient levels of a desired activity such as expression of a gene.

[0127] While several gene therapies have been able to successfully utilize a viral delivery vector (e.g., AAV), lipid-based formulations have been increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and their ease of large-scale production. One of the most significant advances in lipid-based nucleic acid therapies happened in August 2018 when Patisiran (ALN-TTR02) was the first siRNA therapeutic approved by the Food and Drug Administration (FDA) and by the European Commission (EC). ALN-TTRO2 is an siRNA formulation based upon the so-called Stable Nucleic Acid Lipid Particle (SNALP) transfecting technology. Despite the success of Patisiran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still under ongoing development.

[0128] Some art-recognized lipid-formulated delivery vehicles for nucleic acid therapeutics include, according to various embodiments, polymer based carriers, such aspolyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide- containing nanoliposomes, multivesicular liposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, micelles, and emulsions. These lipid formulations can vary in their structure and composition, and as can be expected in a rapidly evolving field, several different terms have been used in the art to describe a single type of delivery vehicle. At the same time, the terms for lipid formulations have varied as to their intended meaning throughout the scientific literature, and this inconsistent use has caused confusion as to the exact meaning of several terms for lipid formulations. Among the several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail and defined herein for the purposes of the present disclosure.

[0129] Liposomes'. Conventional liposomes are vesicles that consist of at least one bilayer and an internal aqueous compartment. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally present as spherical vesicles and can range in size from 20 nm to a few microns. Liposomal formulations can be prepared as a colloidal dispersion or they can be lyophilized to reduce stability risks and to improve the shelf-life for liposome-based drugs. Methods of preparing liposomal compositions are known in the art and would be within the skill of an ordinary artisan.

[0130] Liposomes that have only one bilayer are referred to as being unilamellar, and those having more than one bilayer are referred to as multilamellar. The most common types of liposomes are small unilamellar vesicles (SUV), large unilamellar vesicle (LUV), and multilamellar vesicles (MLV). In contrast to liposomes, lysosomes, micelles, and reversed micelles are composed of monolayers of lipids. Generally, a liposome is thought of as having a single interior compartment, however some formulations can be multivesicular liposomes (MVL), which consist of numerous discontinuous internal aqueous compartments separated by several nonconcentric lipid bilayers.

[0131] Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes, and can be prepared from both natural and synthetic phospholipids (Int J Nanomedicine. 2014; 9: 1833-1843). In their use as drug delivery vehicles, because a liposome has an aqueous solution core surrounded by a hydrophobic membrane, hydrophilicsolutes dissolved in the core cannot readily pass through the bilayer, and hydrophobic compounds will associate with the bilayer. Thus, a liposome can be loaded with hydrophobic and / or hydrophilic molecules. When a liposome is used to carry a nucleic acid such as RNA, the nucleic acid will be contained within the liposomal compartment in an aqueous phase.

[0132] Cationic Liposomes: Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic portion. In addition to the general characteristics profiled above for liposomes, the positively charged moieties of cationic lipids used in cationic liposomes provide several advantages and some unique structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic and thus will direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moiety will associate with aqueous media and more importantly with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being researched for use in gene therapy due to their favorability towards negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed herein below.

[0133] Lipid Nanoparticles: In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0134] For lipid nanoparticle nucleic acid delivery systems, the lipid shell is formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid's negatively charged backbone and loading into the lipid nanoparticle at pH values belowthe pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid- based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0135] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine® reagent, have found considerable utility for in vitro transfection. However, these first- generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (Imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation. In some aspects, nucleic acid molecules provided herein and lipids or lipid formulations provided herein form a lipid nanoparticle (LNP).

[0136] In other aspects, nucleic acid molecules provided herein are incorporated into a lipid formulation (i.e., a lipid-based delivery vehicle).

[0137] In the context of the present disclosure, a lipid-based delivery vehicle typically serves to transport a desired RNA, DNA or polypeptide to a target cell or tissue. The lipid- based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some aspects, the lipid-based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing a self-replicating RNA. In some aspects, the lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and SEQ ID NOs: 1 or 2 and variants thereof, of the disclosure. In some aspects, the lipid bilayer further comprises a neutral lipid or a polymer. In some aspects, the lipid formulation comprises a liquid medium. In some aspects, the formulation further encapsulates a nucleic acid. In some aspects, the lipid formulation further comprises a nucleic acid and a neutral lipid or a polymer. In some aspects, the lipid formulation encapsulates the nucleic acid.

[0138] The description provides lipid formulations comprising one or more SEQ ID NOs: 1 or 2 and variants thereof, of the disclosure encapsulated within the lipid formulation. Insome aspects, the lipid formulation comprises liposomes. In some aspects, the lipid formulation comprises cationic liposomes. In some aspects, the lipid formulation comprises lipid nanoparticles.

[0139] In some aspects, the SEQ ID NOs: 1 or 2 and variants thereof, are fully encapsulated within the lipid portion of the lipid formulation such that the RNA, DNA or polypeptide in the lipid formulation is resistant in aqueous solution to nuclease degradation. In other aspects, the lipid formulations described herein are substantially non-toxic to animals such as humans and other mammals.

[0140] Methods of Production of Liposomes

[0141] DOTAP :Cholesterol Liposomes: DOTAP: cholesterol liposomes are nanoparticle liposomal formulations composed of l,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (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 1 to 8 millimolar (mM) (e.g., 1 mM, 2 to 7 mM, 3 to 6 mM, 4 to 5 mM, 8 mM). In embodiments, the liposomal formulation includes cholesterol or cholesterol derivative or cholesterol mixture in a concentration ranging from 1 to 8 mM (e.g., 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.

