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.
Description
FIELD OF THE DISCLOSURE

The disclosure generally relates to methods of treating ovarian cancer using gene therapy and methods and combinations of methods to deliver gene therapy. It also relates to methods of making DNA MiniVectors and compositions comprising MiniVectors useful in treating ovarian and similar cancers having similar P53 and/or FOXM1 effects.


BACKGROUND OF THE DISCLOSURE

Ovarian cancer refers to any cancerous growth that occurs in the ovary. According to the United States National Cancer Institute, ovarian cancer is the 8th most common cancer among women in the United States (excluding non-melanoma skin cancers). However, it is the 5th most common cause of cancer deaths in women. Each year, more than 22,000 women in the U.S. are diagnosed with ovarian cancer and around 14,000 will die. Tragically, the overall 5-year survival rate is only 46 percent in most developed countries (and survival rate is lower for more advanced stages). However, according to the National Cancer Institute, if diagnosis is made early, before the tumor has spread, the 5-year survival rate is 94 percent.


In addition to the long used chemical and radiological treatments for ovarian cancer, gene therapy is now of clinical interest in treating ovarian cancers, albeit still in its infancy. At least one clinical trial is studying insertion of the p53 gene into a person's cancer cells, which hopefully will improve the body's ability to fight cancer or make the cancer cells more sensitive to treatment. Another group has studied the use of carbonyl reductase 1 (CBR1) overexpression in a mouse model, in which DNA was delivered to ovarian cancer cells via a polyamidoamine (PAMAM) dendrimer and initial results were promising.


One of the most important objectives in gene therapy is the development of highly safe and efficient vector systems for gene transfer to eukaryotic cells. Initially, viral-based vector systems were used, most commonly retroviruses or adenoviruses, to deliver the desired gene. Other viruses used as vectors include adeno-associated viruses, lentiviruses, pox viruses, alphaviruses, and herpes viruses. The main advantage of virus-based vectors is that viruses have evolved to physically deliver a genetic payload into cells and this can be readily exploited. The efficiency of delivery into cells is therefore generally higher than non-viral delivery methods, e.g., plasmid DNAs.


Viral-based vectors can have disadvantages, however. Viruses can usually infect more than one type of cell and can infect healthy cells as well as diseased cells. Another danger is that the new gene might be inserted in the wrong location in the genome, possibly causing cancer or other problems. This has already occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients. In addition, there is a small chance that viral DNA could unintentionally be introduced into the patient's reproductive cells, thus producing changes that may be passed on to children. Another concern is the possibility that transferred genes could be overexpressed, producing so much of the added gene product as to be harmful. Moreover, the viral vector could cause an immune reaction or could be transmitted from the patient to other individuals or even into the environment. Use of viruses is also burdened with concerns of subsequent virus mutation and reactivation. Perhaps most important, most viral vectors can often only be delivered once because of developed immunity; subsequent deliveries produce a strong immune response.


Plasmids could potentially be used instead of viral-based vectors. Plasmids are far less efficient at entering cells than viruses, but have utility because they are straightforward to generate and isolate. In fact, clinical trials using intramuscular injection of “naked” DNA plasmid have occurred with some success. Unfortunately, transfection of plasmids as well as expression from plasmids has been low in comparison to viral vectors-too low to affect disease in many cases.


Numerous studies have shown that the bacterial backbone in plasmids may elicit immune responses as well as cause reduction of transgene expression. Furthermore, the introduction of antibiotic resistance genes, often encoded on plasmids for propagation, is not allowed by some government regulatory agencies. Because of these issues, smaller DNA vectors, such as minicircles and MiniVectors, were developed. These non-viral DNA vectors are small (typically ≤5 kilobase pairs (kb) circular plasmid derivatives that are almost completely devoid of bacterial sequences including the genes for selection (often antibiotic resistance genes) and origins of DNA replication). They have been used as transgene carriers for the genetic modification of mammalian cells, with the advantage that, since they contain no bacterial DNA sequences, they are less likely to suffer from the well documented silencing of transgene expression that often occurs when the transgene is carried on a plasmid containing long bacterial sequences and are also less likely to elicit an immune response. Several studies have demonstrated that minicircles and MiniVectors are safe—they are episomal vectors that enhance transgene expression extent and duration in vivo and in vitro relative to plasmids.


