Methods of treating pet positive cancers

Abstract

The present disclosure provides a method for treating cancer in a patient where the cancer cells are utilizing aerobic glycolysis by administering to the patient a therapeutically effective amount of a polynucleotide construct comprising polynucleotide sequence encoding TUSC2 protein. The cancer cells utilizing aerobic glycolysis can be identified, e.g., by using positron emission tomography (PET) scanning after the patient has been injected with a labeled glucose analog such as 18F-FDG. Thus, the disclosure provides a method for treating PET positive cancer in a patient comprising (i) performing PET on the patient; and (ii) administering to the patient having a PET positive cancer a therapeutically effective amount of a polynucleotide construct comprising polynucleotide sequence encoding TUSC2 protein.

Description

METHODS OF TREATING PET POSITIVE CANCERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Nos. 63 / 550,978, filed February 7, 2024, entitled “METHODS OF TREATING PET POSITIVE CANCERS”, the disclosure of which is incorporated herein by reference in its entirety.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 January 30, 2025, is named SeqList- 198628-46276. xml and is 3161 bytes in size.FIELD

[0003] The field of the present disclosure relates to methods, vectors and compositions for treating PET positive cancers by expressing TUSC2 in the cancer cells. Also contemplated are compositions including nonviral or viral vectors for increasing TUSC2 expression in cancer cells.BACKGROUND

[0004] Even in the presence of oxygen, certain cancer cells demonstrate a distinctive form of cellular metabolism characterized by high levels of glucose uptake and increased conversion of glucose to lactic acid by the glycolytic pathway. This phenomenon, known as aerobic glycolysis, is called the Warburg effect after Otto Warburg who received the Nobel Prize in 1931 for discovery of this effect.

[0005] The “glucose hunger” of many tumors is used clinically to visualize them using positron emission tomography (PET) scanning. In PET patients are injected with18F- fluorodeoxyglucose (18F-FDG), a radiolabeled, nonmetabolizable glucose derivative that is preferentially taken up by cancer cells that are utilizing aerobic glycolysis (as well as normal, actively dividing tissues such as the bone marrow). The18F-FDG then undergoes phosphorylation by hexokinase to FDG-6 phosphate. Unlike glucose, FDG-6 phosphate doesnot undergo further metabolism and so becomes trapped in the cell as the cell membrane is impermeable to FDG-6 phosphate following phosphorylation.

[0006] TUSC2 (TUmor Suppressor Candidate 2, also known as FUS1) is a tumor suppressor gene originally described as a member of the tumor suppressor gene cluster from human 3p21.3 chromosomal region that is frequently deleted in lung cancer. Independently of deletion, reduced expression of TUSC2 is observed in up to 80% of lung cancers, mesothelioma, breast, head-and neck, osteosarcoma, glioblastoma, and other cancers, suggesting a critical anti-tumor role of TUSC2. Human TUSC2 is a small protein (110 amino acids) with an estimated MW of 12 kD. According to a 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.SUMMARY

[0007] In one aspect, the disclosure provides a method for treating cancer in a patient comprising (i) determining that the cancer cells are utilizing aerobic glycolysis; and (ii) administering to the patient a therapeutically effective amount of a polynucleotide construct comprising polynucleotide sequence encoding TUSC2 protein. In embodiments, cancer cells utilizing aerobic glycolysis are identified using positron emission tomography (PET) scanning after the patient has been injected with a labeled glucose analog such as18F-FDG. Thus, the disclosure provides a method for treating positron emission tomography (PET) positive cancer in a patient comprising (i) performing PET on the patient; and (ii) administering to the patient having a PET positive cancer a therapeutically effective amount of a polynucleotide construct comprising polynucleotide sequence encoding TUSC2 protein. The polynucleotide construct may be provided as a nonviral or viral vector as part of a pharmaceutical composition.