[0142] Extrusion Techniques: 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 isotonicsolution. 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.

[0143] Microfluidization Techniques: The DOTAP: cholesterol liposomes described herein may be produced using a microfluidization method. Microfluidization can be used, e.g., when consistently small (50 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 include, for example: 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 efficientwrapping of the two fluids around each other resulting in an exponential enlargement of the interface between the fluids ensuring rapid mixing.

[0144] Nonviral Vectors

[0145] Once manufactured, DOTAP: cholesterol liposomes can be used to encapsulate nucleic acids (e.g., a polynucleotide construct as described herein) resulting in nonviral vectors as 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 above. For example, equal volumes of nucleic acid construct and DOTAP:cholesterol, at a concentration to obtain 100 pg of nucleic acid construct / 5 mM lipids / 100 pl, can be mixed by adding the nucleic acid construct rapidly to the DOTAP:cholesterol solution followed by rapid mixing.

[0146] 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.

[0147] 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).

[0148] Viral Vectors

[0149] 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 TUSC2 protein such as human TUSC2) 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 TUSC2 protein). 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.

[0150] 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 a rAAV genome is encapsidated or packaged into an AAV particle, theparticle can be referred to as a “rAAV.” Such particles or virions include proteins that encapsidate or package the viral genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins (VP1, VP2, VP3). As used herein, the term “serotype” refers to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Recombinant AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8, and variants thereof. Examples of rAAV can include capsid 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.

[0151] A rAAV vector can comprise a genome derived from an AAV serotype distinct from the AAV serotype of one or more of the capsid proteins that package the 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.

[0152] Compositions / Pharmaceutical Formulations

[0153] Compositions including the nucleic acid constructs, nonviral vectors, and viral vectors are described herein. In some embodiments, the composition includes a nonviral vector as described herein and dextrose, e.g., about 5% dextrose in water or 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 compositionincludes a nonviral vector comprising a nucleic acid construct described herein and a combination of about 5% dextrose and about 0.9% sodium chloride.

[0154] The compositions, nucleic acid constructs, nonviral vectors and viral vectors described herein may be administered to a subject 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)). The nucleic acid constructs, nonviral vectors and viral vectors described herein are typically formulated in a composition comprising a pharmaceutically acceptable carrier. 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.

[0155] 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 or viral 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. 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.

[0156] 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 pHmodifying 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.

[0157] The nucleic acid constructs, nonviral vectors, viral vectors and compositions described herein are preferably administered to a subject in a therapeutically effective amount. By the phrases “therapeutically effective amount”, “effective amount” and “effective dosage” is meant an amount sufficient to produce a therapeutically (e.g., clinically) desirable result; for example, the result can include increasing or restoring TUSC2 express! on / signaling to TUSC2-deficient cancer cells, reducing or suppressing aerobic glycolysis in the 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. Unitdoses range from 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1000 pg and higher.

[0158] Methods of Treatment

[0159] 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 TUSC2 to TUSC2-deficient cancer cells, inducing apoptosis of cancer cells, decreasing tumor size or eliminating a tumor in a subject, and / or reducing or preventing metastasis. Methods of treating cancer generally include increasing or restoring TUSC2 signaling / expression to cancer cells that have reduced TUSC2 levels or inhibition of TUSC2 function. In embodiments, expression of TUSC2 from the nucleic acid constructs disclosed herein suppressed aerobic glycolysis in the cancer cell. 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.

[0160] In embodiments, the disclosure provides a method for sensitizing ALK TKI- resistant tumor cells, in a subject, to ALK TKIs by exposing the tumor cells (e.g., by administering a composition described herein) to a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the disclosure provides a method for inducing apoptosis in cancer cells, in a subject, that are ALK+ by exposing the cancer cells (e.g., by administering a composition described herein) to a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide (or variants thereof). In embodiments, the cancer ALK+ cancer cells are resistant to one or more ALK TKIs.

[0161] 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.

[0162] Monitoring of Response to Treatment

[0163] When monitoring a subject having, or suspected of having, cancer as described herein, the monitoring can be before, during, and / or after the course of a cancer treatment, e.g., a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, or a liposome comprising a TUSC2 polypeptide or a TUSC2 polynucleotide. Methods of monitoring provided herein can be used to determine the efficacy of one or more cancer treatments, and / or to select a subject for increased monitoring.

[0164] In some embodiments, the monitoring can include conventional techniques capable of monitoring one or more cancer treatments (e.g., the efficacy of one or more cancer treatments). In some embodiments, a subject selected for increased monitoring can be administered a diagnostic test (e.g., any of the diagnostic tests disclosed herein) at an increased frequency compared to a subject that has not been selected for increased monitoring. For example, a subject selected for increased monitoring can be administered a diagnostic test at a frequency of twice daily, daily, bi-weekly, weekly, bi-monthly, monthly, quarterly, semi-annually, annually, or any at frequency therein.

[0165] In some embodiments, a subject selected for increased monitoring can be administered a one or more additional diagnostic tests compared to a subject that has not been selected for increased monitoring. For example, a subject selected for increased monitoring can be administered two diagnostic tests, whereas a subject that has not been selected for increased monitoring is administered only a single diagnostic test (or no diagnostic tests). In some embodiments, a subject that has been selected for increased monitoring can also be selected for further diagnostic testing. Once the presence of a tumor or a cancer (e.g., a cancercell) has been identified (e.g., by any of the variety of methods disclosed herein), it may be beneficial for the subject to undergo both increased monitoring (e.g., to assess the progression of the tumor or cancer in the subject and / or to assess the development of one or more cancer biomarkers such as mutations), and further diagnostic testing (e.g., to determine the size and / or exact location (e.g., tissue of origin) of the tumor or the cancer).

