USING MINIVECTORS TO TREAT OVARIAN CANCER

Abstract

MiniVectors and compositions containing MiniVectors that target ovarian cancer genes selected from FOXM1, AKT, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16, alone or in any combination, are provided, along with uses in the treatment of ovarian cancer.

Claims

1) A MiniVector, said MiniVector being a double stranded, supercoiled, and circular DNA encoding an ovarian cancer inhibitory sequence (OCi) that can be expressed in a mammalian cell, said MiniVector lacking a bacterial origin of replication and lacking an antibiotic resistance gene.

2) The MiniVector of claim 1, wherein said MiniVector is at least 97% pure.

3) The MiniVector of claim 1, wherein said MiniVector is at least 97% pure, and is separated from a parent plasmid and recombination side-products on the basis of size, and does not use a restriction enzyme cleavage in vivo for preparation of said MiniVector.

4) The MiniVector of claim 1, wherein said OCi encodes an inhibitory RNA for a target gene selected from shRNA, miRNA, lncRNA, piRNA, RNAi, or antisense RNA.

5) The MiniVector of claim 1, wherein said OCi encodes an inhibitory RNA for a target gene selected from FOXM1, AKT, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16, alone, or in combination, and wherein expression of said target gene is reduced at least 10% by said inhibitory RNA when said MiniVector is introduced into mammalian cells and expressed therein.

6) The MiniVector of claim 1, wherein said ovarian cancer inhibitory sequence is an apoptosis gene selected from p53, p16, p21, p27, E2F genes, FHIT, PTEN, and/or CASPASE alone, or in combination, and said apoptosis gene is overexpressed when said MiniVector is introduced into mammalian cells.

7) The MiniVector of claim 1, comprising a promoter operably connected to said OCi operably connected to a terminator.

8) The MiniVector of claim 1, comprising a promoter connected to said OCi operably connected to a terminator, and additionally comprising an enhancer sequence and/or a nuclear localization signal.

9) The MiniVector of claim 1, that is made by: a) engineering a parent plasmid DNA molecule comprising site-specific recombination sites on either side of said OCi; b) transforming said parent plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing a plurality of catenated DNA circles, wherein at least one of the circles in each catenane is a supercoiled DNA MiniVector of less than about 5 kb in length; c) decatenating the catenated site-specific recombination products, thereby releasing the supercoiled DNA MiniVector from the catenanes; and d) isolating the supercoiled DNA MiniVector.

10) The MiniVector of claim 1, wherein said MiniVector is 600 bp in length, excluding said OCi.

11) A composition comprising a MiniVector in a pharmaceutically acceptable excipient, said MiniVector being a double-stranded, supercoiled, nicked, or relaxed circular DNA encoding an OCi and lacking a bacterial origin of replication and lacking an antibiotic resistance gene, wherein said circular DNA is at least 95% free of parent plasmid DNA or recombination side-products, wherein said OCi is expressible in human cells and thereby inhibits the expression of a human target gene selected from FOXM1, AKT, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16, alone, or in any combination.

12) The composition of claim 11, wherein said MiniVectors are 250 bp to 5,000 bp in total length.

13) The composition of claim 11, wherein said MiniVector is 600 bp in length, excluding said OCi.

14) The composition of claim 11, wherein said MiniVector is 250 bp in length, excluding said OCi.

15) The composition of claim 11, wherein said ovarian cancer inhibitory sequence is codon optimized for humans or human cancers.

16) The composition of claim 11, wherein said MiniVector is CpG-free, CpG maximized, or CpG minimized.

17) The composition of claim 11, wherein said MiniVector is supercoiled.

18) The composition of claim 11, wherein said MiniVector has a specific DNA sequence-defined shape.

19) A MiniVector, said MiniVector being a double-stranded, supercoiled, circular DNA encoding an OCi that can be expressed in a mammalian cell, wherein said OCi encodes an inhibitory RNA for a target gene selected from FOXM1, AKT, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16, alone or in any combination, wherein said MiniVector lacks a bacterial origin of replication and lacks an antibiotic resistance gene, and wherein said MiniVector is made by: a) engineering a parent plasmid DNA molecule comprising site-specific recombination sites on either side of said OCi; b) transforming said parent plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing a plurality of catenated DNA circles, wherein at least one of the circles in each catenane is a supercoiled DNA MiniVector of less than about 5 kb in length; c) decatenating the catenated site-specific recombination products, thereby releasing the supercoiled DNA MiniVector from the catenanes; and d) isolating the supercoiled DNA MiniVector.

20) A method of treating ovarian cancer, comprising delivering the MiniVector of claim 19 or cells containing same to a patient having ovarian cancer, wherein said OCi inhibits expression of said target gene in said patient by at least 20%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1. Generation of MiniVector DNA by -integrase-mediated site-specific recombination. Parent plasmid containing the sequence to be delivered flanked by attB and attP, the target sites for recombination. The parent plasmid is propagated in the special E. coli bacterial host strain, LZ54 or LZ31, harboring -integrase (Int) under the control of the temperature sensitive ci857 repressor. When the cells have reached a suitable density, expression of Int is switched on by a temperature switch. Recombination results in a catenated product containing the MiniVector. The products are decatenated, either by endonuclease cleavage of the large circle deletion product ex vivo, or by topoisomerase IV-mediated unlinking subsequent to the removal of topoisomerase inhibitor following the cell harvest. The deletion product containing the undesired bacterial sequences is removed, yielding pure, supercoiled MiniVector product. If desired, the MiniVector can encode attR and the deletion product can contain attL by switching the positions of attB and attP in the parent plasmid. Bla=beta lactamase.

[0046] FIG. 2. Modular design of MiniVectors. On the left is shown the minimal therapeutic unit, consisting only of A) attL or attR site (these sites are the products of recombination by integrase), B) a promoter, C) the therapeutic sequence (e.g., shRNA encoding sequence), and D) a transcriptional terminator. Potential sequences for A, B and C are listed in Tables 1-3. The intervening regions can include any other sequence and can range in length from none to several thousand base pairs. On the right is shown a modified version containing additional modules that may be added to provide long-term persistence and expression, improve transfection, and/or facilitate nuclear localization. Any combination of these additional modules may be added to the essential modules. E) S/MAR sequence, if incorporated into the MiniVector, will be placed upstream of the transcriptional unit to utilize the dynamic negative supercoiling generated by transcription or elsewhere on the molecule. F, G) Enhancer sequences may be positioned in a number of locations, depending on the identity of the enhancer. H) Nuclear localization sequences, if incorporated, will be placed downstream of the transcriptional unit.

[0047] FIG. 3. FOXM1 and downstream gene targets are upregulated in PTEN/DICER1 double cKO tumors. This cancer mouse model spontaneously grows high grade serous ovarian tumors up-regulating the same genes as in humans. Among these genes is FOXM1.

[0048] FIG. 4. Selecting an effective shRNA against FOXM1 in an ovarian cancer cell line. The experiment shown was at 72 h post-transfection with 4 g plasmid DNA encoding the various shRNAs. Gene knockdown was quantified by qPCR. This experiment was repeated twice with three replicates in each experiment. The actin gene was used as an endogenous control. Percentage knockdown is shown relative to the vehicle only control.

[0049] FIG. 5A-B. Effect of shRNA on FOXM1 expression using plasmid delivery in (A) Ovarian cancer cells or (B) non-cancer 293T cells. The experiments shown were from 48 h post-transfection with 2 g DNA. The actin gene was used as an endogenous control. Percentage knockdown is shown from qPCR data compared to the shRNA scrambled control (NTC).

[0050] FIG. 6A: FoxM1 Knockdown via MiniVectors in OVCAR8. FIG. 6B: FoxM1 knockdown via MiniVectors in 293T cells.

DETAILED DESCRIPTION

[0051] The disclosure provides novel MiniVectors used to target and treat ovarian cancers. The invention includes any one or more of the following embodiment(s), in any combination(s) thereof:

TABLE-US-00003 A MiniVector, said MiniVector being a double-stranded, supercoiled, circular DNA encoding an ovarian cancer inhibitory sequence (OCi) that can be expressed in a eukaryotic cell. Said MiniVector typically lacks a bacterial origin of replication and lacks antibiotic resistance genes, but may if desired for some applications. Preferably, the MiniVector without the OCi is <600 bp. The invention also includes ovarian cancer inhibitory (OCi) RNA for a target gene selected from shRNA, miRNA, lncRNA, piRNA, RNAi, or antisense RNA. The OCi RNAs and DNAs encoding same are preferably for a target gene selected from FOXM1, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, AKT1, AKT2, AKT3, and/or PRDM16 alone or in any combination. These OCi RNAs can be delivered by any method, but one preferred method is by delivery of a MiniVector encoding same. The OCi DNA could also be on a plasmid, virus, or minicircle, or other delivery means. Any MiniVector, or composition thereof described herein, wherein said MiniVector is at least 95% pure. It could even be 97% pure, or 99.5% pure, 99.9% pure or higher (purity assessed with respect to contaminating parent plasmid DNA and recombination DNA side products). Any MiniVector, or composition thereof described herein, wherein said MiniVector is at least 95% or 97% or 99% pure, is separated from parent plasmid and recombination side-products on the basis of size, and does not depend upon the use of a restriction cleavage in vivo. Any MiniVector, or composition thereof described herein, wherein said MiniVector is at least 95%, 97%, 99% or 99.5% pure and does not contain detectable parent plasmid or recombination side- products. A MiniVector, or composition thereof, wherein said OCi encodes an inhibitory RNA for a target gene selected from shRNA, miRNA, lncRNA, piRNA, RNAi, and/or antisense RNA alone or in any combination. A MiniVector, or composition thereof, wherein said OCi encodes an inhibitory RNA for a target gene selected from FOXM1, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16, alone or in any combination, and wherein expression of said target gene(s) is reduced by at least 10% by said inhibitory RNA when said MiniVector is introduced into mammalian cells. A MiniVector, or composition thereof, wherein said ovarian cancer inhibitory sequence is an apoptosis gene selected from p53, p16, p21, p27, E2F genes, FHIT, PTEN, and/or CASPASE alone or in any combination, and said apoptosis gene is expressed when said MiniVector is introduced into eukaryotic cells. A MiniVector, or composition thereof, comprising a promoter operably connected to said OCi operably connected to a terminator. A MiniVector, or composition thereof, comprising a promoter connected to said OCi operably connected to a terminator, and additionally comprising an enhancer sequence and/or a nuclear localization signal (NLS). A MiniVector, or composition thereof, wherein said MiniVector is expressible in a human cell and said OCi is for a human target gene. A MiniVector, or composition thereof, that is made by: engineering a parent plasmid DNA molecule comprising site-specific recombination sites on either side of said OCi; transforming said parent plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing a plurality of catenated DNA circles, wherein at least one of the circles in each catenane is a supercoiled DNA MiniVector of typically less than about 5 kb in length; decatenating the catenated site-specific recombination products, thereby releasing the supercoiled DNA MiniVector from the catenanes; and isolating the supercoiled DNA MiniVector. Any MiniVector, or composition thereof, described herein, wherein said MiniVectors are 250 bp to 5,000 bp in total length. Any MiniVector, or composition thereof, described herein, wherein said MiniVectors are <500 bp or <400 bp or <350 bp or <300 bp or <250 bp in length, excluding said OCi. A composition comprising a MiniVector in a pharmaceutically acceptable excipient, said MiniVector being a double-stranded, supercoiled, nicked, or relaxed circular DNA encoding an ovarian cancer inhibitory sequence (OCi) and typically lacking a bacterial origin of replication and lacking an antibiotic resistance gene, wherein said circular DNA is at least 95% free of parent plasmid DNA or recombination side-products, wherein said OCi is expressible in human cells and thereby inhibits the expression of a human target gene selected from FOXM1, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16, alone or in any combination. Any MiniVector, or composition thereof, described herein, wherein said ovarian cancer inhibitory sequence is codon optimized to modify (decrease or increase) expression in humans or expression in specific cell types or specific cancer cell types. Any MiniVector, or composition thereof described herein, wherein said MiniVector is CpG-free, CpG maximized, or CpG minimized. Any MiniVector, or composition thereof described herein, that is supercoiled. Preferably, the DNA is at least 85% supercoiled, 90%, 95% or 99%. A MiniVector, or composition thereof, which is relaxed or nicked. A MiniVector, or composition thereof, said MiniVector being a double-stranded, supercoiled, circular DNA encoding an ovarian cancer inhibitory sequence (OCi) that can be expressed in a eukaryotic cell, wherein said OCi encodes an inhibitory RNA for a target gene selected from FOXM1, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16 alone or in any combination, wherein said MiniVector typically lacks a bacterial origin of replication or antibiotic resistance gene, wherein said MiniVector is separated from a parent plasmid and recombination side-products on the basis of size, and does not use a restriction cleavage in vivo, and does not contain detectable parent plasmid or recombination side-products as measured by gel electrophoresis stain. A MiniVector, or composition thereof, said MiniVector being a double-stranded, supercoiled, circular DNA encoding an ovarian cancer inhibitory sequence (OCi) that can be expressed in a eukaryotic cell, wherein said OCi encodes an inhibitory RNA for a target gene selected from FOXM1, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and/or PRDM16 alone or in combination, wherein said MiniVector typically lacks a bacterial origin of replication or antibiotic resistance genes, wherein said MiniVector is made by: engineering a parent plasmid DNA molecule comprising site-specific recombination sites on either side of said OCi; transforming said parent plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing a plurality of catenated DNA circles, wherein at least one of the circles in each catenane is a supercoiled DNA MiniVector of typically less than about 5 kb in length; decatenating the catenated site-specific recombination products, thereby releasing the supercoiled DNA MiniVector from the catenanes; and isolating the supercoiled DNA MiniVector. A method of treating ovarian cancer, comprising delivering a MiniVector or cell or composition containing same as described herein to a patient having ovarian cancer, in an amount sufficient to reduce the expression of an OCi target gene by at least 10%, at least 20% or at least 30%. A treatment method as described herein, wherein said MiniVector is contained in a gel, a matrix, a solution, or a nanoparticle, and is delivered by injection or by surgery, or by other means. A treatment method as described herein, wherein delivery of said MiniVector may be facilitated by electroporation, sonoporation, electrosonoporation, transfection, mechanical acceleration (gene gun, etc.), or other means, either directly into an ovarian tumor or residual tumor cells or tissue, or into cells ex vivo that are then returned to the patient. A treatment method as described herein wherein said MiniVector is delivered to the lungs of said patient intranasally or via aerosolization; or said MiniVector is delivered intravenously, by intramuscular injection, by intraperitoneal injection; or said MiniVector is delivered topically, intravaginally, and/or rectally. A treatment method as described herein wherein said MiniVector is delivered via a permanent or a temporary device, including but not limited to robot, catheter, shunt, port, arteriovenous fistula, gene gun, needle-free syringe with or without deposition technology, including but not limited to sonoporation, electroporation, electrosonoporation, etc. Ovarian cancer is characterized by cancer causing FOXM1 and P53 levels and/or variants, and the method described herein can be applied to other cancers with the same characteristics. Thus, the invention includes methods of treating a cancer characterized by high FOXM1 levels, comprising delivering a MiniVector as described herein or cells or composition containing said MiniVector to a patient having a cancer characterized by high FOXM1 levels, wherein said sequence encoded by said MiniVector is a FOXM1 shRNA, wherein said FOXM1 shRNA inhibits expression of FOXM1 in said patient (or said cancer) by at least 10%, 15%, 20%, 25%, or 30%. The invention also includes methods of treating a cancer characterized by low P53 levels or P53 mutants, comprising delivering a MiniVector as described herein or cells or composition containing said MiniVector to a patient having a cancer characterized by low P53 levels or P53 mutants, wherein said sequence encoded by MiniVector is a functional P53 or a P53 stimulator, wherein said sequence reduces cancerous cell count vs. controls or increases P53 activity in said patient (or said cancer) by at least 10%, 15%, 20%, 25%, or 30%.