[0008] In another aspect, the disclosure provides a method for suppressing aerobic glycolysis in a PET positive cancer cell comprising delivering to the cancer cell a nucleic acid construct comprising a polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. In embodiments, the nucleic acid construct is delivered to the cancer cell in a nonviral or viral vector.

[0009] In another aspect, the disclosure provides a method for reducing or suppressing mitochondrial ATP production in a cancer cell (e.g., in a PET positive cancer cell) by delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. The nucleic acid construct expressing TUSC2 can be delivered to the cancer cell using the nonviral vectors or viral vectors disclosed herein.

[0010] In another aspect, the disclosure provides a method for reducing or suppressing one or more of basal respiration, maximal respiration, spare respiratory capacity, ATP production, glycolysis, glycolytic capacity, and glycolytic reserve in a cancer cell (e.g., in a PET positive cancer cell) by delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. The nucleic acid construct expressing TUSC2 can be delivered to the cancer cell using the nonviral vectors or viral vectors disclosed herein.

[0011] In embodiments, the nonviral vector comprises DOTAP: cholesterol liposomes. In embodiments, the DOTAP:cholesterol ratio is between about 3: 1 and about 1 :3. In embodiments, the viral vector is an Adeno-Associated Virus (AAV) viral vector.

[0012] In embodiments the cancer is 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 embodiments the cancer, or cancer cell, is lung adenocarcinoma.BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1A top panel is a graph showing Oxygen Consumption Rate (OCR) over time for A549 human lung adenocarcinoma cells transfected with TUSC2-expressing plasmid (A549 TUSC2 mito) or uninserted control (A549 con mito) and subjected to Mitochondrial Stress test. FIG. 1A middle panel is a graph showing Basal OCR and Spare Respiratory Capacity OCR for A549 cells transfected with TUSC2-expressing plasmid or uninserted control, and FIG. 1A lower panel is a graph showing Proton Leak OCR and ATP Production OCR for A549 cells transfected with TUSC2-expressing plasmid or uninserted control plasmid.

[0014] FIG. IB top panel is a graph showing Extracellular Acidification Rate (ECAR) over time for A549 cells transfected with TUSC2-expressing plasmid (A549 TUSC2 glyco) or uninserted control (A549 con glyco). FIG. IB lower panel is a graph showing glycolysis,glycolytic capacity, and glycolytic reserve for A549 cells transfected with TUSC2-expressing plasmid or uninserted control plasmid.

[0015] FIG. 2 A top panel is a graph showing OCR over time for H358 human lung adenocarcinoma cells transfected with TUSC2-expressing plasmid or uninserted control and subjected to Mitochondrial Stress test. Top arrow indicates the H358 control (H358 con mito), bottom arrow indicates H358 TUSC2 (H358 TUSC2 mito). FIG. 2A middle panel is a graph showing Basal OCR and Spare Respiratory Capacity OCR for H538 cells transfected with TUSC2-expressing plasmid or uninserted control, and FIG. 2A lower panel is graph showing Proton Leak OCR and ATP Production OCR for H358 cells transfected with TUSC2-expressing plasmid or uninserted control plasmid.

[0016] FIG. 2B top panel is a graph showing ECAR over time for H358 cells transfected with TUSC2-expressing plasmid (H358 TUSC2 glyco) or uninserted control (H358 con glyco). FIG. 2B lower panel is a graph showing glycolysis, glycolytic capacity, and glycolytic reserve for H358 cells transfected with TUSC2-expressing plasmid or uninserted control plasmid.

[0017] FIG. 3A top panel is a graph showing OCR over time for Beas2B normal human bronchial epithelial cells transfected with TUSC2-expressing plasmid or uninserted control. Top arrow indicates the Beas2B control (beas2b con mito), bottom arrow indicates Beas2B TUSC2 (beas2b TUSC2 mito). FIG. 3A middle panel is a graph showing Basal OCR and Spare Respiratory Capacity for Beas2B cells transfected with TUSC2-expressing plasmid or uninserted control, and FIG. 3A lower panel is a graph showing Proton Leak and ATP Production for Beas2b cells transfected with TUSC2-expressing plasmid or uninserted control plasmid.