[0166] Any of the cancer treatments disclosed herein or known in the art can be administered. For example, a subject that has been selected for increased monitoring can be further monitored, and a cancer treatment can be administered if the presence of the cancer cell is maintained throughout the increased monitoring period. Additionally, or alternatively, a subject that has been selected for increased monitoring can be administered a cancer treatment, e.g., a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, or a liposome comprising a TUSC2 polypeptide or a TUSC2 polynucleotide and further monitored as the cancer treatment progresses.

[0167] When a subject is identified as having cancer as described herein, the identifying can be before and / or during the course of a cancer treatment. Methods of identifying a subject as having cancer provided herein can be used as a first diagnosis to identify the subject (e.g., as having cancer before any course of treatment) and / or to select the subject for further diagnostic testing. In some embodiments, once a subject has been determined to have cancer, the subject may be administered further tests and / or selected for further diagnostic testing.

[0168] In some embodiments, methods provided herein can be used to select a subject for further diagnostic testing at a time period prior to the time period when conventional techniques are capable of diagnosing the subject with an early-stage cancer. For example, methods provided herein for selecting a subject for further diagnostic testing can be used when a subject has not been diagnosed with cancer by conventional methods and / or when a subject is not known to harbor a cancer. In some embodiments, a subject selected for further diagnostic testing can be administered a diagnostic test (e.g., any of the diagnostic tests disclosed herein) at an increased frequency compared to a subject that has not been selected for further diagnostic testing. For example, a subject selected for further diagnostic testing can be administered a diagnostic test at a frequency of twice daily, daily, bi-weekly, weekly, bi-monthly, monthly, quarterly, semi-annually, annually, or any at frequency therein. In some embodiments, a subject selected for further diagnostic testing can be administered a one or more additional diagnostic tests compared to a subject that has not been selected for further diagnostic testing. For example, a subject selected for further diagnostic testing can beadministered two diagnostic tests, whereas a subject that has not been selected for further diagnostic testing is administered only a single diagnostic test (or no diagnostic tests). Additionally, or alternatively, the diagnostic testing method can determine the presence of a different type of cancer as the cancer that was original detected. In some embodiments, the diagnostic testing method is a scan. In some embodiments, the scan is a computed tomography (CT), a CT angiography (CTA), an esophagram (a Barium swallow), a Barium enema, a magnetic resonance imaging (MRI), a PET scan, an ultrasound (e.g., an endobronchial ultrasound, an endoscopic ultrasound), an X-ray, a DEXA scan.

[0169] In some embodiments, the diagnostic testing method is a physical examination, such as an anoscopy, a bronchoscopy (e.g., an autofluorescence bronchoscopy, a white-light bronchoscopy, a navigational bronchoscopy), a colonoscopy, a digital breast tomosynthesis, an endoscopic retrograde cholangiopancreatography (ERCP), an ensophagogastroduodenoscopy, a mammography, a Pap smear, a pelvic exam, a positron emission tomography and computed tomography (PET-CT) scan.

[0170] In some embodiments, a subject that has been selected for further diagnostic testing can also be selected for increased monitoring. Once the presence of a tumor or a cancer (e.g., a cancer cell) has been identified (e.g., by any of the variety of methods disclosed herein), it may be beneficial for the subject to undergo both increased monitoring (e.g., to assess the progression of the tumor or cancer in the subject and / or to assess the development of one or more cancer biomarkers such as mutations), and further diagnostic testing (e.g., to determine the size and / or exact location of the tumor or the cancer). In some embodiments, after a subject that has been selected for further diagnostic testing has been administered a cancer treatment, e.g., ALK inhibitors, the additional testing will reveal whether the cancer is refractory to treatment with ALK inhibitors. In some embodiments, this will provide cause to administer a different cancer treatment (e.g., a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, or a liposome comprising a TUSC2 polypeptide or a TUSC2 polynucleotide).

[0171] When treating a subject having cancer as described herein, the subject can be administered one or more cancer treatments. A cancer treatment can be any appropriate cancer treatment.

[0172] One or more cancer treatments described herein can be administered to a subject at any appropriate frequency (e.g., once or multiple times over a period of time ranging from days to weeks). Examples of cancer treatments include, without limitation immunecheckpoint inhibitors, adjuvant chemotherapy, neoadjuvant chemotherapy, histone deacetylase (HD AC) inhibitors such as 5-azacytidine and entinostat, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy (e.g., chimeric antigen receptors and / or T cells having wild-type or modified T cell receptors), targeted therapy such as administration of kinase inhibitors (e.g., kinase inhibitors that target a particular genetic lesion, such as a translocation or mutation), (e.g. a kinase inhibitor, an antibody, a bispecific antibody), signal transduction inhibitors, bispecific antibodies or antibody fragments (e.g., BiTEs), monoclonal antibodies, immune checkpoint inhibitors, surgery (e.g., surgical resection), or any combination of the above. In some embodiments, a cancer treatment can reduce the severity of the cancer, reduce a symptom of the cancer, and / or to reduce the number of cancer cells present within the subject.

[0173] Combination Therapies

[0174] 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.

[0175] In embodiments, the second anti-cancer therapy comprises one or more ALK inhibitors, including, e.g., the ALK inhibitors described herein. In embodiments, the second anti-cancer therapy comprises one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, an inhibitor of glycolytic glucose metabolism including, e.g., inhibition of glucose transporters (GLUT); inhibitors of hexokinase (HK); inhibitors of pyruvate kinase (PK) and in particular the M2 isoform (PKM2); and / or inhibitors of glutaminase (GLS), radiation, surgery or combinations thereof.