Minivector Targets

[0052] The p53 gene is mutated in 96% of high grade serous ovarian cancer (mucinous, endometrioid, clear cell, and undifferentiated) and negatively regulates FOXM1 (forkhead box M1), a key oncogenic transcription factor implicated in cancer-cell migration, invasion, angiogenesis, and metastasis.

[0053] The Matzuk laboratory has created the only existing mouse model (DICER-PTEN DKO) that develops high-grade serous ovarian cancer that phenocopies the spread and lethality of ovarian cancer in women (Kim 2012). In this genetically engineered mouse model, FOXM1 and its downstream targets [i.e., centromere protein A (Cenpa), polo-like kinase 1 (Plk1), cell division cycle 20 (Cdc20), survivin (Birc5), aurora kinase B (Aurkb), cyclin B1 (Ccnb1), and cyclin-dependent kinase inhibitor 3 (Cdkn3)] are upregulated 9.5-27.7-fold. FOXM1 is, therefore, a novel target in all p53 mutant cancers (both ovarian cancers and other cancers).

[0054] Using transcriptomic and genomic approaches, we have identified two additional novel ovarian cancer targets for novel MiniVector alone or combination therapy. These ovarian cancer-specific gene fusions are BCAM-AKT2 and CDKN2D-WDFY2.

[0055] BCAM-AKT2 is an ovarian cancer-specific fusion between BCAM (basal cell adhesion molecule), a membrane adhesion molecule, and AKT2 (v-Akt homolog 2), a key kinase in the PI3K signal pathway. BCAM-AKT2 is membrane-associated, constitutively phosphorylated, and escapes regulation from external stimuli. BCAM-AKT2 is the only fusion event proven to translate an aberrant yet functional kinase fusion and is detected in 7% of all high-grade serous ovarian cancer and it also significantly alters the PI3K/AKT pathway.

[0056] The inter-chromosomal fusion gene CDKN2D-WDFY2 occurs at a frequency of 20% among sixty high-grade serous cancer samples, but is absent in non-cancerous ovary and fallopian tube samples (Kannan 2014). The CDKN2D-WDFY2 fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumors, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of a cloned fusion construct led to loss of wild type CDKN2D and wild type WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis.

[0057] YM155 (a putative survivin suppressor) is an anticancer drug that is in clinical trials, but requires combination with other drugs for efficacy. The targets of YM155 were unknown. Using a proteomics screen, we identified solute carrier family 25, member 6 (SLC25A6) and cancerous inhibitor of protein phosphatase 2A, PP2A (CIP2A), as two molecular targets of YM155.

[0058] SLC25A6 is a mitochondrial membrane component of the permeability transition pore complex responsible for the release of mitochondrial products that trigger apoptosis and is a unique apoptosis target for anticancer therapy.

[0059] CIP2A, an inhibitor of tumor suppressor PP2A and a stabilizer of the MYC oncogene, is also a unique target for anticancer therapy that includes cancers that are MYC-dependent. Using shRNA delivery to OVCAR8 (human ovarian cancer cells) in culture, we have shown that knockdown of SLC25A6 and CIP2A slows ovarian cancer cell growth and triggers ovarian cancer cell death, thus validating these two targets for MiniVector gene therapy or combination therapy using MiniVectors.

[0060] FOXM1 or FORKHEAD BOX M1 was originally identified in Drosophila, and is a member of a family of transcription factors with a conserved 100-amino acid DNA-binding motif. FOXM1 is normally involved in cell cycle progression and is a master regulator of the DNA damage response. Although the exact mechanism remains unknown, as a proto-oncogene, FOXM1 is involved in the early stages of cancer initiation and is upregulated in many different types of cancer. These cancers include but are not limited to basal cell carcinoma, soft-tissue sarcomas, and cancers of the blood, brain, breast, central nervous system, cervix, colon, colon, rectum, kidney, liver, lung, mouth, ovary, pancreas, prostate, skin, and stomach.

[0061] A broad array of FOXM1-mediated cancers metastasizes to the lungs, including bladder, breast, colon, kidney, and prostate cancers, in addition to neuroblastoma and sarcomas. Many metastatic sarcomas are of soft-tissue origin, such as cartilage, fat, muscle, tendons, lymph vessels, blood vessels, and nerves, or from bone (Ewing sarcoma and osteosarcoma). Where metastases in the lungs occur, therapeutic nebulized MiniVectors (or intranasal application) would be employed for delivery to the lung, either alone or in combination with other therapies. MiniVector, again either alone or in combination with other therapies, may also be delivered to other locations.

Minivector Modifications

[0062] MiniVectors can be labeled, e.g., using a chemical moiety, as desired. Representative labels include fluorescent dyes, biotin, cholesterol, modified bases, and modified backbones. Representative dyes include: 6-carboxyfluorescein, 5-/6-carboxyrhodamine, 5-/6-Carboxytetramethylrhodamine, 6-Carboxy-2-,4-,4-,5-,7-,7-hexachlorofluorescein, 6-Carboxy-2-,4-,7-,7-tetrachlorofluorescein, 6-Carboxy-4-,5-dichloro-2-,7-dimethoxyfluorescein, 7-amino-4-methylcoumarin-3-acetic acid, Cascade Blue, Marina Blue, Pacific Blue, Cy3, Cy5, Cy3.5, Cy5.5, IRDye700, IRDye800, BODIPY dye, Texas Red, Oregon Green, Rhodamine Red, Rhodamine Green, and the full range of Alexa Fluor dyes.

[0063] Additional modifications can also include modified bases (e.g., 2-aminopurine, deoxyuracil, methylated bases), or modified backbones (e.g., phosphorothioates, where one of the non-bridging oxygens is substituted by a sulfur; methyl-phosphonate oligonucleotides).

[0064] Multiple labels, including chemical moieties and/or modified bases and/or modified backbones, can be used simultaneously, if desired. Methods of labeling nucleotides are described, for example, in Luzzietti et al. Nicking enzyme-based internal labeling of DNA at multiple loci, in Nature Protocols (2012), vol. 7, 643-653; Nucleic Acid Probe Technology by Robert E. Farrell; RNA Methodologies (Third Edition), 2005, pp. 285-316; and Enzymatic Labeling of Nucleic Acids by Stanley Tabor and Ann Boyle, in Current Protocols in Immunology 2001 May; Chapter 10: Unit 10.10. The teachings of these references are incorporated herein by reference in their entirety.

Minivector Delivery

[0065] The purified MiniVectors can be transferred into recipient cells or into a differentiated tissue by transfection using, for example, lipofection, electroporation, cationic liposomes, or any other method of transfection, or any method used to introduce DNA into cells or tissues, for instance, jet injection, sonoporation, electroporation, mechanical acceleration (gene gun, etc.), or any other method of transfer.

[0066] MiniVector may be delivered in a gel, a matrix, a solution, a nanoparticle, a cell, or other means directly into an ovarian tumor or residual tumor cells or tissue, or into cells ex vivo that are then returned to a patient. Typically, in vivo studies use injection or surgical introduction, but any method can be used ex vivo. The term cell includes Car T cells or any cell therapy.

[0067] Delivery solutions can be aqueous solutions, non-aqueous solutions, or suspensions. Emulsions are also possible. Delivery solutions can be magnetic, paramagnetic, magnetically resistant, or non-magnetic. Saline is a preferred delivery solution. The MiniVector therapy could optionally be lyophilized.

[0068] The preferred carrier medium for the MiniVector can vary depending on whether it is delivered systemically or locally. The complexity of the carrier could vary as a result. Systemic carriers can be more complex given the need for enlarged circulation times and the need to resist a variety of in vivo processes which might prematurely degrade the carrier and the MiniVector (e.g. opsonization).

[0069] Systemic carrier mediums can have varied in size vs. locally-delivered carriers. Further, the size of the carrier medium can change due to an array of stimuli (e.g. charge, enzyme availability, magnetic field, etc.). Some research suggests that the optimal particle size during the systemic phase is approximately 100 nm and during the local accumulation phase is approximately 40 nm.

[0070] Components which might comprise a carrier medium include but are not limited to: dendrimers, spermine, spermidine, polyethylenimine (PEI), saline, cationic liposomes, phospholipids, cationic lipids, lipoplexes, cationic nanoemulsions, nano or micro-porous silicate nanoparticles, nano or micro-porous silicate microparticles, nano or micro-porous gold nanoparticles, nano or micro-porous gold microparticles, chitosan, cholestoral, hydrazone activated polymers, zwitterionic polymers or co-polymers, amphiphilic polymers or co-polymers, polyplexes modified with PEG or HPMA, polymethacrylate, biodegradable polyesters (e.g. poly-(DL-Lactide) aka PLA or poly-(DL-Lactide-coglycoside) aka PLGA), and microbubbles. Hybrids or combinations of the listed materials and others are possible.

[0071] Optionally the carrier medium can employ a core-shell morphology where the core and the shell optionally have multiple layers and said layers have different charge and contact angle (i.e. hydrophobic, hydrophilic, amphiphilic) and said materials optionally degrade or change due to variations in pH, temperature, shear energy, light, time, the presence of enzymes and the like. Said particles can be conjugated with a broad array of agents which affect shape, size (e.g. shrinking or swelling), immune system activation (e.g. adjuvant), endocytosis, nuclearization and the like. Particles can be nanosized or micro-sized. Such structures can optionally be lamellar. Their interfaces can be graded.

[0072] The carrier medium or its components can be self-assembled.

[0073] Carrier mediums can open to facilitate delivery of the MiniVector when subjected to ultrasound, magnetic field, redox, light, enzymes and the like.

[0074] A carrier medium can be a multi-layer polymer encapsulated particles where the layers degrade over time.

[0075] To reduce the propensity of the body to degrade the employed carrier medium (e.g. via opsonization and phagocytic uptake), it can be optionally coated or treated with a stealth material. Stealth materials are frequently electrostatically neutral and/or hydrophilic. Examples of passive stealth materials include but are not limited to polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyglycerol, poly-N-vinylpryrrolidone, polyozaline, and poly[N-(2 hydroxypropyl)methacrylamide].

[0076] Appreciating that stealth materials can reduce the carrier medium's interaction with cell surfaces and thus cellular uptake, they can be engineered to separate (cleave) from the carrier medium when subjected to stimuli. Examples of such stimuli include but are not limited to light, ultrasound, magnetic field, pH, redox, or enzymes.