[0018] FIG. 3B top panel is a graph showing ECAR over time for Beas2b cells transfected with TUSC2-expressing plasmid (beas2b TUSC2 glyco) or uninserted control (beas2b con glyco). FIG. 3B bottom panel is a graph showing glycolysis, glycolytic capacity, and glycolytic reserve for Beas2B cells transfected with TUSC2-expressing plasmid or uninserted control plasmid.

[0019] FIG. 4 is a schematic of drug injection and OCR calculated upon test completion.

[0020] FIG. 5 is a schematic of drug injection and ECAR calculated upon test completion.DETAILED DESCRIPTION

[0021] Provided herein are methods for treating cancer where the cancer cells are utilizing aerobic glycolysis (e.g., is PET positive due to enhanced glucose demands) by expressingTUSC2 in the cancer cells. TUSC2 can be expressed from a nucleic acid expression construct encoding a TUSC2 protein. Also contemplated are nonviral vectors (e.g., comprising DOTAP: cholesterol liposomes) having the nucleic acid constructs disclosed herein. Additionally contemplated are viral vectors (e.g., recombinant AAV vectors) having the nucleic acid constructs disclosed herein. Further contemplated herein are pharmaceutical compositions having the nonviral vectors or viral vectors disclosed herein.

[0022] Expression of the TUSC2 gene product may facilitate the switch in cellular metabolism in cancer cells from fermentation to aerobic glycolysis. There are several lines of evidence that support the conclusion that TUSC2 gene deletion or protein inactivation facilitates the shift to aerobic glycolysis. First, 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 (NSCLC), 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.

[0023] 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 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.

[0024] 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.

[0025] TUSC2 knock-out mice exhibit a number of characteristics consistent with underlying metabolic abnormalities, including premature aging, aging associated pathologies, and decreased survival. TUSC2 knock-out mice also have an increased incidence of aspecific vascular malignancy (haemangioma / haemangiosarcoma). These findings are consistent with the lack of a gene predisposing to a change in metabolism.

[0026] 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.

[0027] Mitochondria have their own genes, but there are also genes in the nuclear DNA that encode proteins found in the mitochondria, suggesting that these genes developed to allow cells to control metabolic processes in the mitochondria. The TUSC2 gene is located in nuclear DNA, but the TUSC2 protein is located in the inner membrane of the mitochondria. Importantly, the enzymes of oxidative phosphorylation, which are active in aerobic glycolysis, are also located in the inner membrane of the mitochondria. This suggests that TUSC2 may control the activity of the enzymes of oxidative phosphorylation.

[0028] Taken together the above evidence supports that the TUSC2 gene is involved in the switch from cellular fermentation to aerobic glycolysis, and that expression of TUSC2 in cancer cells that are utilizing aerobic glycolysis (e.g., PET positive cancer) will suppress or inhibit this utilization of aerobic glycolysis.

[0029] I. Nucleic Acid Constructs

[0030] 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.

[0031] Expression constructs / plasmids for inclusion in the vectors described herein can be produced in a suitable host producer cells (e.g., E. coif) using suitable methods, e.g., fed- batch fermentation, batch fermentation, etc. For example, the HyperGRO™ inducible fed- batch fermentation process 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).

[0032] In embodiments, the expression construct comprising the polynucleotide sequence encoding a TUSC2 protein is a covalently closed linear DNA (“doggybone DNA” or dbDNA”). dbDNA may be created starting with a circular double-stranded DNA molecule (e.g., a plasmid) containing a TUSC2 encoding sequence flanked on each side by 56 bp palindromic protelomerase recognition sequences. The DNA starting material is then denatured and Phi29 DNA polymerase is primed. Phi29 initiates rolling circle amplification of the template, creating double-stranded concatameric repeats of the original construct. Protelomerase is added, which binds to the recognition sites flanking the TUSC2 encoding sequence and performs a 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.