The use of small vectors less than 1,000 bp is highly promising, but vectors of this small length were initially difficult to produce and purify in significant quantity. Site-specific recombination is inhibited when the recombination sites are closely spaced, and intermolecular recombination between sites on two separate plasmids becomes more favorable than bending such a short sequence on the same plasmid, leading to multimeric products (Fogg 2006). An alternative approach commonly used by experimentalists is the circularization of linear DNA molecules via ligation to form minicircles. However, yields are very low and intermolecular ligation contaminants are prevalent when the linear DNA molecules short enough to generate minicircles are used.


U.S. Pat. No. 7,622,252 overcame the problem of MiniVector yield and purity by transforming the plasmid into a cell suitable for site-specific recombination to occur, under conditions such that topoisomerase IV decatenation activity is inhibited, thereby producing catenated DNA circles wherein at least one of the circles in each catenane is a supercoiled DNA minicircle of less than about 1 kb in size.


MiniVectors are minimized, non-viral DNA vectors similar to minicircles but with some important differences. Like minicircles, MiniVectors are synthesized from a parent plasmid via site-specific recombination. Encoding only the genetic payload and short integration sequences, MiniVectors can be engineered as small as ˜250-350 base pairs (bp) and generated in high yields (in comparison, the smallest reported minicircle length is 650 bp although the yields of minicircles of that length are unreported). As before, unwanted bacterial sequences are on a discarded miniplasmid. The recombination and purification system used to make MiniVectors is highly optimized, resulting in as much as 100-fold less plasmid contamination than is recommended by health regulatory agencies (0.015%). MiniVector preparation usually follows the basic procedure shown in FIG. 1.


The different DNA species in the MiniVector purification process are typically engineered to be of sufficiently different lengths to be readily separated by size-exclusion chromatography (gel-filtration). This step is a unique and major advantage of the MiniVector system and enables the recovery of a highly pure preparation of MiniVector. By contrast, a minicircle, although similarly made initially, cannot be made as small as a MiniVector and is typically less pure, carrying along up to 10% of plasmid and other circle contaminants in the final product, which is a yield well above the 1.5% allowed by some health regulatory agencies.


What is needed in the art are better methods of conducting gene delivery, especially delivery of sequences specific for treating ovarian cancers. We are now using the materials and methods described in U.S. Pat. No. 7,622,252 to develop and test a variety of sequences for this use.


SUMMARY OF THE DISCLOSURE

This application focuses on the treatment of ovarian cancer and improving the survival rate of patients by creating MiniVectors to specifically target key ovarian cancer targets and pathways. Below are some of the ovarian cancer targets for which we have data from cell culture and/or animal models testing novel therapeutics.


The MiniVectors are designed to target ovarian cancer inhibiting sequences, such as FOXM1, CENPA, PLK1, CDC20, BIRC5, AURKB, CCNB1, CDKN3, BCAM-AKT2, CDKN2D-WDFY2, SLC25A6, CIP2A, CD133, ALDH1A1, CD44, SALL4, and PRDM16, to name just a few.


The MiniVectors can contain a single ovarian cancer inhibitory gene, or it can contain more than one such sequence, although with increasing size, efficiency advantages start to be lost. Thus, in some embodiments, MiniVectors are used that are encoding single or multiple DNA sequences against one or multiple targets or multiple MiniVectors encoding single or multiple DNA sequences against one or multiple targets delivered in combination to reduce the expression of one or more targets simultaneously. In other embodiments, MiniVectors can be combined with other gene delivery vectors and/or other therapeutic agents.


While minicircles, plasmid, viruses, micelles, cationic lipids, and the like could be used herein for delivery of ovarian cancer inhibitory gene(s) or RNA sequences, MiniVectors are preferred, being easier to manufacture in purity and quantity and thus safer for human use. MiniVectors are small, circular, supercoiled DNA constructs ideal for RNA interference (e.g., shRNA knockdown), RNA activation, long term gene and RNA expression, cell labeling, and molecular studies of DNA structure and binding. In contrast to plasmids, MiniVectors are stripped down to the very essence of what one hopes to deliver in therapeutics: just the DNA sequence of interest.