[0077] The carrier medium can employ targeting agents (frequently attached as ligands) which enhance accumulation at target sites. Targeting agents include but are not limited to antibodies, aptamers, peptides, and small molecules that bind to receptors on the cell.

[0078] Peptide-types include but are not limited to those which exhibit high affinity for a targeted cell surface receptor (e.g. cell targeted peptides or CTPs such as arginine-glycine-aspartic acid aka RGD tripeptide). Other peptides can exhibit properties which afford the ability to non-specifically interact with cell surfaces and enhance cell entry (e.g. protein transduction domains or PTDs and cell penetrating peptides or CPPs).

[0079] Example non-peptide targeting agents include cholera toxin B, folic acid, low-density lipoprotein, nicotinic acid, riboflavin, and transferrin (e.g. Arrowhead Pharmaceutical's CALAA-01).

[0080] Solutions of all types may be combined with other phases such as gasses for purposes of delivery. A typical example would be for the purpose of atomization and more specifically control of droplet size and droplet size distribution.

[0081] MiniVector delivery can be facilitated by an ex vivo or in vivo device that meters out delivery quantities locally or systemically, but preferably locally. Said devices can control or influence other desired properties such as temperature, pH, shear, and dispersion uniformity. Said devices will likely comprise microelectromechanical (MEM) or nanoelectromechanical (NEM) components. Said devices could afford multiple purposes (Combination Devices) ex vivo or in vivo. Functions afforded by a Combination Device could include therapeutic dispensing and optionally therapeutic atomizing, pH control, heating, cooling, magnetic potential control, sensing of these and other activities, and wireless communication amongst others.

[0082] Solutions of MiniVectors can be delivered locally into the peritoneal cavity via a needle, a minimally invasive surgical device, or a surgical device during surgery more generally. Said surgical devices could comprise an atomizer and the solution of MiniVectors could optionally be atomized. Atomization could be achieved via control of nozzle aperture, pressure, or the introduction of a second phase (e.g., a gas). Droplet size and size distribution could be controlled in similar fashion.

[0083] MiniVector therapies could be stored in powder form, gel form, as an emulsion, or as a solution, or as a precipitate under alcohol. To maximize the shelf-life of any MiniVector therapy a variety of preservatives can be employed. Example preservatives include but are not limited to ethyleneglycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), a nuclease inhibitor, a protease inhibitor, or any other chelating agent.

Combination Therapies

[0084] To improve the efficacy of MiniVector-based therapies, they may be administered in combination with other MiniVectors or with other FDA approved therapies. Thus, MiniVectors can be administered before, concurrently with, and/or following treatment with other MiniVectors, small molecule drugs, peptides, antibodies, siRNA, minicircles, ministrings, plasmids, viruses, surgery, or radiation, or any combination and/or timing of administration of two, three or more of these individual approaches.

[0085] Combination therapy is regularly used in cancer treatment because of the heterogeneity and complexity of the disease, and the tendency of cancer cells to become resistant to certain drugs. There are many potential benefits to using such an approach with MiniVectors. Primarily, although resistance to current frontline small molecules and radiation occurs, the genetic basis of cancer (e.g., p53 downregulation) remains. Therefore, although it remains a theoretical possibility, there is no reason to believe that resistance to gene therapy would emergeindeed, to date this has never happened. Therefore, a MiniVector therapy should be able to block growth of any chemoresistant or radiation-resistant cancer and the genetic basis for the resistant tumors are easily checked by sequencing prior to MiniVector treatment. To the extent that the adjunct therapy and the MiniVector target different pathways, this should increase the success of cancer eradication by targeting two different pathways. Furthermore, because of the increased efficacy, combination therapy may allow lower doses of each therapy to be used, and consequently lower toxicity and reduced side effects.

[0086] PARP inhibitors have shown great promise for ovarian cancer treatment and a number of drugs that target this protein have recently been approved. PARP is involved in the repair of single-strand breaks in DNA. When PARP is inhibited these single-strand breaks become double-strand breaks and must be repaired by homologous recombination. A significant proportion of ovarian cancers have impaired homologous recombination. The inability of the cells to repair the DNA breaks ultimately leads to death of the cancer cells. PARP inhibitors have been assayed in combination with chemotherapy or radiation because the inhibition of DNA repair by PARP inhibitors should enhance the efficacy of these treatments.

[0087] One potential combinatorial therapy will be the concurrent administration of a MiniVector encoding an shRNA to FoxM1 and a Poly ADP-ribose polymerase (PARP) inhibitor, for example, Rucaparib, Niraparib, Lynparza, Olaparib, and/or Talazoparib. PARP inhibitors we can thus target two distinct characteristics of the cancer cells. This combinatory approach may lack the synergistic power of PARP inhibitors combined with chemotherapy or radiation, but should have lower toxicity and also a reduced incidence of resistance. The most likely application for such an approach is to target any remaining cancer cells following surgery. The lower toxicity and reduced incidence of resistance should be useful for the prolonged treatment necessary to eliminate the cancer cells and prevent relapse.

[0088] In an alternative approach, MiniVectors and PARP inhibitors could be combined with chemotherapy and/or radiation for the purpose of maximizing patient longevity, and minimizing treatment-induced toxicity and drug resistance. It is understood that quantities of each therapy type would be optimized combinatorially in animals and humans for the purpose of maximizing net benefit.

[0089] Chemotherapy agents available for combination therapy with MiniVectors include but are not limited to Paclitaxel, Capecitabine, Cyclophosphamide, Etoposide, Gemcitabine, Topotecan, Doxorubicin, Cisplatin, Carboplatin, Vinorelbine, Ifosfamide, Etoposide, Fluorouracil, Docetaxel, or combinations thereof. Tyrosine kinase inhibitors, such as Pazoparib, can also be combined with MiniVectors.

[0090] Radiation therapies available for combination with MiniVectors include but are not limited to proton therapy, two-dimensional proton therapy, three dimensional conform radiation therapy, brachytherapy, intensity modulated radiation therapy, image guided radio-therapy, stereotactic radiation, radio surgery, orthovoltage radiation, electron radiation, or combinations thereof.

[0091] Biological approaches, including hormone therapies, available for combination with MiniVectors include but are not limited to Aromassin, Femara, Arimidex, Megace, Farletuzumab, Tamoxifen, Rucaparib, Niraparib, Olaparib, and/or Talozaparib.

[0092] Surgical procedures that can be combined with MiniVectors include but are not limited to tumor debulking, oophorectomy, salpingectomy, hysterectomy, omentectomy, and second look surgery where the efficacy of assorted treatments is interrogated in vivo.

[0093] Another ovarian cancer therapy approach available to be combined with MiniVectors is that of tumor ablation. During tumor ablation micro- or nano-sized particles of varied composition are delivered adjacent to cancers. The particles typically preferentially ablate tumors when subjected to thermal energy, radiofrequency, ultrasound, microwave and/or a cryoablation source, amongst others. The acting particles often concurrently protect or minimize damage to healthy tissues adjacent to the tumor. Examples of candidate particles suitable for ablation include but are not limited to gold (as undergoing commercialization by NanoSpectra), platinated nanoparticles, copper sulfide, and polydopamine. MiniVectors could be delivered by the methods described herein before, during, or after ablation therapies.

[0094] Stem cell-derived therapies afford great promise in a variety of medical domains including ovarian cancer. Stem cell-derived cell therapy approaches frequently augment patients' immune systems by awakening the body to the presence of assorted cancers or cancer-driving mutations. In one embodiment, adult stem cells are repurposed to express a T-cell receptor known to NY-ESO 1. When the repurposed stem cells are re-injected into the patient a portion of said cells expand and self-renew. These surviving repurposed T-cells alert the body to the presence of certain ovarian cancer types and thereby initiating a series of processes that result in the destruction of the tumor cell. Stem cell-derived immunotherapy approaches and others could be combined with MiniVector-derived therapies.

[0095] Another combination therapy approach would be to deliver multiple MiniVectors, each encoding a different sequence. Delivering MiniVectors compositions that inhibit more than one target may produce a synergistic effect and be more effective than a single-target MiniVector. In addition, delivering multiple MiniVectors against targets on different pathways may reduce toxicity and likelihood of resistance. Both approaches will be tested for feasibility.

[0096] A key context in which MiniVectors could be used following other therapies is ovarian cancer tumor recurrence. More than 50% of ovarian cancer patients that undergo surgical tumor resection experience recurrence and most of these recurring cancers are resistant to the previous chemotherapeutic or radiation treatment(s). There are only limited drugs or treatments available such that there remains no choice left but to stop treatment. In addition, the human body has limited capacity to undergo repeated surgical, chemical, and radiation treatments; however, ovarian cancer recurrence is frequently fatal, particularly for high grade serous ovarian cancer. Although recurring ovarian cancer is typically resistant to the previous treatments, the genetics behind the cancer phenotype (e.g., p53 downregulation) remains. Therefore, MiniVectors can still be used in these cases. Furthermore, the cassette encoded by MiniVectors (see FIG. 2) can be changed repeatedly and treatments continue.

[0097] MiniVectors afford the ability to treat ovarian cancer patients repeatedly and perhaps on a long-term basis, even when the patient has undergone other treatments (that may or may not have been combined with MiniVector therapies). Reasons for the possibility of repeated MiniVector treatment include the fact that they can be delivered locally vs. systemically, their lack of toxicity as a consequence of their small size and their naked DNA composition (limited cytotoxicity and immune response), the lack of unwanted delivery or integration (high shear strength of the closed circular particles), and the facile switching of MiniVector cassettes.

[0098] These attributes in combination afford the ability to modify (tune) MiniVector composition and its cassette to address both spatial and temporal tumor heterogeneity and to mitigate or obviate tumor recurrence. It is specifically possible to employ said tuned MiniVectors either alone, combinations of MiniVectors, or in combination with other therapies (e.g., small molecule, other gene therapy approaches, cell therapy agents, other MiniVectors, and biologics).

Personalized Minivector Therapies

[0099] Although our proof-of-concept work will continue to proceed with commercial sequences that target common mutations, we contemplate developing personalized target sequences for each patient. Cancer is highly heterogenous in nature. No two cancers are identical and cancer cells may even vary within the same patient. The DNA sequence and gene expression profiles of an individual patient's cancer can be readily determined through high-throughput DNA sequencing, microarrays, qPCR, RNA-Seq, and other methods on patient tissue samples. These tests reveal which sequences and/or gene product(s) are present or absent, and which genes are abnormally expressed in the cancer cells, so that a custom MiniVector can be developed encoding targets specifically tailored to a particular patient. This type of approach can be readily modified as needed and according to treatment outcomes by altering one or more of the sequences encoded on the personalized MiniVector or MiniVectors.

[0100] In ovarian cancer, 7% of patients with high-grade serous ovarian cancer have the gene fusion BCAM-AKT2 and 20% have the gene fusion CDKN2D-WDFY2. These gene fusions are absent from normal cells and therefore these targets are unique to this subset of ovarian cancers. For a personalized therapy approach, individuals presenting these fusions would be treated with sequences that target these proteins specifically.

[0101] An additional example of personalization of MiniVector/MiniVectors treatment is to target specific isoforms of a protein that are present in certain cancer patients. The rationale for which isoform is targeted will rely on those determined from ovarian cancer tissue samples. Once the isoform(s) is determined, shRNA(s) can be designed to specifically knockdown that isoform(s), based on identified unique sequences of exons included in the isoform. FoxM1b and FoxM1c, for example, are two isoforms of FoxM1 that are upregulated and transcriptionally active in many cancers, including ovarian cancer. FoxM1b has a greater transforming potential than FoxM1c. FoxM1a is not transcriptionally active in cancer cells. As part of alternative splicing, FoxM1b lacks exon Va, a stretch of 15 amino acids, whereas FoxM1a and FoxM1c contain this exon. An shRNA construct will be designed to specifically target the FoxM1b isoform using the unique sequence that connects exons IVa and VIa. This personalization will allow specific knockdown of FoxM1b for treatment and will not target the FoxM1a or FoxM1c isoforms.

Model Ovarian Cancer Stem Cell Line

[0102] Although women with ovarian cancer initially respond well to surgical debulking and chemotherapy, there is a high cancer recurrence rate that is hypothesized to arise secondary to the chemoresistant population of cancer stem cells (CSC). We have identified, for the first time, an ovarian CSC-like cell line (called OV90) that tends to form spheroids (spherical 3 dimensional cultures) under standard culture conditions, greatly expressing stem-like markers Prominin 1 (CD133), Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1), CD44 Molecule (CD44), Spalt Like Transcription Factor 4 (SALL4), and PR/SET Domain 16 (PRDM16), as well as FOXM1. OV90 cells are resistant to many National Cancer Institute oncology library compounds.

[0103] The OV90 line will allow us to test the effects of novel treatments in vitro and in vivo (in cell line xenograft models) and make advances toward treatment of cancer patients. Because of the role of FOXM1 in stemness, the OV90 cells will be particularly useful for testing MiniVectors or combination therapies involving MiniVectors that target FOXM1 for their ability to kill these ovarian cancer stem cells. Ovarian cancer stem cell markers (e.g., CD133, ALDH1A1, CD44, SALL4, and PRDM16) are also potential targets for MiniVector therapy.