[0033] i. TUSC2 Polynucleotide and Amino Acid Sequences

[0034] The nucleic acid constructs described herein include a polynucleotide sequence encoding a TUSC2 protein. In embodiments, the TUSC2 protein is human TUSC2. The human TUSC2 amino acid sequence is provided as SEQ ID NO: 1, below.SEQ ID NO: 1 MGASGSKARGLWPFASAAGGGGSEAAGAEQALVRPRGRAVPPFVFTRRGSMFYDE DGDLAHEFYEETIVTKNGQKRAKLRRVHKNLIPQGIVKLDHPRIHVDFPVILYEV

[0035] 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: 1. 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: 1.

[0036] In embodiments, the polynucleotide sequence encoding human TUSC2 is SEQ ID NO: 2, or is a polynucleotide sequence having greater than 85%, greater than 90%, or greater than 95% sequence identity to SEQ ID NO: 2. The human TUSC2 DNA coding sequence is provided as SEQ ID NO: 2, belowSEQ ID NO: 2 ATGGGCGCCAGCGGGTCCAAAGCTCGGGGCCTGTGGCCCTTCGCCTCGGCGGCC GGAGGCGGCGGCTCAGAGGCAGCAGGAGCTGAGCAAGCTTTGGTGCGGCCTCGG GGCCGAGCTGTGCCCCCCTTCGTATTCACGCGCCGCGGCTCTATGTTCTATGATG AGGATGGGGATCTGGCTCACGAGTTCTATGAGGAGACAATCGTCACCAAGAACG GGCAGAAGCGGGCCAAGCTGAGGCGAGTGCATAAGAATCTGATTCCTCAGGGCA TCGTGAAGCTGGATCACCCCCGCATCCACGTGGATTTCCCTGTGATCCTCTATGA GGTGTGA

[0037] In 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: 1, 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: 1. As used herein, the term “sequenceidentity” refers to the degree of which two sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits.

[0038] ii. Control / Regulatory Sequences

[0039] 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.

[0040] 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 enhancement of mRNA translation efficiency and increasing transgene expression. Nuclear targeting sequences, which promote shuttling of the nucleic acid construct into the nucleus, can be included 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.

[0041] iii. Selectable Markers

[0042] 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.

[0043] II. Nonviral Vectors

[0044] 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 liposomeformulations 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 nonviral vectors 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.

[0045] 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, 190nm, 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.

[0046] i. DOTAP:Cholesterol Liposomes

[0047] 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.

[0048] ii. Extrusion Techniques

[0049] The DOTAP:cholesterol liposomes described herein may be prepared, for example, by an extrusion method including the steps of heating, sonicating, and sequential extrusion of the lipids through filters of decreasing pore size, thereby resulting in the formation of small, stable liposome structures. In such methods, the production of liposomes often is accomplished by sonication or serial extrusion of liposomal mixtures after (i) reverse phase evaporation (ii) dehydration-rehydration (iii) detergent dialysis and (iv) thin film hydration. Methods of producing liposomes via extrusion are described in Templeton et al. (Nat. Biotechnol., 1997 15(7):647-52) and US Patent No. 10,293,056. In these methods, DNAlipid complexes are prepared by diluting a given nucleic acid and lipids in 5% dextrose in water to obtain an appropriate concentration of nucleic acid and lipids in an isotonic solution. For example, DOTAP (cationic lipid) is mixed with cholesterol (neutral lipid) at about equimolar concentrations. This mixture of powdered lipids is then dissolved with a solvent such as chloroform. The lipid solution is dried to a thin film at 30°C for 30 minutes(using, e.g., a rotary evaporator). The thin film is further freeze dried under vacuum for 15 minutes. The film is hydrated with water containing 5% dextrose (w / v) to give a final concentration of about 20 mM DOTAP and about 20 mM cholesterol. The hydrated lipid film is rotated in a 50°C water bath for 45 minutes and then at 375°C for an additional 10 minutes. The mixture is left standing at room temperature overnight. The following day the mixture is sonicated for 5-8 minutes at 50°C. The sonicated mixture is transferred to a new vessel and is heated for 10 minutes at 50° C. This mixture is sequentially extruded through filters (e.g., syringe filters) of decreasing pore size (e.g., 1 pm, 0.45 pm, 0.2 pm, and 0.1 pm). The 0.2 pm and 0.1 pm filters can be, e.g., Whatman Anotop filters (Cat. #: 6809-2122 or equivalent). The filtrate can be stored at, e.g., 4°C under argon gas.