[0176] Chemotherapy: Cancer therapies in general also include a variety of combination therapies with both chemical and radiation-based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, famesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous form of DTIC), or any analog or derivative variant of the foregoing. The combination of chemotherapy with biological therapy is known as biochemotherapy. The chemotherapy may also be administered at low, continuous doses which is known as metronomic chemotherapy.

[0177] Yet further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophy cin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine andansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with histone deacetylase inhibitors. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevec (e.g., from about 400 to about 800 mg / day of Gleevec may be administered to a patient). In certain embodiments, one or more chemotherapeutic may be used in combination with the compositions provided herein.

[0178] Radiotherapy: Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also known such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0179] Immunotherapy: Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actuallyeffect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells as well as genetically engineered variants of these cell types modified to express chimeric antigen receptors. Mda-7 gene transfer to tumor cells causes tumor cell death and apoptosis. The apoptotic tumor cells are scavenged by reticuloendothelial cells including dendritic cells and macrophages and presented to the immune system to generate anti-tumor immunity.

[0180] It will be appreciated by those skilled in the art of cancer immunotherapy that other complementary immune therapies may be added to the regimens described above to further enhance their efficacy including but not limited to GM-CSF to increase the number of myeloid derived innate immune system cells, low dose cyclophosphamide or PI3K inhibitors (e.g., PI3K delta inhibitors) to eliminate T regulatory cells that inhibit innate and adaptive immunity and 5FU (e.g., capecitabine), PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells. For example, PI3K inhibitors include, but are not limited to, LY294002, Perifosine, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136. In some aspects, the PI3K inhibitor is a PI3K delta inhibitor such as, but not limited to, Idelalisib, RP6530, TGR1202, and RP6503. Additional PI3K inhibitors are disclosed in U.S. Patent Application Nos. US20150291595, US20110190319, and International Patent Application Nos. WO2012146667, WO2014164942, WO2012062748, and WO2015082376. The immunotherapy may also comprise the administration of an interleukin such as IL-2, or an interferon such as INFa.

[0181] Examples of immunotherapies that can be combined with the p53 and / or MDA-7 gene therapy and immune checkpoint inhibitor are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds) (U.S. Pat. No. 5,801,005; U.S. Pat. No. 5,739,169), cytokine therapy (e.g., interferons a, P and y; interleukins (IL-1, IL-2), GM-CSF and TNF), gene therapy (e.g., TNF, IL-1, IL-2, p53) and monoclonal antibodies (e.g., anti -ganglioside GM2, anti-HER-2, anti-pl 85. Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It possesses anti-tumor activity and has been approved for use in the treatment ofmalignant tumors. Combination therapy of cancer with herceptin and chemotherapy has been shown to be more effective than the individual therapies.

[0182] Additional immunotherapies that may be combined with the p53 and / or MDA-7 gene therapy and immune checkpoint inhibitor include a co-stimulatory receptor agonist, a stimulator of innate immune cells, or an activator of innate immunity. The co-stimulatory receptor agonist may be an anti-OX40 antibody (e.g., MEDI6469, MEDI6383, MEDI0562, and MOXR0916), anti-GITR antibody (e.g., TRX518, and MK-4166), anti-CD137 antibody (e.g., Urelumab, and PF-05082566), anti-CD40 antibody (e.g., CP-870,893, and Chi Lob 7 / 4), or an anti-CD27 antibody (e.g., Varlilumab, also known as CDX-1127). The stimulators of innate immune cells include, but are not limited to, a KIR monoclonal antibody (e.g., lirilumab), an inhibitor of a cytotoxicity-inhibiting receptor (e.g., NKG2A, also known as KLRC and as CD94, such as the monoclonal antibody monalizumab, and anti-CD96, also known as TACTILE), and a toll like receptor (TLR) agonist. The TLR agonist may be BCG, a TLR7 agonist (e.g., polyOICLC, and imiquimod), a TLR8 agonist (e.g., resiquimod), or a TLR9 agonist (e.g., CPG 7909). The activators of innate immune cells, such as natural killer (NK) cells, macrophages, and dendritic cells, include IDO inhibitors, TGFP inhibitor, IL-10 inhibitor. An exemplary activator of innate immunity is Indoximod. In some aspects, the immunotherapy is a stimulator of interferon genes (STING) agonist.

[0183] Other immunotherapies contemplated for use in methods of the present disclosure include suppression of T regulatory cells (Tregs), myeloid derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs). In some embodiments, the immunotherapy is a tumor vaccine (e.g., whole tumor cell vaccines, peptides, and recombinant tumor associated antigen vaccines), or adoptive cellular therapies (ACT) (e.g., T cells, natural killer cells, TILs, and LAK cells). The T cells may be engineered with chimeric antigen receptors (CARs) or T cell receptors (TCRs) to specific tumor antigens. As used herein, a chimeric antigen receptor (or CAR) may refer to any engineered receptor specific for an antigen of interest that, when expressed in a T cell, confers the specificity of the CAR onto the T cell. Once created using standard molecular techniques, a T cell expressing a chimeric antigen receptor may be introduced into a patient, as with a technique such as adoptive cell transfer. In some aspects, the T cells are activated CD4 and / or CD8 T cells in the individual which are characterized by y-IFN- producing CD4 and / or CD8 T cells and / or enhanced cytolytic activity relative to prior to the administration of the combination. The CD4 and / or CD8 T cells may exhibit increased release of cytokines selected from the group consisting of IFN-y, TNF-a and interleukins. The CD4 and / or CD8 T cells can be effector memory T cells. Incertain embodiments, the CD4 and / or CD8 effector memory T cells are characterized by having the expression of CD44hlghCD62Llow.