Proof of Concept FOXM1 Experiments

[0104] For our initial work, we tested FOXM1 shRNA (targeting 5-ATAATTAGAGGATAATTTG-3) in plasmid vectors against the ovarian cancer cell line OVCAR8. In these initial experiments, we were able to show 25% knockdown after 72 hours post-transfection and significant cancer cell inhibition (FIG. 5A). In a control non-cancerous cell culture line, 293T cells, we achieved 60% knockdown of FOXM1 (the gene is not overexpressed in non-cancer cell lines, so there was a lower amount to start with) but there was no inhibition of growth (FIG. 5B).

[0105] Since that early experiment, we have also confirmed proof of concept using MiniVectors. FOXM1 shRNA (targeting 5-ATAATTAGAGGATAATTTG-3) in MiniVector was tested against the ovarian cancer cell line OVCAR8 (FIG. 6A). In these initial experiments, we were able to show between 16 to 48% knockdown of FoxM1 when MiniVectors were delivered in a dose response manner, 72 hours post-transfection. In a control non-cancerous cell culture line, 293T cells, we achieved between 25 to 50% knockdown of FOXM1 with no inhibition of cell growth (FIG. 6B). Therefore, we have a good indication that our approach using MiniVector therapy will be far safer for use in humans.

Prophetic FOXM1 Experiments

[0106] The following prophetic examples can apply equally to all cancers described herein. For brevity however, they are written relative to ovarian cancer. Other described cancers could be substituted provided the appropriate cancer specific cell line (see Table 6) and animal model (i.e., xenografted or patient-derived xenografted mice) are employed. Also, while FOXM1 is relevant to all p53 related cancers described herein, other named targets specific to a relevant cancer (e.g., ovarian cancer) can be substituted or added to the FOXM1 example in the prophetic experiments without limitation. In addition, although e.g., FOXM1 may be the primary target, similar effects may be obtained by targeting up- or downstream genes in the same pathway.

[0107] Knockdown efficiency of the de novo shRNAs will be validated using synthetic small interfering RNAs (with the same RNA sequence as the shRNA transcripts) that will be transfected into different cell lines (e.g., OV90 or OVCAR8 cell lines for ovarian cancer; see below) using lipofection, electroporation, sonoporation, or any other method of nucleic acid delivery for cell culture. Knockdown will be assayed using SYBR Green PCR Master mix to measure levels of the target mRNA in cell lysates. Knockdown efficiencies of the siRNAs will be compared to validated shRNA sequences (encoded on pGIPZ plasmid vectors) obtained from Dharmacon (see Table 1).

[0108] shRNA sequences that demonstrate effective levels of knockdown efficiency (>10% reduction in mRNA levels as determined by quantitative RT-PCR (Q-RT-PCR) analysis and/or >10% reduction in protein levels as determined by western blot analysis) will be cloned between the attB and attP recombination sites on the MiniVector, generating parent plasmid using standard, well-established molecular cloning techniques. MiniVectors are the products from an intramolecular recombination reaction as described above and below. In the most basic embodiment, the resulting MiniVector will comprise elements include a promoter, the therapeutic sequence, a terminator, and the hybrid site from recombination (attL or attR). Accessory sequences can be added to improve efficiencies, such as enhancers, DNA targeting sequences (DTS's), etc. (see Tables 2-5).

Minivector Synthesis

[0109] MiniVectors are generated using engineered Escherichia coli strains (examples include but are not limited to LZ31 and LZ54), in which a small aliquot of this strain is transformed with the relevant parent plasmid, growth, and then is used to inoculate a fermenter containing modified terrific broth medium. Cells are grown at 30 C. with maintaining the pH at 7 and the dissolved oxygen concentration above 60%. Once cells have reached mid-exponential phase, -Int expression is induced by shifting the culture to 43 C. Norfloxacin is added to prevent decatenation by topoisomerase IV, and the culture is shifted down to 28 C. to allow recombination to proceed for about an hour (1-4 hrs). Cells are harvested by centrifugation.

[0110] MiniVectors are purified by first resuspending the cells in buffer and lysozyme, and further lysed using alkaline lysis. Nucleic acids are precipitated by isopropanol and treated with RNase A and Proteinase K. Parent plasmid is removed through precipitation with polyethyleneglycol. MiniVectors are further purified from their parent plasmid using anion exchange purification kits and gel filtration. Endotoxin is removed using commercially available purification kits.

FOXM1 SHRNA Evaluation

[0111] For ovarian cancer, an ovarian cancer stem-cell like cell line (OV90) or another ovarian cancer cell line (e.g., OVCAR8) will be transfected with MiniVectors encoding a single or a combination of multiple shRNAs for the same target or for different targets with the purpose of assessing the effect of single or combination shRNA for blocking cancer growth. As described above, MiniVectors will encode one or multiple shRNA sequences that have been screened to avoid off-target effects based upon sequence homology. When using more than one shRNA against the same or multiple different target genes, combinatorial knockdown of the target gene or genes may increase either the overall knockdown efficiency or the inhibition of cancer cell growth. Consequently, lower doses of each shRNA-encoding MiniVector may be required with the added benefit of minimizing potential off-target effects of each specific shRNA sequence.

[0112] To test knockdown of the MiniVector-encoded shRNA gene targets in vitro, cells will be plated and transfected with Lipofectamine3000 transfection reagent once they reach 80% confluency. We may also use other methods of DNA delivery in culture (electroporation, sonoporation, etc.). 24 and 72 hours post-transfection, cells will be trypsinized and harvested for RNA extraction and cDNA synthesis. Q-RT-PCR will be conducted to quantify knockdown of FOXM1 or other target genes using SYBR Green PCR Master mix. We anticipate knockdown efficiencies to show a >10% reduction in mRNA levels as determined by Q-RT-PCR analysis and/or >10% reduction in protein levels as determined by western blot analysis. The effects of these knockdown levels will be further measured by assessing the phenotype of the cultured cancer cells.

[0113] The phenotype from the knockdown of FOXM1 will be assessed in culture by measuring cell apoptosis, cell cycle arrest, and cell proliferation. We will use flow cytometry and commercially available kits to measure these variables. We predict that the therapy or combination therapies that result in sustained knockdown of the targets will have the best ability to block cancer growth.

[0114] Off-target effects and cytotoxicity resulting from the knockdown of FOXM1 will be measured concurrently with the experiments outlined above by transfecting a non-cancer cell line (i.e., 293T cells). mRNA from cell lysates of cancer cells, and/or 293T cells will be used to do microarray analysis or RNAseq to further confirm the lack of off-target effects of the therapies. Cytotoxicity will be measured using cell viability and apoptosis assays. Any potential shRNA candidates that display any deleterious level of off-target effects or cytotoxicity will not be pursued in vivo.

[0115] An alternative therapy to block growth of cancer cells will be transfection with MiniVectors encoding genes that promote apoptosis (e.g., p53, p16, p21, p27, E2F genes, FHIT, PTEN, or CASPASE). In the most basic embodiment (see FIG. 2), the resulting MiniVector encoding such genes will have a promoter, the apoptosis-promoting sequence, a terminator, and the hybrid recombination site (attL or attR). The benefit of this approach will be assessed by measuring cell apoptosis, cell cycle arrest, and cell proliferation in the target cancer cells.

[0116] MiniVectors encoding the best shRNA candidates for single and combinatorial therapies against FOXM1, and with demonstrated efficient knockdown and corresponding phenotype in cell culture, will be pre-clinically screened in vivo first in cell line xenograft and then patient derived xenograft mouse models to assess cancer cell-death (via apoptosis or another mechanism) or slowdown of cancer cell growth, and also to further optimize and formulate treatment therapies against different cancers, some of which are based on metastatic models.

[0117] Bioluminescent cell lines for cell line xenografting will be generated by stably transfecting various cancer cell lines with a vector encoding a Luciferase reporter (pGL4.51) using lipid transfection or any other method of nucleic acid delivery. Selection of cells stably transfected with pGL4.51 will be achieved with the antibiotic G418. Mice will be injected with D-luciferin (the luciferase substrate) by intraperitoneal (IP) injection. Bioluminescence from the luciferase enzyme will allow in vivo imaging to be used to detect and quantify any changes in the size of the tumors. Alternative imaging methods include making a stable cell line with a different fluorescence-encoding reporter plasmid (e.g., GFP, RFP, etc.).

[0118] Cells stably expressing the luciferase or fluorescence reporter will be transfected in vitro with MiniVectors encoding the shRNA candidates (using transfection or any other method of nucleic acid delivery), followed by IP injection of the transfected cells to female immunodeficient eight-week athymic nude (Foxn1nu) mice (or other appropriate mouse models) to generate the cell line xenografts.

[0119] Growth or proliferation of these cancer cells as well as the size of the tumors in mice will be tracked in real-time by in vivo bioluminescence (after IP injection of the D-luciferin substrate) or fluorescence imaging. Negative controls in these experiments will be vehicle only (no MiniVector) or MiniVectors encoding control (for example, scrambled validated control sequences) shRNA sequences. We predict that shRNAs shown to result in efficient knockdown of the target genes will successfully attenuate growth of the tumors relative to mice xenografted with vehicle only or with MiniVectors encoding control shRNA sequences. At the completion of the bioluminescence or fluorescence studies, mice will be sacrificed and dissected, and tumors evaluated.

[0120] Tumors as well as other organs will be harvested for gene expression and histological analysis. Q-RT-PCR and western blot will be done in tumor homogenates to quantify knockdown of cancer targets. Histology of the organs will be assessed, and any cytotoxicity or off-target effects of the therapy will be noted. We will re-formulate the therapy if needed.

[0121] Bioluminescent cancer cells will be injected into mice to generate a xenograft mouse model. MiniVectors will be delivered subsequently either immediately or by varying the time following tumor engraftment by IP injection and any changes in tumor size will be quantified using bioluminescent imaging compared to control xenograft mice treated with control MiniVectors or untreated. Other possible routes of in vivo MiniVector delivery in mice include intranasal, intravenous (tail vein, face vein, or other), intramuscular, topical applications or other methods to reach tumors. Either naked DNA MiniVectors or the use of delivery vehicles (lipofectamine, lipid polymers, etc.) will be tested, and optimized if needed, by measuring tumor reduction and knockdown of the target. In the case where metastases to the lungs are being interrogated, nebulized (or intranasally instilled) MiniVectors would be employed for delivery to the lung alone or in combination with other delivery mechanisms and locations.

[0122] Dosage, treatment frequency, as well as duration of the therapy will be assessed by measuring and monitoring tumor size, measuring knockdown of the target mRNAs, and assessing toxicity.

Minivectors with Specific Shapes

[0123] MiniVector DNA backbone sequence can be modified to engineer DNA sequence and supercoiling-dependent bends to affect DNA 2-dimensional (if planar) or 3-dimensional shape. Geometries such as, but not limited to, rod-shaped, two, three, four, and five or more-leafed clover-shaped, triangle-shaped, square-shaped, rectangle-shaped, trapezoid-shaped, kite-shaped, both regular and irregular pentagon-shaped, hexagon-shaped, other polygon-shaped, star-shaped, disc-shaped, sphere-shaped, ellipse-shaped, cylinder-shaped, cone-shaped, crescent-shaped, obelisk-shaped, tetrahedron-shaped, hexahedron, octahedron-shaped, dodecahedron-shaped, icosahedron-shaped, pointed shapes, shapes that mimic viral capsids, hybrids of these shapes, convex and concave versions as well of each of these geometries, and the like can be engineered to improve transfection or preferentially target one cell type over another. MiniVector shapes may change or be induced over time or with specific condition (encounter with proteins, salts, cell compartment-specific environment, temperature, pH, etc.) from one to another shape.

Additional Experiments

[0124] Novel therapeutic shRNA sequences (at least 5) against each of the primary targets, FOXM1, BCAM-AKT2, and CDKN2D-WDFY2, will be designed using freely available, open access, algorithms (e.g., siRNA Wizard Software, siDESIGN Center, etc.) and then screened for off targets effects using NCBI-BLAST. Alternatively, commercially available sequences can be used for initial proof of concept work.

[0125] Knockdown efficiency of the de novo shRNAs will be validated as described above, and shRNA sequences that demonstrate effective levels of knockdown efficiency will be cloned between the attB and attP recombination sites on the MiniVector, as above. The MiniVectors will then be tested in OV90 or OCVAR8.

[0126] Knockdown (or increases) in targets can be measured by measuring mRNA levels or protein activity in e.g., biopsy or patient fluids or in cell culture or in animal models. The phenotype from the knockdown of targets, such as FOXM1 or BCAM-AKT2 or CDKN2D-WDFY2, may also be assessed in cell culture by measuring cell apoptosis, cell cycle arrest, and cell proliferation. We will use flow cytometry and commercially available kits to measure these variables. We predict that the therapy or combination therapies that result in sustained knockdown of the targets will have the best ability to block cancer growth.