[0050] iii. Microfluidization Techniques

[0051] 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 efficient wrapping of the two fluidsaround each other resulting in an exponential enlargement of the interface between the fluids ensuring rapid mixing.

[0052] iv. Methods of Making Nonviral Vectors

[0053] 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.

[0054] 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.

[0055] 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).

[0056] III. Viral Vectors

[0057] 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.

[0058] 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, the particle can be referred to as a “rAAV.” Such particles or virions include proteins that encapsidate or package the viral genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins (VP1, VP2, VP3). As used herein, the term “serotype” refers to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Recombinant AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8, and variants thereof. Examples of rAAV can include capsid 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.

[0059] 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.

[0060] IV. Compositions / Pharmaceutical Formulations

[0061] 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 otherembodiments, the composition includes a nonviral vector as described herein and about 0.9% (e.g., 0.8%, 0.9%, 1.0%, etc.) sodium chloride. In additional embodiments, the composition includes a nonviral vector comprising a nucleic acid construct described herein and a combination of about 5% dextrose and about 0.9% sodium chloride.

[0062] The compositions, nucleic acid constructs, nonviral vectors and viral vectors described herein may be administered to mammals (e.g., rodents, humans, nonhuman primates, canines, felines, ovines, bovines) in a suitable formulation according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2000) and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (1988-1999)). 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.

[0063] 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.

[0064] In other embodiments, the compositions described herein may be in a form suitable for intranasal administration. In one embodiment, the intranasal formulation is an aqueous formulation including a nucleic acid construct, nonviral vector or composition as described herein, a pH modifying agent, and a thickening agent. In the intranasal formulation, the pH modifying agent may provide or adjust the pH of the formulation to a suitable pH, e.g., a pH that assists in solubilizing an active agent in solution. In some embodiments, the intranasal formulation is administered as a stable intranasal spray that provides sufficient residence time on the nasal mucosa to allow trans-nasal absorption of the active agent(s). The thickening agent of the intranasal formulations described herein may modify the viscosity of the formulation to provide improved adherence of the formulation to the nasal mucosa without adversely affecting the ease of administration as an intranasal spray. The thickening agent may additionally increase the residence time of the formulation on the nasal mucosa, reduce loss of the formulation via mucociliary clearance of the nasal passages and / or improve the trans-nasal absorption. Such intranasal formulations may provide a sustained release of a nonviral vector as described herein.

[0065] The nucleic acid constructs, nonviral vectors, viral vectors and compositions described herein are preferably administered to a mammal (e.g., human) in a therapeutically effective amount. By the phrases “therapeutically effective amount”, “effective amount” and “effective dosage” is meant an amount sufficient to produce a therapeutically (e.g., clinically) desirable result; for example, the result can include increasing or restoring 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 agentcalculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. A unit dose as described herein may be described in terms of nucleic acid mass (pg) of the nucleic acid construct in the lipid complex. Unit doses range from 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1000 pg and higher.