[0184] Human Subjects

[0185] 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.

[0186] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present disclosure should not be limited by any of the abovedescribed embodiments.

[0187] The following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0188] Definitions

[0189] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0190] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0191] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, recitation of “a cell”, for example, includes a plurality of the cells of the same type. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0192] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / -20%, + / — 10%, + / -5%, + / -1%, or + / — 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0193] The term, “amino acid” includes the residues of the natural a-amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Lys, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) in D or L form, as well as P-amino acids, synthetic and unnatural amino acids. Many types of amino acid residues are useful in the adipokine polypeptides and the disclosure is not limited to natural, genetically-encoded amino acids. Examples of amino acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the reference cited therein. Another source of a wide array of amino acid residues is provided by the website of RSP Amino Acids LLC.

[0194] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements — or, as appropriate,equivalents thereof — and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0195] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0196] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0197] Unless otherwise specified, a “nucleotide sequence encoding” an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0198] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0199] “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0200] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

[0201] The term “polynucleotide” is a chain of nucleotides, also known as a “nucleic acid”. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art and include both naturally occurring and synthetic nucleic acids.

[0202] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. Aprotein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The peptides provided herein for use in the described and claimed methods and compositions can be cyclic.

[0203] The term “percent sequence identity” or having “a sequence identity” refers to the degree of identity between any given query sequence and a subject sequence.

[0204] The terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.

[0205] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0206] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0207] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0208] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0209] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0210] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0211] The term “transfected” or “transformed” or “transduced” means to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The transfected / transformed / transduced cell includes the primary subject cell and its progeny.

[0212] “ Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Accordingly, “treating” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0213] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions,deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0214] As used herein, “vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cv.s-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term includes an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0215] Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, are intended to encompass homologous and / or orthologous genes and gene products from other species.

[0216] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical valueswithin that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0217] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.EXAMPLES

[0218] EXAMPLE 1: TUSC2 UPREGULATION IN ALK POSITIVE NON-SMALL CELL LUNG CARCINOMA CAN INDUCE CELLULAR APOPTOSIS

[0219] Tumor Suppressor Candidate 2 (TUSC2) is a tumor suppressor gene that has low endogenous expression in NSCLC in general, but data on TUSC2 in ALK+lung cancers are not available. A TUSC2 expression plasmid encapsulated in non-viral lipid nanoparticles (quaratusugene ozeplasmid, Reqorsa) upregulates TUSC2 expression in cancer cells by delivering the functional TUSC2 gene. TUSC2 expression was evaluated in three ALK+cell lines (cell lines having the EML4-ALK fusion, namely DFCI-032, NCI-H2228, and NCI- 143122), both before and after exposure to quaratusugene ozeplasmid and to a TUSC2- expressing plasmid. Controls were non- ALK? cell lines (NCI-H2170 and A549) and transfection with a plasmid containing no insert. It was shown herein that that overexpressing TUSC2 using quaratusugene ozeplasmid in the treatment of treatment of ALK+lung cancer cell lines, is able to suppress colony formation by 50%, the effect being more significant than using a TUSC2-containing plasmid. Furthermore, a robust pro- apoptotic response to TUSC2 expression in ALK? NSCLC was observed, as both quaratusugene ozeplasmid and TUSC2-containing plasmid induced an increase in caspase 3 / 7 activity in the cancer cells, accompanied by an increase in cleaved PARP expression (see FIGs 4, 5). Taken together, our data indicate that overexpression of TUSC2 in ALK? NSCLC cell lines with quaratusugene ozeplasmid or with TUSC2-containing plasmid is effective in decreasing growth and proliferation through the activation of apoptotic pathways and providing evidence for use as an anti-ALK NSCLC strategy.

[0220] In FIGS. 1-3, A549 parental, DFCI-032, NCI-H2170, NCLH3122 and NCLH2228 cell lines were transiently transfected with 3.5 pg of pcDNA3.1, Reqorsa and TUSC2plasmid (as a positive control). 24 hours later, the cells (A549 and DFCI-032) were trypsinized, counted and seeded for colony formation assay with 100, 200 and 500 cells being seeded in three 60 mm dishes for each of the groups. The remaining cells were put back in culture. After another 48 hours, i.e., 72 hours post transfection, the cells were collected, and RNA was isolated using the Qiagen kit. 2 pg of RNA was used to prepare cDNA in a 20 pl reaction volume. The cDNA was diluted 4 times and used to determine the mRNA expression of TUSC2 using GAPDH as a housekeeping control.

[0221] In FIGS. 4-6, A549 parental, DFCI-032, NCI-H2170, NCI-H3122 and NCI-H2228 cell lines were transiently transfected with 3.5 pg of pcDNA3.1, Reqorsa or TUSC2 plasmid (as a positive control). 24 hours later, the cells were trypsinized, counted and seeded for caspase 3 / 7 assay in a 96-well plate at a density of 10,000 cells / well in adherent condition. The remaining cells were put back in culture. After another 48 hours, i.e., 72 hours posttransfection, the cells were collected, and protein was isolated using RIPA buffer. The cells seeded in the 96-well plates were used to determine the caspase 3 / 7 activity 24 hours after seeding (i.e., 48 hours post transfection) using the Promega kit.