[0127] Off-target effects and cytotoxicity resulting from the knockdown of FOXM1 or BCAM-AKT2 or CDKN2D-WDFY2 will be measured concurrently with the experiments outlined above by transfecting a non-ovarian cancer cell line (i.e., 293T cells). mRNA from cell lysates of OV90, OVCAR8, and/or 293T cells will be used to do microarray analysis or RNAseq to further confirm the lack of off-target effects of the therapies. Cytotoxicity will be measured using cell viability and apoptosis assays. Any potential shRNA candidates that display any deleterious level of off-target effects or extreme cytotoxicity will no longer be pursued in vivo.

[0128] A therapy to further sensitize cancer cells for shRNA treatment will include the co-transfection with MiniVectors-encoding genes that promote apoptosis (e.g., p53, p16, p21, p27, E2F genes, FHIT, PTEN, or CASPASE). In the most basic embodiment, the resulting MiniVector-encoding such genes will have a promoter, the apoptosis-promoting sequence, a terminator, and the hybrid recombination site (attL or attR). The benefit of this approach will be assessed by measuring cell apoptosis, cell cycle arrest, and cell proliferation in OV90 and/or OVCAR8 cells.

[0129] MiniVectors encoding the best shRNA candidates for single and combinatorial therapies against FOXM1 or BCAM-AKT2 or CDKN2D-WDFY2, and with demonstrated efficient knockdown and corresponding phenotype in cell culture, will be pre-clinically screened in vivo in a bioluminescent or fluorescent cell-line xenograft mouse model (mice injected with bioluminescent or fluorescent human high-grade serous ovarian cancer cell lines) to assess cell-death (via apoptosis or another mechanism) or slowdown of cancer cell growth, and also to further optimize and formulate treatment therapies against ovarian cancer.

[0130] Bioluminescent cell lines for xenografting with be generated by stably transfecting ovarian cancer cells (OVCAR8 or OV90) and non-ovarian cancer cells (U2OS) with a vector encoding a Luciferase reporter (pGL4.51) using lipid transfection or any other method of nucleic acid delivery. Selection of cells stably transfected with pGL4.51 will be achieved with the antibiotic G418. Mice will be injected with D-luciferin (the luciferase substrate) by IP injection. Bioluminescence from the luciferase enzyme will allow in vivo imaging to be used to detect and quantify any changes in the size of the tumors.

[0131] Cells stably expressing the Luciferase reporter will be first transfected in vitro, with MiniVectors-encoding the shRNA candidates (using lipid transfection or any other method of nucleic acid delivery), followed by IP injection of the transfected cells to female immunodeficient eight-week athymic nude (Foxn1nu) mice to generate the xenografts. Growth or proliferation of these cancer cells as well as the size of the xenograft in mice will be tracked in real-time by in vivo bioluminescence imaging after IP injection of the D-luciferin substrate. We predict that shRNAs shown to result in efficient knockdown of the target genes will successfully attenuate growth of the tumors, relative to mice xenografted with MiniVectors encoding control shRNA sequences. At the completion of the bioluminescence studies, mice will be sacrificed and dissected to ensure that tumor size corresponds to bioluminescence data.

[0132] Tumors as well as other organs will be harvested for gene expression and histological analysis. Quantitative real-time PCR and western blot will be done in tumor homogenates for validation of the cancer targets in vivo. Histology of the organs will be done to assess cytotoxicity or any off-target effects of the therapy. This will allow us to re-formulate the therapy if needed.

[0133] Bioluminescent ovarian cancer cells will be injected into mice to generate a xenograft mouse model. MiniVectors-will be delivered separately by IP injection and any changes in tumor size will be detected and quantified using bioluminescent imaging and compared to control xenograft mice treated with control MiniVectors. Other routes of in vivo delivery in mice (intranasal, tail vein injection, intramuscular, topical applications to tumors) as well as delivery vehicles (naked DNA, lipofectamine, lipid polymers, etc.) will be tested, and optimized if needed, by measuring tumor reduction and knockdown of the target.

[0134] Dosage, treatment frequency, as well as duration of the therapy will be assessed by measuring and monitoring tumor size and also by measuring knockdown of the target mRNAs, and toxicity.

[0135] Note that if the therapeutic sequence is shRNA, the promoter will likely be U6 or H1 or another promoter recognized by mammalian RNA polymerase III. If said therapeutic sequence is a gene (p53, p16, p21, p27, E2F genes, PTEN, caspase, or another apoptosis inducing gene), the promoter will be CMV, EF1, or another promoter for mammalian RNA polymerase II.

TABLE-US-00004 TABLE1 TherapeuticsequencestobeencodedonMiniVector SEQ Dharmacon IDNO Gene Description Cat.No. MatureAntisense 1. AKT2 RAC-betaserine/threonine- V2LHS_237948 AAATTCATCATCGAAGTAC proteinkinase(geneAKT2) P31751 2. V2LHS_132502 TGACAAAGGTGTTGGGTCG 3. V3LHS_636396 GTGTGAGCGACTTCATCCT 4. V3LHS_646518 TGATGCTGAGGAAGAACCT 5. V3LHS_636398 CATCATCGAAGTACCTTGT 6. V3LHS_636400 TTGATGACAGACACCTCAT 7. V3LHS_325557 TCTTTGATGACAGACACCT 8. ALDH1A1 Retinaldehydrogenase1 V2LHS_112035 TTATTAAAGATGCCACGTG P00352 9. V2LHS_265598 AAAGACAGGAAATTTCTTG 10. V2LHS_112039 ATGTCTTTGGTAAACACTC 11. V2LHS_112037 ATCCATGTGAGAAGAAATG 12. V3LHS_398453 ACTTTGTCTATATCCATGT 13. V3LHS_398455 AATTCAACAGCATTGTCCA 14. AURKB AurorakinaseBQ96GD4 V2LHS_28602 TAAGGGAACAGTTAGGGAT 15. V2LHS_28606 ATGACAGGGACCATCAGGC 16. V2LHS_28601 TTCTCCATCACCTTCTGGC 17. V3LHS_341839 TCAAGTAGATCCTCCTCCG 18. V3LHS_341836 ATGTCTCTGTGAATCACCT 19. V3LHS_341841 TCGATCTCTCTGCGCAGCT 20. V3LHS_341840 AGAGCATCTGCCAACTCCT 21. V3LHS_341837 TTTCTGGCTTTATGTCTCT 22. BCAM BasalCellAdhesionMolecule V2LHS_62437 ATAATGGTCGTGGGTTCCC P50895 23. V2LHS_62435 TTGCAAACACGTTGAGCCG 24. V3LHS_323253 AATCCTCCACTCTGCAGCC 25. V3LHS_323254 TCCGCTGTCTTTAGCTCTG 26. V3LHS_323256 TGAGTGTGACTTCGTCTCC 27. V3LHS_323255 GTGACTTCGTCTCCTTCCC 28. V3LHS_323251 AGAGGTAAGGAAAGCACCT 29. BIRC5 BaculoviralIAPrepeat- V2LHS_94585 ATCAAATCCATCATCTTAC containingprotein5O15392 30. V2LHS_94582 TAAACAGTAGAGGAGCCAG 31. V2LHS_262796 AGCAGAAGAAACACTGGGC 32. V2LHS_262484 TTCCTAAGACATTGCTAAG 33. V2LHS_230582 TCTTGAATGTAGAGATGCG 34. V3LHS_350788 AATTCTTCAAACTGCTTCT 35. V3LHS_350789 TGTTCTTGGCTCTTTCTCT 36. V3LHS_383705 TGAAGCAGAAGAAACACTG 37. V3LHS_383704 GAAGCAGAAGAAACACTGG 38. CCNB1 G2/mitotic-specificcyclin-B1 V3LHS_369356 TTACCATGACTACATTCTT P14635 39. V3LHS_369358 TGCTTGCAATAAACATGGC 40. V3LHS_369355 TAATTTTCGAGTTCCTGGT 41. V3LHS_369360 AAAGCTCTTAGAATCTTCA 42. V3LHS_369359 AGAATCTTCATTTCCATCT 43. CD133 Prominin-1O43490 V2LHS_71816 ATCATTAAGGGATTGATAG 44. V2LHS_71820 TTATACAAATCACCAACAG 45. V2LHS_71818 TAGTAGACAATCTTTAGAC 46. V2LHS_71819 TGTTCTATAGGAAGGACTC 47. V3LHS_407402 TTCATTTTAGAACACTTGA 48. V3LHS_352745 ATAGGAAGGACTCGTTGCT 49. V3LHS_352742 ATAGTTTCAACATCATCGT 50. V3LHS_352743 ATTATTATACAAATCACCA 51. CD44 CD44antigen,Receptorfor V2LHS_111680 TATATTCAAATCGATCTGC hyaluronicacid(HA)P16070 52. V2LHS_111682 ATATGTGTCATACTGGGAG 53. V2LHS_111684 AATGGTGTAGGTGTTACAC 54. V3LHS_334831 AGAGTTGGAATCTCCAACA 55. V3LHS_334830 TGGGTCTCTTCTTCCACCT 56. V3LHS_334834 TGTGCTTGTAGAATGTGGG 57. V3LHS_334832 TGTCTGAAGTAGCACTTCC 58. CDC20 Celldivisioncycleprotein20 V2LHS_112883 TTCCAGATGCGAATGTGTC homologQ12834 59. V2LHS_112884 ATAACTAGCTGGTTCTGTG 60. V3LHS_640507 AACTAGCTGGTTCTGTGCA 61. V3LHS_640508 CAGGTAATAGTCATTTCGG 62. V3LHS_645717 AAACAACTGAGGTGATGGG 63. V3LHS_645716 AATAAAAAACAACTGAGGT 64. V3LHS_640514 ACTTCCAAATAACTAGCTG 65. V3LHS_363298 TCTGCTGCTGCACATCCCA 66. CDKN2D Cyclin-dependentkinase4 V2LHS_262156 AATAAATAGAATCCATTTC inhibitorDP55273 67. V3LHS_401207 ATGAATAACTCATAACTCA 68. V3LHS_310385 CCACTAGGACCTTCAGGGT 69. V3LHS_310386 CGGGATGCACCAGCTCGCG 70. V3LHS_310389 AGGACCTTCAGGGTGTCCA 71. V3LHS_310387 GAACTGCCAGATGGATTGG 72. CDKN3 Cyclin-dependentkinase V2LHS_262397 TATAGTAGGAGACAAGCAG inhibitor3Q16667 73. V2LHS_201585 TGCTTGATGGTCTGTATTG 74. V3LHS_386043 TGATTGTGAATCTCTTGAT 75. V3LHS_386040 ATCTTGATACAGATCTTGA 76. V3LHS_386041 TGATACAGATCTTGATTGT 77. CENPA HistoneH3-likecentromeric V2LHS_150535 ATATGATGGAAATGCCCAG proteinAP49450 78. V2LHS_150534 TATTACCTCTGTTACAGAG 79. V2LHS_150531 TAACACATATTTCTCTTGC 80. V3LHS_403419 AAAGCAACACACACATACT 81. V3LHS_403420 AGACTGACAGAAACACTGG 82. V3LHS_403421 TGTCTCATATATTACCTCT 83. V3LHS_403422 TATCTGAAAATTATTTTCA 84. V3LHS_313522 TTGGGAAGAGAGTAACTCG 85. CIP2A CIP2A(geneKIAA1524) V2LHS_206422 TACTCAATGTCTTTATGTG Q8TCG1 86. V3LHS_308568 TGAATGTGATCTATCAGGA 87. V3LHS_308569 TGTTCTCTATTATCTGACG 88. V3LHS_308565 TTCATTTCATATACATCCA 89. V3LHS_308566 TGAACAGAAAGATTGTGCC 90. FOXM1 ForkheadboxproteinM1 V2LHS_283849 ATAATTAGAGGATAATTTG Q08050 91. V3LHS_396939 ATTGTTGATAGTGCAGCCT 92. V3LHS_396937 TGAATCACAAGCATTTCCG 93. V3LHS_396941 TGATGGTCATGTTCCGGCG 94. V3LHS_396940 AATAATCTTGATCCCAGCT 95. PLK1 Serine/threonine-protein V2LHS_19709 ATTCTGTACAATTCATATG kinasePLK1P53350 96. V2LHS_19711 ATAGCCAGAAGTAAAGAAC 97. V2LHS_241437 TGCGGAAATATTTAAGGAG 98. V2LHS_19708 GTAATTAGGAGTCCCACAC 99. V2LHS_262328 AATTAGGAGTCCCACACAG 100 V3LHS_311459 TTCTTGCTCAGCACCTCGG 101 V3LHS_311462 TTGACACTGTGCAGCTGCT 102 V3LHS_311463 TAGGCACAATCTTGCCCGC 103 PRDM16 PRdomainzincfingerprotein V2LHS_215636 TAAAGCCTCAGAATCTAAG 16Q9HAZ2 104 V2LHS_251390 TAAATTACGACTCTGACAC 105 V3LHS_300082 ATTATTTACAACGTCACCG 106 V3LHS_300078 TTCTCGTCTAAAAGTGCGT 107 V3LHS_300081 AAAAGTGCGTGGTTGTCCG 108 SALL4 Sal-likeprotein4Q9UJQ4 V3LHS_363661 TAGCTGACCGCAATCTTGT 109 V3LHS_363659 TAGTGAACTTCTTCTGGCA 110 V3LHS_363662 TCGGCTTGACTATTGGCCG 111 V3LHS_363664 TTCTGAGACTCTTTTTCCG 112 SLC25A6 ADP/ATPtranslocase3(gene V3LHS_314256 TGTACTTATCCTTGAAGGC SLC25A6)P12236 113 V3LHS_314257 TGCCCGCAAAGTACCTCCA 114 WDFY2 WDrepeatandFYVEdomain- V2LHS_118254 TATCCCACAACTTAATAAC containingprotein2Q96P53 115 V2LHS_118249 TAACCAAACACGAACTGTC 116 V3LHS_405758 ATTGTATGAACAAGTTGGA 117 V3LHS_341295 TTCACAGGAGTCATCTTGT 118 V3LHS_405756 TATATTGTATGAACAAGTT 119 CBCP1 cyclinY V2LHS_243158 ATACTTGGCATAGACACTG 120 V3LHS_314369 TACTGAGGAATATTGTGCT 121 V3LHS_314371 TAATGAAGAGACTCTTGCG