[0066] V. Methods of Treatment

[0067] Methods of treating cancer in a human subject are described herein, in particular wherein the cancer PET positive, or otherwise utilizing aerobic glycolysis. 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.

[0068] 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 otherembodiments, 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.

[0069] Also contemplated is a method for suppressing aerobic glycolysis in a PET positive cancer cell by delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. The nucleic acid construct expressing TUSC2 can be delivered to the cancer cell using the nonviral vectors or viral vectors disclosed herein.

[0070] Further contemplated is a method for reducing or suppressing mitochondrial ATP production in a cancer cell (e.g., in a PET positive cancer cell) by delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. The nucleic acid construct expressing TUSC2 can be delivered to the cancer cell using the nonviral vectors or viral vectors disclosed herein. In embodiments of the methods disclosed herein, reducing or suppressing (e.g., aerobic glycolysis and / or mitochondrial ATP production) refers to levels relative to a cancer cell that has not been delivered the nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter.

[0071] i. Combination Therapies

[0072] 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.

[0073] In embodiments, the second anti-cancer therapy comprises 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).

[0074] VI. Human Subjects

[0075] The terms “patient,” “subject,” and “individual” are used interchangeably herein, and mean a mammalian (e.g., human) subject in need of treatment for cancer, in particular PET positive cancer (e.g., cancer utilizing aerobic glycolysis and having increased glucose demands compared to normal tissue from which the cancer is derived). Human subjects suffering from cancer include individuals suffering from various types of cancers, such ascolon 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.

[0076] VII. Methods of Use

[0077] In another aspect, the disclosure provides a method for suppressing aerobic glycolysis in a PET positive cancer cell comprising delivering to the cancer cell a nucleic acid construct comprising a polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. In embodiments, the nucleic acid construct is delivered to the cancer cell in a nonviral or viral vector.

[0078] In another aspect, the disclosure provides a method for reducing or suppressing mitochondrial ATP production in a cancer cell (e.g., in a PET positive cancer cell) by delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. The nucleic acid construct expressing TUSC2 can be delivered to the cancer cell using the nonviral vectors or viral vectors disclosed herein.

[0079] In another aspect, the disclosure provides a method for reducing or suppressing one or more of basal respiration, maximal respiration, spare respiratory capacity, ATP production, glycolysis, glycolytic capacity, and glycolytic reserve in a cancer cell (e.g., in a PET positive cancer cell) by delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter. The nucleic acid construct expressing TUSC2 can be delivered to the cancer cell using the nonviral vectors or viral vectors disclosed herein.

[0080] Reducing or suppressing as used herein may be in comparison to an appropriate control, for example, an untreated cancer or cancer cell. Suppression or reduction may be 20 %, 30 %, 40 %, 50%, 60 %, 70% or more compared to untreated cancer or cancer cell.

[0081] VIII. Glucose Uptake Tests

[0082] The disclosure provides methods of treating cancers (or cancer cells) that have elevated glucose uptake due to the Warburg Effect by administering an effective amount of a polynucleotide expressing TUSC2, as disclosed herein. The cancer cells utilize aerobic glycolysis deriving most of their energy from glycolysis (glucose converted to lactate followed by lactate fermentation) even when oxygen is available, rather than utilizing oxidative respiration.

[0083] The enhanced glucose demand of these cancers can be detected using [18F] 2- fluoro-2-deoxy-D-glucose (18F-FDG) PET imaging (e.g., PET / computerized tomography (CT) imaging).18F-FDG (a glucose analog) is administered to the patient and is taken up by cells via glucose transporter proteins. The glucose analog then undergoes phosphorylation by hexokinase to FDG-6 phosphate. Unlike glucose, FDG-6 phosphate does not undergo further metabolism and so becomes trapped in the cell as the cell membrane is impermeable to FDG- 6 phosphate following phosphorylation. PET positive cancers are those that are identified as having increased glucose demand and thus increased accumulation of the radiolabeled glucose analog using PET imaging.