[0222] In FIGS. 7-15, NCI-H2228 cell line was earlier treated with different doses of Alectinib to generate Alectinib resistant cell lines by three independent methods (referred to as Cmax, Start low and Start IC50 here). Corresponding Alectinib resistant cell lines for NCI- 143122 were also generated (referred to as Cmax here). These cells were transiently transfected with 3.5 pg of pcDNA3.1, Reqorsa or TUSC2 plasmid (as a positive control). 48 hours later, the cells were trypsinized, counted and seeded for caspase 3 / 7 assay in a 96-well plate at a density of 10,000 cells / well (2000 cells / well in attempt 1) in adherent condition.The cells were also seeded for cell viability assays at a density of 2000 cells / well in a 96-well plate. The remaining cells were collected, and RNA was isolated. Cell viability, cellular proliferation, and caspase 3 / 7 assay were performed as described herein.

[0223] EXAMPLE 2: MATERIALS AND METHODS

[0224] Cell Culture and Reagents

[0225] The human lung cancer cell line, NCI-H3122 was obtained from Frederick National Laboratory for Cancer Research and DFCI-032 from Dana-Farber Cancer Institute, Boston. NCI-H2228, NCI-H2170 and A549 cells were purchased from ATCC. While NCI- 113122, NCLH2228 and DFCI-032 bear the EML4-ALK fusion, NCLH2170 and A549 serve as non-ALK controls. All cell lines were authenticated by DNA (short tandem repeat) profiling and tested for Mycoplasma contamination routinely.

[0226] NCI-H3122 and NCLH2170 cells were cultured in RPMI 1640 medium (ATCC) supplemented with 10% FBS, 1% Penicillin-Streptomycin, 0.05% Gentamicin and 1% L- Glutamine. NCI-H2228 cells were cultured in RPMI 1640 medium (ATCC) supplemented with 10% FBS, 1% Penicillin-Streptomycin and 0.05% Gentamicin. A549 cells were cultured in F-12K medium (ATCC) supplemented with 10% FBS, 1% Penicillin-Streptomycin and 0.05% Gentamicin. DFCI-032 cells were cultured in RPMI 16440 medium (Gibco) supplemented with 10% FBS, 1% Penicillin-Streptomycin, 0.05% Gentamicin along with specific concentrations of ITS, Hydrocortisone, EGF, Triiodothyronine, Phosphoryl ethanolamine, Ethanolamine, BSA, Hepes Buffer, L-Glutamine, Sodium Pyruvate and Sodium Bicarbonate. All cell lines were maintained at 37°C and 5% CO2.

[0227] Lipofectamine 3000 from Invitrogen was used for transfecting the vectors into the cells. The anti-TUSC2 antibody was purchased from Abeam and the pre-designed TUSC2 primer was bought from Applied Biosystems. The caspase 3 / 7 activity kit was purchased from Promega.

[0228] Plasmid transfection

[0229] The ALK+NSCLC cell lines as well as the non-ALK cells were transfected with 3.5 pg of pcDNA 3.1 (negative control), Quaratusugene ozeplasmid or TUSC2 plasmid (positive control) separately using Lipofectamine 3000. 48 hours post-transfection, the cells were used to confirm the over-expression of TUSC2 via qRT-pcr and Western blot.

[0230] RNA Isolation and qPCR Analysis

[0231] The total RNA was extracted using the Qiagen RNeasy mini kit and reverse transcription was performed to generate cDNA using the Promega Reverse Transcription System in a 20 pL reaction containing 2 pg of total RNA. A 2 pL aliquot of cDNA was amplified by Fast Taqman Master Mix from Applied Biosystems in each 20 pL reaction. PCR reactions were run on the QuantStudio 3 Real-Time PCR system (Applied Biosystems).

[0232] Immunoblotting

[0233] Whole-cell lysates were prepared by scraping the adherent cells in RIPA lysis buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail from Thermo Fisher Scientific. Protein quantification was performed using a Pierce BCA Protein Assay kit from Thermo Fisher Scientific. Equal amounts of protein were loaded and separated on a polyacrylamide gel. The proteins on the gel were transferred to a PVDF membrane, blocked with 5% nonfat milk in TBST and incubated with appropriate antibodies at 4 °C overnight.

[0234] After extensive washing, the membrane was incubated with the goat anti-rabbit secondary antibody conjugated with HPR from Cell Signaling Technologies for 1 h at roomtemperature. After washing, the protein bands were detected with chemiluminescence. The protein expressions were analyzed with respect to the expression of the house keeping gene, Tubulin.

[0235] Colony formation assay

[0236] The cells were transiently transfected with pcDNA3.1 (negative control), Quaratusugene ozeplasmid (lipid nanoparticles encapsulating TUSC2 expression construct) and the TUSC2 plasmid (positive control) separately and 48 hours post-transfection, the cells were trypsinized and counted. Cells from each group were seeded in each well of a 6-well plate at a density of 100 cells / well, 200 cells / well and 500 cells / well in their respective media and maintained at 37°C and 5% CO2. The cells were monitored for 3 weeks, post which the cells were fixed in 4% paraformaldehyde, stained with Toluidine Blue and counted to determine their colony formation ability.

[0237] Caspase 3 / 7 activity

[0238] The cells were transiently transfected with pcDNA3.1, Quaratusugene ozeplasmid or the TUSC2 plasmid separately and 48 hours post-transfection, the cells were trypsinized and counted. They were seeded in a 96 well plate at a density of 10,000 cells / well in their respective media and 24 hours later, the caspase 3 / 7 activity of the cells was recorded using the specific kit from Promega.