MiniVectors offer one or more of the following advantages over other gene delivery vehicles. 1) MiniVector transfection efficiency is equal to siRNA and better than plasmid in several cell types tested. MiniVector DNA transfects every cell type we have tried, including: aortic smooth muscle cells, suspension lymphoma cells, and other difficult to transfect cell types. 2) MiniVector knockdown efficiency lasts longer than siRNA or plasmid because siRNA, shRNA, or miRNA are all rapidly degraded and thus require constant replenishment. Unlike therapeutically delivered RNAs, which typically rapidly degrade, MiniVectors are long-lasting. Unlike plasmids, which are silenced, MiniVectors are not silenced. 3) Smaller therapeutic MiniVectors survive exposure to human serum for at least three times longer than a typical larger sized therapeutic plasmid (there is a strong length dependence on digestion). Plasmids are typically too big to penetrate cells, so they require potentially toxic delivery methods. Finally, longer plasmids are highly susceptible to shear forces from nebulization. Resulting degraded linear DNA can trigger DNA repair and/or activate apoptosis. 4) MiniVectors withstand nebulization, making them an ideal delivery vector to lungs via aerosol. 5) MiniVectors successfully transfect T-cells, stem cells, and cancer cells.


A significant difference between MiniVectors and minicircles lies in the method of their purification. Minicircle purification relies upon (never 100% efficient) cleavage of the parent vector, leading to its degradation inside the bacterial cell, and fails to separate remaining uncleaved parent or the other recombination product from minicircle. Indeed, a recent review (Hardee et al. (2017) provides the following comparison:














Type of DNA vector
Advantages
Disadvantages







Minicircle
Vectors have been designed that are appropriate
Some plasmid and other DNA



for mammalian mitochondrial gene therapy
contaminants can remain in




the final product


MiniVector
Smallest circular DNA vector
Not well-known in the field



Most supercoiled DNA vector




Greatest purity




Naked MiniVector < 1200 bp resists




nebulization shear forces









MiniVector is a double-stranded, supercoiled circular DNA typically lacking a bacterial origin of replication or an antibiotic selection gene, and having a length of about 250 bp up to about 5 kb. It is usually obtained by site-specific recombination of a parent plasmid to eliminate plasmid sequences outside of the recombination sites, but the sizes of the various components are designed to facilitate separation, and the separation is not in vivo restriction enzyme based by definition herein. Purity levels of MiniVectors are typically much higher than a minicircle preparation and there is usually, by gel electrophoresis analysis, no detectible contamination from catenanes, the other circular recombination product, or the parent plasmid. MiniVectors of very short lengths do sometimes become dimerized and sometimes trimerized (or higher multimers). These multimers do not constitute “contaminants” and they still contain only the therapeutic sequence but are merely double (or triple, etc.) the desired therapy. Slightly longer MiniVectors decrease the likelihood of multimers forming (Fogg et al. 2006). Furthermore, if a short MiniVector is desired, an extra gel filtration step typically separates higher multimers from single unit-sized MiniVector, if needed.



FIG. 2 schematizes the modularity of MiniVectors. On the left is shown the simplest embodiment of a MiniVector consisting of (A) the hybrid DNA recombination sequences, attL or attR, that are products of the site-specific recombination, (B) a mammalian promoter, (C) the therapeutic DNA sequence to be expressed, and (D) a transcriptional terminator.


The MiniVector contains, for example, DNA encoding merely the transgene expression cassette (including promoter and a sequence of interest, wherein the nucleic acid sequence may be, for example, a gene, or a segment of a gene, a sequence encoding an interfering RNA (e.g., shRNA, lhRNA, miRNA, shRNA-embedded miRNA, lncRNA, piRNA), or a template for e.g., homology-directed repair, alteration, or replacement of the targeted DNA sequence). Importantly, the MiniVector is almost completely devoid of bacterial-originated sequences.


MiniVectors are designed to contain limited or no homology to the human genome. They are also typically shorter in length than plasmids. Therefore, the integration is at least as low as the 5×10−6 rate of plasmid integration and likely is lower. Designed to be delivered locally, any non-target would have to have MiniVector in the non-target cells/tissue to cause an off-target effect. In that way, then, MiniVectors should not have off-target effects. In contrast, viruses are designed to integrate into the genome, and therefore there is a major risk of off-target integration.


As used herein, “shape” encompasses the basic geometric shapes, such as star, rod, disc, and the like, as well as including features such as aspect ratio, local surface roughness, features in all three-dimensions, varied surface curvatures, the potential for creative and diverse biomimicry, numbers of surface appendages, extreme geometries, etc. “Shape” is best assessed by electron microscopy.