[0084] Other methods for detecting elevated glucose demand of cancer cells that are utilizing aerobic glycolysis can be used in the disclosed methods as an alternative to18F-FDG PET imaging. For example, glucose uptake by cancer cells may be measured using labeled glucose or glucose analogs including 2-deoxy-D-[l,2-3H]-glucose, 2-deoxy-D-[l-14C]- glucose, and 2-[7V-(7-nitrobenz-2-oxa-l,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). In addition, glycolytic flux can be determined by measuring metabolites of glycolysis and in particular, lactate production. Other methods for detecting elevated glucose demand of cancer cells that are utilizing aerobic glycolysis include bioanalytic methods such as glycolytic rate assay (e.g., Agilent Seahorse XF Glycolytic Rate Assay; see also the Agilent Seahorse XF Cell Mito Stress Test). Elevated glucose demand and elevated utilization of aerobic glycolysis may be as compared to an appropriate control, for example, non-cancerous tissue or cells of the same cell type as the cancer cells.EXAMPLESExample 1: TUSC2 Expression in Normal and Cancer Cells

[0085] If and how human lung adenocarcinoma cells (A549 and H358) that have -80-90% decrease in TUSC2 expression change their energy metabolism in response to re-introduction of TUSC2 as compared to a normal human bronchial epithelial cell line (Beas2B) wasinvestigated. This example demonstrates that reintroduction of TUSC2 into TUSC2-deficient lung cancer cell lines suppressed both glycolytic and mitochondrial ATP production, while overexpression of TUSC2 in a normal lung epithelium cell line strengthens both glycolytic and mitochondrial metabolism. The results show that one mechanism through which TUSC2 promotes its antitumorigenic activities in lung cancer cells is by regulating energy homeostasis in epithelial cells.

[0086] Methods: The cell lines used in this study were NCI 549 “A549” and NCI 358 “H358,” which are human lung adenocarcinoma cell lines that have no detectable TUSC2 protein; and the Beas-2B cell line, which is a normal human bronchial epithelial cell line that expresses the TUSC2 protein. All were obtained from the ATCC. The cell lines were tested for expression of TUSC2 RNA via qPCR using TUSC2 specific primers. A plasmid containing the TUSC2 gene and uninserted control plasmid were transiently transfected into the cell lines using TransIT-X2 transfection kit (MirusBio). Seventy-two hours posttransfection, cells were tested for expression of TUSC2 RNA via qPCR. A metabolic analysis of energy production by these cells, including Oxidative phosphorylation and anaerobic glycolysis, was evaluated using the Agilent Seahorse XF Mitostress Test and Glycolysis Test Assay Kits. Based on the captured Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) under different conditions, the analyzer calculates main metabolic parameters such as basal respiration, ATP production, maximal mitochondrial respiration, spare respiratory capacity, coupling efficiency, glycolysis, glycolytic capacity, and glycolytic reserve. Experiments were repeated four times; Student T-test was used to calculate statistical significance.

[0087] Agilent Seahorse XF 96-well analyzer measures in real life and reports the OCR, PER or ECAR, as well as ATP production rates of live cells in a 96-well format. Cells were seeded into a 96-well XF culture plate with test-specific media. Drugs were automatically injected at specific time points by the analyzer. The drug cartridge contains sensor sleeves with polymer embedded fluorophores on the tip; one measures OCR and the other measures protons for ECAR. Drugs were loaded into the 4-wells surrounding the sensor sleeve and were released into media one by one during analysis. After drug injection into culture media, sensor probes slide into the sleeve to mix the drug and emit light to activate the fluorophores. Fluorophores are activated depending on the concentration of oxygen or protons in the chamber. Using these measurements, the instrument then calculates OCR and ECAR.