[0239] EXAMPL E 3: QUARATUSUGENE OZEPLASMID MEDIATED TUSC2 UPREGULATION IN EML4-ALK BEARING NON-SMALL CELL LUNG CARCINOMA CAN INDUCE CELLULAR APOPTOSIS

[0240] Materials

[0241] Cell lines: EML4-ALK+NSCLC cell lines NCI-H2228, NCI-3122, DFCI-032; Non- ALK NSCLC cell lines A549, NCI-H2170.

[0242] Results

[0243] Anaplastic Lymphoma Kinase (ALK), a potent oncogenic driver in Non-Small Cell Lung Carcinoma (NSCLC), is found to be rearranged and fused to Echinoderm Microtubule- associated protein-Like 4 (EML4), contributing to approximately 5% of NSCLC. Tumors bearing this fusion are sensitive to ALK Tyrosine Kinase Inhibitors (TKIs). However, emerging resistance to ALK TKIs demands new treatment strategies. Quaratusugene Ozeplasmid (QO), is an immunogene therapy that upregulates Tumor Suppressor Candidate 2 (TUSC2) expression in cancer cells by delivering a functional TUSC2 gene in non-viral lipid nanoparticles. TUSC2 expression was evaluated in three ALK+cell lines, both before andafter exposure to QO and to a TUSC2-containing plasmid. Controls were non-ALK+cell lines and transfection with a plasmid containing no insert. Our studies reveal that overexpressing TUSC2 using QO in ALK+lung cancer cell lines can suppress colony formation by 50%, the effect being more significant than using a TUSC2-containing plasmid. Furthermore, a robust pro-apoptotic response to TUSC2 expression in ALK+NSCLC was observed, as both QO and TUSC2-containing plasmid induced an increase in caspase 3 / 7 activity in the cancer cells, accompanied by an increase in cleaved PARP expression. Taken together, the data demonstrate that overexpression of TUSC2 in ALK+NSCLC cell lines with QO or with TUSC2-expressing plasmid is effective in decreasing growth and proliferation through the activation of apoptotic pathways.

[0244] Conclusion

[0245] The results demonstrate that upregulation of TUSC2 in ALK positive NSCLC cells by contacting the cells with a TUSC2 expression construct (TUSC2 lipid nanoparticles, e.g., QO, or TUSC2 plasmid) can induce cellular apoptosis in ALK+ NSCLC cells, and in the corresponding ALK inhibitor resistant cell lines, as seen by increased caspase 3 / 7 activity, increased protein expressions of cP ARP and cCaspase 3 and decreased colony formation ability.

[0246] Interestingly, TUSC2 lipid nanoparticle (QO) mediated overexpression of TUSC2 was found to be more effective in promoting apoptosis in comparison to the TUSC2 plasmid alone in ALK positive lung cancer and thus appears to be a treatment approach in NSCLC dominated by ALK.

[0247] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0248] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their disclosure.

Claims

What is claimed:

1. A method of treating cancer in a subject comprising administering to the subject in need thereof a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, wherein the cancer cells are anaplastic lymphoma kinase (ALK) positive, thereby treating the cancer.

2. The method of claim 1, wherein the cancer is non-small cell lung carcinoma (NSCLC),3. The method of claim 1 or claim 2, wherein the composition comprises a therapeutically effective amount of a liposome encapsulating the polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide.

4. The method of any one of claims 1-3, wherein the subject has previously received treatment comprising administration of ALK inhibitors.

5. The method of any one of claims 1-4, wherein the cancer is refractory to treatment with ALK inhibitors.

6. The method of claims 5, wherein ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X- 396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-d]pyrimidine or combinations thereof.

7. The method of any one of claims 1-6, wherein expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct.

8. The method of any one of claims 1-7, wherein the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2.

9. The method of claim 8, wherein the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2.

10. The method of any one of claims 1-9, wherein the polynucleotide construct comprises non-viral vectors or viral vectors.

11. The method of claim 10, wherein the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors.

12. The method of any of claims 3 or 11, wherein the liposome comprises a DOTAP: cholesterol liposome.

13. The method of claim 12, wherein the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3.

14. The method of claim 10, wherein the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof.

15. The method of claim 1, wherein the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreally (i.v.i.), intra-ci sterna magna (i.c.m.), or intrastemally.

16. The method of any one of claims 1-15, further comprising administering to the subject one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, radiation, surgery or combinations thereof.

17. A method of diagnosing and treating an ALK positive cancer in a subject, comprising: assaying a cancer sample obtained from the subject for cells that are ALK positive; administering to the subject having an ALK positive cancer a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, thereby diagnosing and treating ALK positive cancer in the subject.

18. The method of claim 17, wherein the cancer is non-small cell lung carcinoma (NSCLC), and optionally wherein the method further comprises assaying the cancer cells for one or more NSCLC specific biomarkers, and diagnosing the subject as having NSCLC when one or more NSCLC specific biomarkers are detected.

19. The method of claim 17 or claim 18, wherein the composition comprises a therapeutically effective amount of a liposome encapsulating the polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide.

20. The method of any one of claims 17-19, wherein the subject has previously received treatment comprising administration of ALK inhibitors.

21. The method of claim 20, wherein the cancer is refractory to treatment with ALK inhibitors.

22. The method of any one of claims 20-21, wherein ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X-396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-J]pyrimidine or combinations thereof.

23. The method of claim 17, wherein expression of the TUSC2 protein in the cancer cells is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct.

24. The method of claims 17 or 18, wherein the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2.

25. The method of claim 24, wherein the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2.

26. The method of any one of claims 17-25, wherein the polynucleotide construct comprises non-viral vectors or viral vectors.