TABLE-US-00005 TABLE2 MiniVectorelements Table2.MiniVectorelements Module Element Description Use A -attL attLfromthe-integrasesystem Recombination -attR attRfromthe-integrasesystem sites(productof -attB attBfromthe-integrasesystem site-specific -attP attPfromthe-integrasesystem recombination loxP loxPsiteforCrerecombinase usedtogenerate -res ressiteforthe (Tn1000)resolvase MiniVector). FRT FRTsiteforFlprecombinase Sequenceslisted hixL hixLsiteforHinrecombinase inTable3. hixR hixRsiteforHinrecombinase Tn3res ressiteforTn3resolvase Tn21res ressiteforTn21resolvase cer cersiteforXerCDsystem psi psisiteforXerCD B Tissue-specificpromoterofalcohol Initiationof dehydrogenase1(ALDH1) transciption. AMY1C Tissue-specificpromoterofhumanamylase Includes alpha1C(AMY1C) promotersfor -actin Promoterfromthe(human)betaactingene RNApolymerase CaMKII Ca2+/calmolulin-dependentproteinkinaseII IIandRNA alphapromoter polymeraseIII. CMV Promoterfromthehumancytobegalovirus(CMV) Fullsequences MiniCMV MinimizedversionofCMV ofselected CAG CMVearlyenhancer/chicken actinpromoter(CAG). promoters Synthetichybridpromotermadefroma)theCMV providedin earlyenhancerelement,b)thepromoter,thefirst Table4. exonandthefirstintronofchickenbeta-actin gene,andc)thespliceacceptoroftherabbit beta-globingene Cyto- Cell-specificpromotersofthehumankeratin18 keratin18 and19genes and19 EF1 Strongexpressionpromoterfromhumanelongation factor1alpha GFAP Tissue-specificpromoteroftheglialfibrillary acidicprotein(GFAP) PromoterfromthehumanpolymeraseIIIRNA promoter Kallikrein Tissue-specificpromoterofthekallikreingene. NFK- Nuclearfactorkappa-light-chain-enhancerof activatedBcells(NF-K) PGK1 Promoterfromhumanormousephosphoglycerate kinasegene(PGK) RSV Longterminalrepeat(LTR)oftheroussarcoma virus(RSV) SV40 Mammalianexpressionpromoterfromthesimian vacuolatingvirus40 UBC PromoterofthehumanubiquitinCgene(UBC) U6 PromoterfromthehumanU6smallnuclearpromoter C shRNA (DNA)sequenceencodingshorthairpinRNA(shRNA) Knockdownof transcript.Sequencesforuseintargetvalidation geneexpression arelistedinTable1.Potentialtherapeutic throughRNA sequenceswillbedesigneddenovoandoptimized interference forknockdownefficiency. miRNA (DNA)sequenceencodingmicro-RNA(miRNA) transcript. lhRNA (DNA)sequenceencodinglonghairpinRNA(lhRNA) transcript lncRNA (DNA)sequenceencodinglongnon-codingRNA (lncRNA)transcript piRNA (DNA)sequenceencodingpiwi-interacting(piRNA) RNAtranscript D Transcriptionalterminatorsequence E S/MAR Scaffold/matrixattachedregionfromeukaryotic Episomal chromosomes(SequencesinTable5) replication F/G -globin Intronofthehuman globingene(130bp) intron HGH Intronofthehumangrowthhormonegene(262bp) Geneexpression intron enhancer H SV40 Simianvirus40earlypromoter(351bp) Nuclear early localization promoter NF-K Bindingsiteofnuclearfactorkappa-light-chain- enhancerofactivatedBcells(55bp(5repeats ofGGGGACTTTCCSEQIDNO122)) p53NLS Bindingsiteoftumorprotein53(p53): AGACTGGGCATGTCTGGGCASEQIDNO123 p53NLS Bindingsiteoftumorprotein53(p53): GAACATGTCCCAACATGTTGSEQIDNO124 Adeno- GGGGCTATAAAAGGGSEQIDNO125 virus majorlate promoter

TABLE-US-00006 TABLE3 CompletesequencesforelementA(recombinationsites) SEQIDNO Site Sequence(5-3) 126. -attL TCCGTTGAAGCCTGCTTT TAAGTTGGCATTATAAAAAAGCATTGC TTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTA TT 127. -attR AGATGCCTCAGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCA TAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGC AAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAG CTTT TAACTTGAGCGAAACG 128. -attB TCCGTTGAAGCCTGCTTT TAACTTGAGCGAAACG 129. -attP AGATGCCTCAGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCA TAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGC AAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAG CTTT TAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTG CAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATT 130. loxP ATAACTTCGTATAGCATACATTATACGAAGTTAT 131. -res ATTTTGCAACCGTCCGAAATATTATAAATTATCGCACACATAAAAACAGTGC TGTTAATGTGTCTATTAAATCGATTTTTTGTTATAACAGACACTGCTTGTCC GATATTTGATTTAGGATACATTTTTA 132. FRT GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC 133. hixL TTCTTGAAAACCAAGGTTTTTGATAA 134. hixR TTTTCCTTTTGGAAGGTTTTTGATAA 135. Tn3res CAACCGTTCGAAATATTATAAATTATCAGACATAGTAAAACGGCTTCGTTTG AGTGTCCATTAAATCGTCATTTTGGCATAATAGACACATCGTGTCTGATATT CGATTTAAGGTACATTT 136. Tn21res GCCGCCGTCAGGTTGAGGCATACCCTAACCTGATGTCAGATGCCATGTGTAA ATTGCGTCAGGATAGGATTGAATTTTGAATTTATTGACATATCTCGTTGAAG GTCATAGAGTCTTCCCTGACAT 137. GGTGCGTACAATTAAGGGATTATGGTAAAT 138. GGTGCGCGCAAGATCCATTATGTTAAAC

TABLE-US-00007 TABLE4 CompletesequencesforelementB(promoters) SEQIDNO Promoter Sequence(5-3) 139. CMV GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGT TCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCG CCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTA TTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGT ACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG GGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT ATAAGCAGAGCT 140. mini-CMV CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT 141. RSV GGTGCACACCAATGTGGTGAATGGTCAAATGGCGTTTATTGTATCGAGCTAG GCACTTAAATACAATATCTCTGCAATGCGGAATTCAGTGGTTCGTCCAATCC ATGTCAGACCCGTCTGTTGCCTTCCTAATAAGGCACGATCGTACCACCTTAC TTCCACCAATCGGCATGCACGGTGCTTTTTCTCTCCTTGTAAGGCATGTTGC TAACTCATCGTTACCATGTTGCAAGACTACAAGAGTATTGCATAAGACTACA TT 142. CAG GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGA CGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGG TGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGG GGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCG GGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGC GCGCGGCGGGCG 143. EF1a GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAA GTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGG GTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTG GGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAA CGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCT GGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCC TGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGG AGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGA GGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGC GCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGAC CTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGA TCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGT GCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGA GAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCT CGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCA CCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCT CAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACA AAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAG TACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGT CGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGA GTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTG GAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAG TGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA 144. EFS ATCGATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTC CCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGT GGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCC CGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT TTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGACGCG 145. Human GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGC -actin GAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCC CGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCC AGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACT GGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGC GATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGG ACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCG GTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTG GGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTG TGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGA ACTGGGGGTTGGGGGGAGCGCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAA TGGAAGACGCTTGTGAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGC ATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAG CTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAA GTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGCGGGGGCGGCAGT TATGGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCCTC GTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCA CCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCG GGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGG AGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCT TAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTG TTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATAT GTAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTT GGCTTTTTTGTTAGAC 146. NFK- GCTAGCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCAGATCT GCCGCCCCGACTGCATCTGCGTGTTCGAATTCGCCAATGACAAGACGCTGGG CGGGGTTTGTGTCATCATAGAACTAAAGACATGCAAATATATTTCTTCCGGG GACACCGCCAGCAAACGCGAGCAACGGGCCACGGGGATGAAGCAGAAGCTTG GCA 147. Ubiquitin-C GTCTAACAAAAAAGCCAAAAACGGCCAGAATTTAGCGGACAATTTACTAGTC TAACACTGAAAATTACATATTGACCCAAATGATTACATTTCAAAAGGTGCCT AAAAAACTTCACAAAACACACTCGCCAACCCCGAGCGCATAGTTCAAAACCG GAGCTTCAGCTACTTAAGAAGATAGGTACATAAAACCGACCAAAGAAACTGA CGCCTCACTTATCCCTCCCCTCACCAGAGGTCCGGCGCCTGTCGATTCAGGA GAGCCTACCCTAGGCCCGAACCCTGCGTCCTGCGACGGAGAAAAGCCTACCG CACACCTACCGGCAGGTGGCCCCACCCTGCATTATAAGCCAACAGAACGGGT GACGTCACGACACGACGAGGGCGCGCGCTCCCAAAGGTACGGGTGCACTGCC CAACGGCACCGCCATAACTGCCGCCCCCGCAACAGACGACAAACCGAGTTCT CCAGTCAGTGACAAACTTCACGTCAGGGTCCCCAGATGGTGCCCCAGCCCAT CTCACCCGAATAAGAGCTTTCCCGCATTAGCGAAGGCCTCAAGACCTTGGGT TCTTGCCGCCCACCATGCCCCCCACCTTGTTTCAACGACCTCACAGCCCGCC TCACAAGCGTCTTCCATTCAAGACTCGGGAACAGCCGCCATTTTGCTGCGCT CCCCCCAACCCCCAGTTCAGGGCAACCTTGCTCGCGGACCCAGACTACAGCC CTTGGCGGTCTCTCCACACGCTTCCGTCCCACCGAGCGGCCCGGCGGCCACG AAAGCCCCGGCCAGCCCAGCAGCCCGCTACTCACCAAGTGACGATCACAGCG ATCCACAAACAAGAACCGCGACCCAAATCCCGGCTGCGACGGAACTAGCTGT GCCACACCCGGCGCGTCCTTATATAATCATCGGCGTTCACCGCCCCACGGAG ATCCCTCCGCAGAATCGCCGAGAAGGGACTACTTTTCCTCGCCTGTTCCGCT CTCTGGAAAGAAAACCAGTGCCCTAGAGTCACCCAAGTCCCGTCCTAAAATG TCCTTCTGCTGATACTGGGGTTCTAAGGCCGAGTCTTATGAGCAGCGGGCCG CTGTCCTGAGCGTCCGGGCGGAAGGATCAGGACGCTCGCTGCGCCCTTCGTC TGACGTGGCAGCGCTCGCCGTGAGGAGGGGGGCGCCCGCGGGAGGCGCCAAA ACCCGGCGCGGAGGC 148. SV40 GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCA TCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCC TAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCC CCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCC TCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTT GCAAA 149. PGK CCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTC TACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGC AGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACA GCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCA GCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGG GGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCG GAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTC CTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCC 150. H1 AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGT CTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACAGATCCC 151. U6 GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGT GGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCC TAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTT TAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAA ATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTG TAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTG TT

TABLE-US-00008 TABLE5 CompletesequencesforelementsE,FandG(accessorysequences) SEQIDNO Element Sequence(5-3) 152. 250bpS/MAR TCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAG AATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTT AGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATAT TTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATAT ATTTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAAT ATATTTAGAA 153. 439bpS/MAR TCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAG AATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTT AGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATAT TTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATAT ATTTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAAT ATATTTAGAA 154. (45bp)TypeA GGTGCATCGATGCAGCATCGAGGCAGGTGCATCGATACAGGGGGG Cpgmotif 155. (24bp)TypeB TCGTCGTTTTGTCGTTTTGTCGTT Cpgmotif 156. (21bp)TypeC TCGTCGAACGTTCGAGATGAT CpGmotif 157. -globinintron GTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTG GGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACT CTCTCTGCCTATTGGTCTATTTTCCCACCCTTAG 158. Humangrowth TTCGAACAGGTAAGCGCCCCTAAAATCCCTTTGGGCACAATGTGTCCT hormoneintron GAGGGGAGAGGCAGCGACCTGTAGATGGGACGGGGGCACTAACCCTCA GGTTTGGGGCTTCTGAATGTGAGTATCGCCATGTAAGCCCAGTATTTG GCCAATCTCAGAAAGCTCCTGGTCCCTGGAGGGATGGAGAGAGAAAAA CAAACAGCTCCTGGAGCAGGGAGAGTGCTGGCCTCTTGCTCTCCGGCT CCCTCTGTTGCCCTCTGGTTTC