[0088] The drugs that were used to interrogate mitochondrial respiration and anaerobic glycolysis include oligomycin, FCCP, antimycin + rotenone, and 2-Deoxy-D-glucose (2-DG). Oligomycin is an inhibitor of ATP synthase (Complex V), which significantly reduces electron flow through the electron transport chain and prevents ATP production. FCCP is an uncoupling agent that disrupts ATP synthesis by transporting protons (H+) through the mitochondrial membrane before they can be used to provide the energy for oxidative phosphorylation. FCCP evaluates maximal respiratory capacity of the cell. Antimycin + Rotenone inhibit complex III and I of the mitochondrial electron transport chain (mETC), thus effectively mitochondrial respiration. 2-DG is a non-metabolizing glucose molecule, used to shut down glycolysis.

[0089] Results: All three cell lines demonstrated high transfection efficiency, confirmed by qPCR analysis. Seahorse analysis revealed that TUSC2 re-introduction to TUSC2- deficient cancer cells consistently suppressed both glycolytic and mitochondrial ATP production at 72h after transfection. This left cells without sufficient energy to support their vital functions (FIGS. 1 A, IB, 2A, 2B). A significant decrease in basal respiration, maximal respiration, spare respiratory capacity, ATP production, glycolysis, glycolytic capacity, and glycolytic reserve was detected in both cancer cell lines, A549 and H358, transfected with TUSC2-expressing plasmid (see FIGs.lA, IB, 2A, 2B).

[0090] Surprisingly, unlike cancer cells, both glycolytic and mitochondrial metabolism of the normal epithelial cell line Beas-2B were significantly strengthened after the introduction of TUSC2 (see FIGs.3A, 3B), indicating a beneficial role of TUSC2 for the metabolic health of normal cells. The experiments were repeated four times; Student T-test was used to calculate statistical significance. See FIGS. 4 and 5 for schematics of drug injection and mitochondrial parameters calculated upon test completion.

[0091] Conclusion: These experiments demonstrate that TUSC2 plasmid delivery to cancer patients, and thus therapeutic use of quaratusugene ozeplasmid (“quar oze”, TUSC2- containing plasmid encapsulated in lipid nanoparticles), may target and disrupt the metabolism of cancer cells (e.g., PET positive cancer cells), triggering either senescence or apoptotic pathways, while on the other hand, supporting the metabolism of normal epithelial cells. These data have high therapeutic significance, indicating that one of the anti-tumor mechanisms of quaratusugene ozeplasmid action in patients is the suppression of cancer cell metabolism resulting in cancer cell death.

Claims

We Claim:

1. A method for treating positron emission tomography (PET) positive cancer in a patient comprising (i) performing a PET scan; and (ii) administering to the patient having a PET positive cancer a therapeutically effective amount of a polynucleotide construct comprising polynucleotide sequence encoding TUSC2 protein.

2. The method of claim 1, wherein the polynucleotide construct is administered in a nonviral vector.

3. The method of claim 2, wherein the nonviral vector comprises a DOTAP: cholesterol liposome.

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

5. The method of claim 1, wherein the polynucleotide construct is administered in a viral vector.

6. The method of claim 5, wherein the viral vector is an Adeno-Associated Virus (AAV) viral vector.

7. A method for suppressing aerobic glycolysis in a PET positive cancer cell comprising delivering to the cancer cell a nucleic acid construct comprising polynucleotide sequence encoding TUSC2 protein operably linked to a promoter.

8. The method of claim 7, wherein the polynucleotide construct is administered in a nonviral vector.

9. The method of claim 8, wherein the nonviral vector comprises a DOTAP: cholesterol liposome.

10. The method of claim 9, wherein the DOTAP:cholesterol ratio is between about 3 : 1 and about 1 :3.

11. The method of claim 7, wherein the nucleic acid construct delivered to the cancer cell in a viral vector.

12. The method of claim 11, wherein the viral vector is an Adeno-Associated Virus (AAV) viral vector.

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