As used herein, “a defined geometric shape” means that the MiniVector has a particular geometric shape that is either transient or non-transient, e.g., is formed in or retained in solution in vivo, such as e.g., a rod, a star, a hexagon, a cube or rhomboid, or a tetrahedron, and other specific shape. It expressly excludes linear or nicked DNAs that freely change shape in solution or ordinary supercoiled DNAs lacking a non-transient shape imposed thereon by design. Furthermore, the shape is a function of the DNA sequence, and is not only externally imposed thereon, e.g., by histones, capsid proteins, ligands, or micelles, and the like.


As used herein, when we say that ˜50% of said MiniVectors have a specific shape, we mean that when visualized we see that more than half of the vectors have the same shape, although that shape appears differently when viewed from different angles.


As used herein, “non-transient” means that the shape is retained for a time long enough to be measured in solution. “Transient” shapes may also be useful such that cells may specifically take up such shapes when they transiently appear. These sequence-engineered shapes contrast from the various forms that a circular or linear DNA without engineered shape may take in solution, such as random linear DNA shapes or circles (nicked or supercoiled).


As used herein a “rod” is a generally cylindrical shape that is elongated and has an aspect ratio (AR) of >2.4. A “microrod” is a rod that is at least 1 micron long in the long axis. Shape has been shown to influence cellular localization and uptake of synthetic nanoparticles (e.g., gold nanorods, sugar structures, etc.). Particles with diameters on the order of microns (interestingly about the same size as a platelet) preferentially displace to the cell free layer (CFL) in the presence of red blood cells (RBCs), while smaller particles do not experience this enhanced localization. Although not yet directly tested with DNA, we anticipate that shape and size will similarly affect the cellular localization and uptake of DNA nanoparticles generated by incorporation of sequence-directed bends into the MiniVector DNA. A “nanorod” by contrast is of length in the long axis <1 micron. Nanorods may accumulate significantly in the spleen.


As used herein, a “star” shape has a plurality of generally evenly sized and distributed projections, e.g., six armed stars have been shown to be preferentially delivered to pulmonary tissue when delivered intravenously.


As used herein, the term “RNA interference,” or “RNAi,” refers to the process whereby sequence-specific, post-transcriptional gene silencing is initiated by an RNA that is homologous in sequence to the silenced gene. RNAi, which occurs in a wide variety of living organisms and their cells, from plants to humans, has also been referred to as post-transcriptional gene silencing and co-suppression in different biological systems. The sequence-specific degradation of mRNA observed in RNAi is mediated by small (or short) interfering RNAs (siRNAs).


As used herein, the term “interfering RNA” means an RNA molecule capable of decreasing the expression of a gene having a nucleotide sequence at least a portion of which is substantially the same as that of the interfering RNA. As known in the art, interfering RNAs can be “small interfering RNAs,” or siRNAs, composed of two complementary single-stranded RNAs that form an intermolecular duplex. Interfering RNAs can also be “long hairpin RNAs,” or lhRNAs, which are shRNA-like molecules with longer intramolecular duplexes and contain more than one siRNA sequence within the duplex region.


As used herein, the term “gene silencing” refers to a reduction in the expression product of a target gene. Silencing may be complete, in that no final gene product is detectable, or partial, in that a substantial reduction in the amount of gene product occurs.


As used herein, “shRNA” is short hairpin RNA or small hairpin RNA, and “lhRNA” is long hairpin RNA, both of which can be used to silence target gene expression via RNAi.


As used herein, “miRNA” is microRNA—a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression. Alternative to a contiguous duplex shRNA is an shRNA sequence embedded in a microRNA stemloop (e.g., MiRE), which may be used because it can be processed more efficiently in mammalian cells leading to more robust knockdown of the expression of the target gene. The more efficient processing of the microRNA stemloop relies on both Drosha and Dicer, whereas the contiguous duplex shRNA relies only on Dicer to cut the guide RNA that will be inserted into the RNA-induced silencing complex.


As used herein, “lncRNA” are long non-coding RNAs. These lncRNAs are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins (or lack >100 amino acid open reading frame). lncRNAs are thought to encompass nearly 30,000 different transcripts in humans, hence lncRNA transcripts account for the major part of the non-coding transcriptome. lncRNA discovery is still at a preliminary stage. There are many specialized lncRNA databases, which are organized and centralized through RNAcentral (rnacentral.org). lncRNAs can be transcribed as whole or partial natural antisense transcripts to coding genes, or located between genes or within introns. Some lncRNAs originate from pseudogenes. lncRNAs may be classified into different subtypes (Antisense, Intergenic, Overlapping, Intronic, Bidirectional, and Processed) according to the position and direction of transcription in relation to other genes.