TABLE-US-00009 TABLE 6 cancers and suitable test cell lines Breast cancer: MDA-MB 231, SKBR3, SUM 102, SUM 149, and MCF-7. Cervical cancer: Nos NC104, NC105, HeLa, SiHa, CasKi, C33A, and C4-1. Colorectal cancer: LoVo, SW480, and SW116. Stomach cancer: NCI-N87, AGS, HTB 103, HTB 135, SNU1, SNU16, Sk-GT5, BGC-823, HGC-27, and KATO-III. Glioma: Hs683, U118MG, LN 229, U87 MG, HF-U251 MG, SW1783, and U87 MG. Pancreatic cancer: AsPC-1, BxPC-3, COLO-357, HPAC, L3.6PI, MIAPaCa, PANC1, CaPan-1, MiaPaca- 2, MDAPanc-28, and MDAPanc-48. Prostate cancer: PC-3, DU145, C4-2B, and LNCaP. Kidney/renal cancer: 786-O and Caki-1. Liver cancer: SK-Hep1, MHCC-LM3, and SMMC-7721. Lung cancer: A549 and H1299. Ovarian cancer: OVCAR8 and OV90. Prostate cancer: PC3, DU145, and LNCaP. Chondrasarcoma: HS-819.T, SW1353. Dermatofibrosarcoma: Hs295.Sk, Hs357.T, Hs63.T. Ewing sarcoma: SK-N-MC, TC-268, CHP-100S, CHP-100L, IMR-32, SK-ES1, ES4, WE68, RD-ES, HS863.T, HS822.T. Fibrosarcoma: FC83.Res. Giant cell sarcoma: Hs706.T, Hs737.T, Hs821.T, Hs127.T. Leiomyosarcoma: HS5T, SK-LMS-1, DDT1 MF-2. Liposarcoma: SW872. Lung sarcoma: Hs57.T, LL86 Lymphosarcoma: LB9.Bm, BL3.1. Osteosarcoma: SK-ES-1. Pagetoid sarcoma: Hs 925.T. Reticulum cell sarcoma: J774A.1, HS324.T. Rhabdomyosarcoma: TE381.T, TE441.T, RD, A-673, HS729, A-204, SJCRH30, TE159.T. Uterine sarcoma: MES-SA, MES-SA/DX-5, MES-SA/MX2. Lung sarcoma: HS-57.T, MiCl1 (S + L) Other Sarcomas: FB2.K, DoCl1, CV-1, QNR/D, QNR/K2, 10.014 pRSV-T, 2.040pRSV-T, XMMCO-791, Hs925.Sk, Hs707(B).Ep, Sarcoma 180, EHS.

TABLE-US-00010 TABLE 7 Other Gene Therapy Targets Target Type of cancer AKAP12 Glioma AKT Sarcomas AKT1 Lung cancer ALK Glioma Lung cancer AR Prostate Cancer ARID1A Cervical Cancer, Liver cancer ARID2 Liver cancer AXIN1 Liver cancer AXL Kidney/Renal Cancer BARD1 Glioma BASP1 Stomach Cancer BCAR4 Cervical, Colorectal, Stomach Cancer Bcl-2 Prostate Cancer BCL-9 Liver cancer BIRC5 (Survivin) Cervical Cancer BLM Glioma Bmi1 Stomach Cancer BMP2 Glioma BRAF Stomach. Lung cancer Glioma BRCA1 Breast Cancer, Glioma BRCA2 Breast Cancer BRIP1 Glioma BUB1 Glioma c-met Prostate Cancer c-Myc Stomach Cancer CASP8 Cervical Cancer CAV1 Kidney/Renal Cancer CAV2 Kidney/Renal Cancer CBX7 Stomach Cancer CCNA2 Glioma CCND1 Liver, Lung cancer CCND2 Glioma CD274 (also known as PD-L1) Cervical Cancer CD44 Stomach Cancer CDC20 Glioma Cdc42 Stomach Cancer CDCA5 Prostate Cancer CDCA8 Prostate Cancer CDH1 Stomach Cancer CDK1 Glioma CDK2 Glioma CDK4 Glioma CDKN2A Liver cancer CDT-1 Liver cancer CDX1 Stomach Cancer CDX2 Stomach Cancer CEP55 Glioma CHEK1 Glioma COX-2 Stomach Cancer CTLA4 Liver cancer CTNNB1 aka Beta Catenin Liver cancer CyclinD2 Stomach Cancer DAB2IP Glioma DHX57-TMEM178-MAP4K3 Glioma DNMT Liver cancer DTMT1 Stomach Cancer DUSP26 Glioma E2F1 Glioma E2F2 Prostate Cancer ECOP Stomach Cancer EGF Liver cancer EGFR Cervical, Colorectal, Lung Cancer, Glioma EGFR-PSPH Glioma EGFR-SEPT14 Glioma EGR1 Prostate Cancer EML4-ALK Lung cancer EMSY Breast Lung Cancer EpCAM Breast Cancer ERBB3 Cervical Cancer ERG Prostate Cancer ERK Liver cancer Sarcomas Estrogen receptor (ER) Breast cancer FANCD2 Glioma Fat Specific Protein 27 aka DFFA-like Liver cancer effector aka CIDEC FGF19 Liver cancer FGF5 Glioma FGFR1 Breast Cancer FGFR1-4 Liver cancer FGFR1-TACC1 Glioma FGFR3-TACC3 Glioma FOXO1 Stomach Cancer G12C Lung cancer G12V Lung cancer GNAI1 Kidney/Renal Cancer GNAO1 Kidney/Renal Cancer GPSM2 Kidney/Renal Cancer GRPEL 1 Liver cancer HDAC Liver cancer HDAC Sarcomas HER-2 Breast, Glioma, Liver, Lung cancer HEYL Glioma HGESS Sarcomas HGF Glioma HIP1 Prostate cancer hK2 Prostate Cancer HLA-A Cervical Cancer HMGB2 Glioma HOXA1 Cervical Cancer ID4 Glioma IDH Glioma, Sarcomas IRF2 Liver cancer IRX5 Glioma ITGB3 Glioma JAK1 Liver cancer JAK2 Stomach Cancer KEAP1 Liver cancer KIT Liver cancer KRAS Breast, Cervical, Colorectal, Stomach, Lung Cancer LIMD1 Glioma LIN9 Glioma LKB1 Cervical Cancer LKB1 Lung cancer LPHN2 Stomach Cancer MAFG Stomach Cancer MAST1 Ovarian cancer Mcl-1 Stomach Cancer MDM2 Sarcomas MDM4 Lung cancer MeCP2 Stomach Cancer MEF2C Glioma MEIS2 Glioma MEK Cervical, Liver Cancer, Glioma, Sarcomas MEK1 Lung cancer MET Glioma, Kidney/Renal Cancer, Liver cancer, Lung cancer MET fusions: Glioma TFG-MET CLIP2-MET PTPRZ1(exon1)-MET PTPRZ1(exon2)-MET PTPRZ1(exon8)-MET MFSD2A Glioma mir-125b, mir-145, mir-21, and mir-155. Breast Cancer miR-203 Cervical Cancer miR-21, miR-17-5p, miR-191, miR-29b-2, Colorectal Cancer miR-223, miR-128b, miR-24-1, miR-24-2, miR-155 miR-21, miR-17-5p, miR-191, miR-29b-2, Prostate Cancer miR-223, miR-128b, miR-199a-1, miR-146, miR-181b-1 miR-21, miR-17-5p, miR-191, miR-29b-2, Pancreatic Cancer miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-181b-1 mlR-21, miR-17-5p, miR-191, miR-128b, miR-199a-1, Lung cancer miR-155 miR-21, miR-17-5p, miR-29b-2, miR-146, miR-155, miR- Breast Cancer 181b-1 miR-21, miR-191, miR-223, miR-24-1, miR-24-2 Stomach Cancer miR-221-3p Cervical Cancer miR-30b Cervical Cancer miR122 Liver cancer MN/CA9 Cervical Cancer mTOR Kidney/Renal Cancer mTOR, mTORC Sarcomas MYBL2 Glioma Myc Prostate Cancer NET1 Glioma NF1 Glioma NFE2L2 Liver cancer NOTCH4 Stomach Cancer NRAS Lung cancer NR3C4 (Androgen receptor) Prostate cancer NT53 Glioma NTRK fusions: Glioma TPM3-NTRK1 BTBD1-NTRK3 ETV6-NTRK3 VCL-NTRK2 AGBL4-NTRK2 OLFM4 Stomach Cancer p53 Sarcomas, Ovarian cancer Parp + CDK12, EWS/FLI, Sarcomas PCDH10 Glioma, Breast, Colorectal, Stomach, Kidney/Renal Cancer Liver cancer PD-L1 Stomach. Kidney/Renal. Liver Cancer PD1 Cervical Cancer PDCD1LG2 (also known as PD-L2), Cervical Cancer PDGF Sarcomas PDGFR Glioma, Liver cancer PDGFR(Alpha), PDGFR(Beta) Liver cancer PDL-1 Breast, Colorectal, Lung Cancer PHF10 Stomach Cancer PIP2 Sarcomas PIP3 Sarcomas PI3K Sarcomas PL3KCA Cervical, Liver Cancer PLAS3 Stomach Cancer PLK1 Glioma PLK3CA Lung cancer Prohibitin Stomach Cancer PTEN Cervical, Lung, Stomach, Liver Cancer PTPN3 Glioma PTPRZ1-MET Glioma PTTG1 Glioma, Prostate Cancer RAB40C Stomach Cancer RAF Liver cancer Raf Sarcomas RARB Cervical Cancer Ras Stomach Cancer RAS Liver cancer, Sarcomas RB Sarcomas RB1 Glioma RBL1 Glioma RECK Stomach Cancer RELA fusion Glioma RET Lung cancer RMBXL1 Stomach Cancer ROS-1 Glioma Lung cancer RPS6KA3 Liver cancer RRM2B Liver cancer RTK Colorectal Cancer, Sarcomas RUNX3 Stomach Cancer S-100A9 Liver cancer SHKBP1 Cervical Cancer SLC52A2 Liver cancer SMARCD1 Stomach Cancer SOX2 Stomach Cancer SPHK1 Stomach Cancer STAP-2 Prostate Cancer STARD13 Glioma STAT3 Breast cancer STMN1 Stomach Cancer STX6 Liver cancer TBX2 Glioma TERT Liver cancer TF Prostate Cancer TFP12 Glioma TGF-BetaR Liver cancer TGFBR2 Cervical Cancer THBS2 Cervical Cancer TIE2 Liver cancer TK1 Prostate Cancer TMEFF2 Glioma TMPRSS2-ERG Prostate Cancer TP53 Liver cancer TP53 Lung cancer UBE2C Prostate Cancer VEGF Colorectal Cancer, Glioma, Sarcomas VEGFA Liver cancer VEGFR Kidney/Renal Cancer VEGFR1-4 Liver cancer WDR79 Breast Cancer WDR79 Colorectal Cancer WDR79 Lung cancer YWAE/FAM22A/B Sarcomas ZEB1/ZEB2 Stomach Cancer ZFHX3 Glioma ZXH2 Kidney/Renal cancer