27. The method of claim 26, wherein the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors.

28. The method of claim 27, wherein the liposome comprises a DOTAP: cholesterol liposome.

29. The method of claim 28, wherein the DOTAP:cholesterol ratio is between about 3 : 1 and about 1 :3.

30. The method of claim 26, wherein the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof.

31. The method of claim 17, wherein the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra-ci sterna magna (i.c.m.), or intrastemally.

32. The method of any one of claims 17-31, further comprising administering to the subject one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, radiation, surgery or combinations thereof.

33. A method of treating tumors in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, or a liposome comprising a TUSC2 polypeptide or a TUSC2 polynucleotide, wherein the tumors are resistant to treatment with ALK inhibitors.

34. The method of claim 33, wherein the tumors comprise non-small cell lung carcinomas (NSCLCs).

35. The method of claim 34, wherein the NSCLC cells are anaplastic lymphoma kinase (ALK) positive.

36. The method of any one of claims 33-35, wherein ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X-396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-J]pyrimidine or combinations thereof.

37. The method of any one of claims 33-36, wherein expression of the TUSC2 protein in the tumor is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct.

38. The method of claim 33, wherein the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2.

39. The method of claim 38, wherein the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2.

40. The method of any one of claims 33-39, wherein the polynucleotide construct comprises non-viral vectors or viral vectors.

41. The method of claim 40, wherein the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors.

42. The method of claims 33 or 41, wherein the liposome comprises a DOTAP:cholesterol liposome.

43. The method of claim 42, wherein the DOTAP:cholesterol ratio is between about 3 : 1 and about 1 :3.

44. The method of claim 40, wherein the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof.

45. The method of claim 33, wherein the composition is administered intratumorally, subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intravitreallly (i.v.i.), intra-ci sterna magna (i.c.m.), or intrastemally.

46. The method of any one of claims 33-45, further comprising administering to the subject one or more ALK inhibitors, chemotherapeutic agents, immunotherapeutic agents, radiation, surgery or combinations thereof.

47. A method of inducing cytotoxicity in a cancer cell comprising contacting the cancer cell with a composition comprising a therapeutically effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, wherein the cancer cells are anaplastic lymphoma kinase (ALK) positive, thereby treating the cancer.

48. The method of claim 47, wherein the cancer cell is non-small cell lung carcinoma (NSCLC) cell.

49. The method of claim 47 or claim 48, wherein the composition comprises a therapeutically effective amount of a liposome encapsulating the polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide.

50. The method of any one of claims 47-49, wherein the cancer cell is refractory to treatment with ALK inhibitors.

51. The method of claims 50, wherein ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X- 396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-d]pyrimidine or combinations thereof.

52. The method of any one of claims 47-51, wherein expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct.

53. The method of any one of claims 47-52, wherein the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2.

54. The method of claim 53, wherein the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2.

55. The method of claim 47, wherein the polynucleotide construct comprises non-viral vectors or viral vectors.

56. The method of claim 55, wherein the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors.

57. The method of claim 49, wherein the liposome comprises a DOTAP: cholesterol liposome.

58. The method of claim 57, wherein the DOTAP:cholesterol ratio is between about 3 : 1 and about 1 :3.

60. The method of claim 44, wherein the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof.

61. A method for inducing apoptosis or sensitizing an anaplastic lymphoma kinase (ALK) positive cancer cell to a composition, comprising: contacting the cancer cell with the composition comprising an effective amount of a polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide or variants thereof, wherein the cancer cells are anaplastic lymphoma kinase (ALK) positive, thereby sensitizing or inducing apoptosis of the cell by the composition.

62. The method of claim 61, wherein the cancer cell is non-small cell lung carcinoma (NSCLC) cell.

63. The method of claim 61 or claim 62, wherein the composition comprises an effective amount of a liposome encapsulating the polynucleotide construct comprising a polynucleotide sequence encoding a tumor suppressor candidate 2 (TUSC2) polypeptide.

64. The method of any one of claims 61-63, wherein the cancer cell is refractory to ALK inhibitors.

65. The method of claim 64, wherein ALK inhibitors comprise ceritinib (LDK378), alectinib (RG7853 / AF-802 / RO5424802 / CH5424802), brigatinib (AP26113), entrectinib (RXDX-101, NMS-E628), lorlatinib (PF-06463922), ASP3026, TSR-011, X-376 / X-396, CEP-28122 / CEP-37440, 5H-pyrrolo[3,2-d]pyrimidine or combinations thereof.

66. The method of any one of claims 61-65, wherein expression of the TUSC2 protein in the NSCLC cells is increased as compared to a baseline level in NSCLC cells prior to administering the polynucleotide construct.

67. The method of any one of claims 61-66, wherein the polynucleotide construct encodes a TUSC2 polypeptide having at least an 80% sequence identity to a human TUSC2 protein, SEQ ID NO: 2.

68. The method of claim 67, wherein the polynucleotide construct encodes a TUSC2 polypeptide comprising SEQ ID NO: 2.

69. The method of claim 68, wherein the polynucleotide construct comprises non-viral vectors or viral vectors.

70. The method of claim 69, wherein the non-viral vectors comprise plasmids, phages, synthetic constructs, liposomes or bacterial vectors.

71. The method of claim 70, wherein the liposome comprises a DOTAP:cholesterol liposome.

72. The method of claim 71, wherein the DOTAP:cholesterol ratio is between about 3 : 1 and about 1 :3.

73. The method of claim 69, wherein the viral vector comprises an adenovirus vector, an adeno-associated viral vector (AAV), a lentivirus vector or derivatives thereof.

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