TABLE-US-00011 TABLE8 OtherOvarianCancerTargets(someincludingtargetsequences) SEQIDNO Target Detail/TargetingSequence 4EBP1 shRNAagainsthuman4E-BP1:(hshBP1)(sigma:TRCN0000040203): 159. CCGGGCCAGGCCTTATGAAAGTGATCTCGAGATCACTTTCATAAGGCCTGGCTTTTTG 160. AKT AKT1TRCN0000010174GGACTACCTGCACTCGGAGAA 161. AKT2TRCN0000000564CTTCGACTATCTCAAACTCCT 162. AKT3TRCN0000010187CTGCCTTGGACTATCTACATT ANG1/ANG2andTie,ATM,AXL,BCL-2,BET,bFGF,BRAF,BRIPI,CDC42,c-KIT CD40,CD184,CDCP1,CHK1,TNNB1,CXCL1akaFractalkine,CX.sub.3CR1aka Fractalkine,CXCR4 163. DXL1 ThetargetingsequenceforDlx1was5-AACCGGAGGTTCCAACAAACT-3 164. (sensestrand:5-CCGGAGGTTCCAA-CAAACTTT-3; 165. antisensestrand:5-AGTTTGTTGGAACCTCCGGTT-3) EGFR 166. elF4E shRNA1 5-CACCGCCAAAGATAGTGATTGGTTATTTCAAGAGAATAACCAATCACTATCTTTGGTTTTTT G-3 167. shRNA2 5-CACCGGAGGACGATGGCTAATTACATTCAAGAGATGTAATTAGCCATCGTCCTCCTTTTTT G-3 168. shRNA3 5-CACCGTGGCGCTGTTGTTAATGTTATTCAAGAGATAACATTAACAACAGCGCCACTTTTTT G-3 EMSY,ERBB1,Erb2,ETS1,EZH2,FAK,FAS,FER,FGF,FOS,FR-alpha,Flt3, FRA,GAB1,GADD45B,Grb2,HER2,HER3,HER4,ICOS,IDO,IGF,IGF-2, Insulin,IGF-1R,IR,IL-6,JAK1,JAK2,JAK3,JAK4,KLF6,KRAS,MAPK 169. March7 shRNA1forMARCH7(NM_022826)AAGTGCTAGGATGATGTCTGGAA 170. shRNA2forMARCH7(NM_022826)AAGAACAGATTCCTCTATTAGTA 171. shRNA3forMARCH7(NM_022826)AAGATCTAGTCAGGATTCCTTGA 172. shRNA4forMARCH7(NM_022826)AAGAGATGAATCTTCAAGGATAC MAST1,MDM2,MEK,MET,MMP9 173. mTOR V2LHS_262100MatureAntisense:TAGGAGGCAGCAGTAAATG;manyothers listedfromcompany NOX1,OX40,PAK1,PARP,PDGF,PD-1,PDL1,PICT-akaGLTSCR2 174. PI3K shRNA-1(ccggccacttatgctttaccttctactcgagtagaaggtaaagcataagtggttttt) TRCN0000002228, 175. shRNA-2(ccgggctagtgtgaaggtctccattctcgagaatggagaccttcacactagcttttt) (TRCN0000002229 176. shRNA-3(ccggcaaagaagtatggaacgagtactc-gagtactcgttccatacttctttgttttt) TRCN0000002231, 177. shRNA-4(ccggcgagcagtagatcaataattctcgagaattacttgatctactgctcgttttt) TRCN0000002230 PIK3CA 178. PRAS40 TheshRNAsequenceofPRAS40wasGCTGAGTTCTAAGCTCTAA(sense) PRMT5,Raf,RAS,RB,Ror1,Ror2,Shp2,SLIT3,STAT,SYK,TGFalpha,TLR4, TNF-alpha,TNF,TOP1,TOP2,TP53,TRAIL,TYK2 179. USP7 USP7(GCGATTACAAGAAGAGAAA)throughDhamracon 180. USP15 shRNA-1 5-CCGGCCGTAATCAATGTGGGCCTATCTCAGATAGGCCCACATTGATTACGGTTTTT-3; VEGF,WDR77,WDR79,WEE1,XIAP,Y1349

TABLE-US-00012 TABLE 9 List of Cancer Gene Targets. Gene Chromosome Locus ABCB1 7 7q21.1 ACVR1B 12 12q13 AGTR1 3 3q21-q25 AKT1 14 14q32.32 AKT2 19 19q13.1-q13.2 ALOX12 17 17p13.1 ALOX5 10 10q11.2 ALOX5AP 13 13q12 ANG 14 14q11.1-q11.2 ARHC 1 1p21-p13 ARMET 3 3p21.1 B3GALT5 21 21q22.3 BAG3 10 10q25.2-q26.2 BAG4 8 8p22 BAX 19 19q13.3-q13.4 BHLHB2 3 3p26 BIRC5 17 17q25 BRCA2 13 13q12.3 CCK 3 3p22-p21.3 CCKAR 4 4p15.1-p15.2 CCKBR 11 11p15.4 CCND3 6 6p21 CD44 11 11p13 CD9 12 12p13 CDH1 16 16q22.1 CDK4 12 12q14 CDKN1A 6 6p21.2 CDKN2A 9 9p21 CDKN2B 9 9p21 CLDN4 7 7q11.23 DAB2 5 5p13 DCC 18 18q21.3 DUSP6 12 12q22-q23 EGF 4 4q25 EGFR 7 7p12 EP300 22 22q13.2 EPHB2 1 1p36.1-p35 ERBB2 17 17q21.1 EREG 4 4 FGFR1 8 8p11.2-p11.1 FHIT 3 3p14.2 FRAP1 1 1p36.3-p36.2 GAS 17 17q21 GLRX 5 5q14 GNG7 19 19p13.3 GPI 19 19q13.1 GUSB 7 7q21.11 HK2 2 2p12 HPSE 4 4q21.3 ID2 2 2p25 IL8 4 4q13-q21 IRS1 2 2q36 IRS2 13 13q34 KAI1 11 11p11.2 KRAS2 12 12p12.1 KRT20 17 * MADH4 18 18q21.1 MADH6 15 15q21-q22 MAP2K4 17 17p11.2 MAP3K1 5 5 MAPK14 6 6p21.3-p21.2 MDM2 12 12q14.3-q15 MADH7 18 18 MEN1 11 11q13 MET 7 7q31 MMP11 22 22q11.23 MMP2 16 16q13-q21 MMP3 11 11q22.3 MMP7 11 11q21-q22 MTA1 * * MUC1 1 1q21 MYC 8 8q24.12-q24.13 NFKB ** ** NME1 17 17q21.3 NTRK1 1 1q21-q22 NTSR1 20 20q13-20q13 P8 16 * PCD1 13 13q21.33 PLAUR 19 19q13 PPARG 3 3p25 PRG1 19 19q13.2 PRKCA 17 17q22-q23.2 PSCA 8 8q24.2 PTGS2 1 1q25.2-q25.3 RABIF 1 1q32-q41 RAD51 15 15q15.1 RB1 13 13q14.2 RELA 11 11q13 RNASE1 14 14 RPS6KB2 11 11cen-q12.1 SLC16A7 12 12q13 SCYB14 5 5q31 SDC1 2 2p24.1 SERPINB5 18 18q21.3 SERPINI2 3 3q26.1-q26.2 SLC2A1 1 1p35-p31.3 SPINT2 19 19q13.1 SST 3 3q28 STK11 19 19p13.3 TDG 12 12q24.1 TEM7 17 * TFF1 21 21q22.3 TFF2 21 21q22.3 TGFA 2 2p13 TGFB1 19 19q13.1 TSFBR1 9 9q22 TGFBR2 3 3p22 TIMP1 x xp11.3-p11.23 TJP1 15 15q13 TM4SF5 17 17p13.3 TMPRSS4 11 11q23.3 TNFRSF6 10 10q24.1 TP53 17 17p13.1 TSLC1 * * TXN 9 9q31 UCHL1 4 4p14 VEGF 6 6p12 VEGFC 4 4q34.1-q34.3 ZNF146 19 19q13.1

[0136] The following references are incorporated by reference in their entirety for all purposes. [0137] ATCC Sarcoma Cell Lines. atcc. org//media/PDF s/Cancer %20and %20Normal %20cell %20lines %20tables/Sarcoma %20cel 1%20lines.ashx [0138] Catanese, D. J., et al., Supercoiled MiniVector DNA resists shear forces associated with gene therapy delivery, Gene Ther. 19(1): 94-100 (2012). [0139] Chan D. W., et al., Overexpression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis, J. Pathol. 215(3): 245-252 (2008). [0140] Darquet A. M., et al., Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer, Gene Ther., 6: 209-218 (1999). [0141] Douard R., et al., Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer, Surgery. 139(5): 665-670 (2006). [0142] ES4: Cellosaurus, web.expasy.org/cellosaurus/CVCL_1200. [0143] Fogg, J. M., et al., Exploring writhe in supercoiled minicircle DNA. J. Phys.-Condes. Matter, 18: S145-S159 (2006). [0144] Hardee, C. L., Advances in Non-Viral DNA Vectors for Gene Therapy, Genes 8, 65 (2017) [0145] Hornstein, B. D., et al., Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells, PLoS One. 11(12): e0167537 (2016). [0146] Jaiswal N., et al., Biology of FOXM1 and its emerging role in cancer therapy, J. Proteins Proteomics 5(1): 1-24 (2014). [0147] Kalin T. V., et al., Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice, Cancer Res. 66(3): 1712-1720 (2006). [0148] Kalinichenko V. V., et al., Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor, Genes Dev., 18(7): 830-850 (2004). [0149] Kannan K., CDKN2D-WDFY2 is a cancer-specific fusion gene recurrent in high-grade serous ovarian carcinoma, PLoS Genet. 10(3):e1004216 (2014). [0150] Kannan K., et al., Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma, P.N.A.S. USA. 17; 112(11):E1272-7 (2015). [0151] Kelleher F. C. and O'sullivan H., FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways, Oncotarget 7(27): 42792-42804 (2016). [0152] Kim I. M., et al., The Forkhead Box ml transcription factor stimulates the proliferation of tumor cells during development of lung cancer, Cancer Res. 66(4): 2153-2161 (2006). [0153] Kim J., et al., High-grade serous ovarian cancer arises from fallopian tube in a [Dicer-Pten double-knockout] mouse model, P.N.A.S. USA. 109(10):3921-6 (2012). [0154] Liu M., et al., FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells, Cancer Res. 66(7): 3593-3602 (2006). [0155] Lokody, I., Signalling: FOXM1 and CENPF: co-pilots driving prostate cancer, Nature Reviews Cancer 14: 450-451 (2014). [0156] Mitsiades, N., Fas-Mediated Apoptosis in Ewing's Sarcoma Cell Lines by Metalloproteinase Inhibitors, Journal of the National Cancer Institute, Volume 91, Issue 19: 1678-1684 (1999). [0157] SK-ES-1: Human Ewing Sarcoma Cell Line, Memorial Sloan Kettering Cancer Center, https://www.mskcc.org/research-advantage/support/technology/tangible-material/sk-es-1-human-ewing-sarcoma-cell-line [0158] Teh M. T., et al., FOXM1 is a downstream target of Gli1 in basal cell carcinomas, Cancer Res., 62(16): 4773-4780 (2002). [0159] US20150376645, US20140056868, 61/653,279, filed May 30, 2012, Supercoiled MiniVectors as a tool for DNA repair, alteration and replacement [0160] Wang Z., et al., Down-regulation of Forkhead Box M1 transcription factor leads to the inhibition of invasion and angiogenesis of pancreatic cancer cells, Cancer Res. 67(17): 8293-8300 (2007). [0161] WE68: Cellosaurus, web.expasy.org/cellosaurus/CVCL_9717. [0162] Wei, Ping et al., FOXM1 promotes lung adenocarcinoma invasion and metastisis by upregulating snail, International Journal of Biological Sciences 11(2): 186-198 (2015). [0163] Wonsey D. R., et al., Loss of the forkhead transcription factor FoxM1 causes centrosome amplification and mitotic catastrophe, Cancer Res., 65(12): 5181-5189 (2005). [0164] Zhang X, et al., Knockdown of CIP2A sensitizes ovarian cancer cells to cisplatin: an in vitro study, Int. J. Clin. Exp. Med. 8(9):16941-16947 (2015). [0165] Zhang X. et al., A novel FOXM1 isoform, FOXM1D, promotes epithelial-mesenchymal transition and metastasis through ROCKs activation in colorectal cancer, Oncogene 36: 807-819 (2017). [0166] Zhao N., et al., Transfection of shRNA-encoding MiniVector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells, Gene Ther. 18(3):220-4 (2011). [0167] Zona S., et al., FOXM1: An emerging master regulator of DNA damage response and genotoxic agent resistance, Biochim Biophys Acta., 1839(11): 1316-1322 (2014). [0168] U.S. Pat. Nos. 8,460,924, 8,729,044, 9,267,150, US20110160284, US20120302625, US20130316449, 61/252,455, filed Oct. 16, 2009, Supercoiled MiniVectors for gene therapy applications [0169] U.S. Pat. No. 7,622,252, US20070020659, 60/689,298, filed Jun. 10, 2005, Generation of minicircle DNA with physiological supercoiling [0170] US20060211117 Methods of making minicircles [0171] WO1994009127 Supercoiled minicircle DNA as a unitary promoter vector [0172] WO2002083889 Methods for the production of minicircles [0173] 62/470,997, Mar. 14, 2017, TARGETING MINIVECTORS TO SPECIFIC TISSUE USING SHAPE [0174] IMPROVING NON-VIRAL GENE DELIVERY: POLYMER CARRIERS FOR SPATIAL AND TEMPORAL CONTROL OF NUCLEIC ACID RELEASE, Abbygail A. A. Foster, University of Delaware Chemical Engineering Ph.D. thesis dissertation, Spring 2014 [0175] Ramamoorth M., & Narvekar, A., Non Viral Vectors in Gene TherapyAn Overview, J. Clinical & Diagnostic Res. 2015 January, Vol-9(1): GE01-GEO6. [0176] Hidai C., & Kitano, H., Nonviral Gene Therapy for Cancer: A Review, Diseases 2018, 6, 57. [0177] Lijun Qian et al, The present and future role of ultrasound targeted microbubble destruction in preclinical studies of cardiac gene therapy, J. Thoracic Dis., 2018; 10(2):1099-1111.