Piwi-interacting RNA or “piRNA” is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with PIWI family proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, particularly those in spermatogenesis. They are distinct from miRNA in size (26-31 nt rather than 21-24 nt), lack of sequence conservation, and increased complexity.


The term “treating” includes both therapeutic treatment and prophylactic treatment (reducing the likelihood of disease development). The term means decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a disease or disorder delineated herein), lessen the severity of the disease, or improve the symptoms associated with the disease.


As described herein, MiniVector for use in gene therapy is present in an effective amount. As used herein, the term “effective amount” refers to an amount which, when administered in a proper dosing regimen, is sufficient to treat (therapeutically or prophylactically) the target disorder or symptoms of the target disorder. For example, an effective amount is sufficient to reduce or ameliorate the severity, duration, or progression of the disorder being treated, prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.


By “reducing” the expression of a target protein, we mean a reduction of at least 10%, as the body's own immune response may thereby be sufficient to target and kill the cancer cells, particularly in a combination therapy combined with an immune-boosting treatment, such as CpG motifs, cytokines (chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors). Preferably the reduction is at least 20%, 30% or 40%, but typically a complete knockout is not required, and indeed, can contribute to unwanted side effects.


“Nanoparticles” are understood to comprise particles in any dimension that are less than 1,000 nanometers, more preferably less than 500 nanometers, and most preferably less than 300 nanometers. The nanoparticle can be a viral vector, a component of a viral vector (e.g., a capsid), a non-viral vector (e.g., a plasmid or RNA or MiniVector), a cell, a fullerene and its variants, a small molecule, a peptide, metal and oxides thereof, etc.


The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means either one or more than one, unless the context dictates otherwise. The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 25% if no method of measurement is indicated. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.


The terms “comprise,” “have,” “include,” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim. The phrase “consisting of” is closed, and excludes all additional elements. The phrase “consisting essentially of” excludes additional material elements but allows the inclusions of non-material elements that do not substantially change the nature of the invention, such as instructions for use, buffers, and the like.


The following abbreviations are used herein. Description includes UniProt accession numbers, when appropriate (NA, not applicable).
















AKT2
RAC-beta serine/threonine-protein kinase
P31751



(gene AKT2)



ALDH1A1
Retinal dehydrogenase 1
P00352


AURKB
Aurora kinase B
Q96GD4


BCAM
Basal Cell Adhesion Molecule
P50895


BIRC5
Baculoviral IAP repeat-containing protein 5
O15392


CCNB1
G2/mitotic-specific cyclin-B1
P14635


CD133
Prominin-1
O43490


CD44
CD44 antigen, Receptor for hyaluronic acid
P16070



(HA)



CDC20
Cell division cycle protein 20 homolog
Q12834


CDKN2D
Cyclin-dependent kinase 4 inhibitor D
P55273


CDKN3
Cyclin-dependent kinase inhibitor 3
Q16667


CENPA
Histone H3-like centromeric protein A
P49450


CIP2A
CIP2A (gene KIAA1524)
Q8TCG1


FOXM1
Forkhead box protein M1
Q08050


MAR
Matrix attachment region
NA


NLS
Nuclear localization signal
NA


PLK1
Serine/threonine-protein kinase PLK1
P53350


PRDM16
PR domain zinc finger protein 16
Q9HAZ2


S/MAR
Scaffold/matrix attachment region
NA


SALL4
Sal-like protein 4
Q9UJQ4


SLC25A6
ADP/ATP translocase 3 (gene SLC25A6)
P12236


WDFY2
WD repeat and FYVE domain-containing
Q96P53



protein 2












BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1. Generation of MiniVector DNA by λ-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 λ-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.



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.



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.



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 μg 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.



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 μg DNA. The actin gene was used as an endogenous control. Percentage knockdown is shown from qPCR data compared to the shRNA scrambled control (NTC).



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





DETAILED DESCRIPTION

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:














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

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.


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).


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.


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.


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.


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.


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.


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.


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.


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

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.


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).


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

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.


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.


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.


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).


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.


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.


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.


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


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


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


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].


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.


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.


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).


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).


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.


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.


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.


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

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.


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 emerge—indeed, 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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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

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.


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.


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

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.


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

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).


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

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.


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).


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

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, λ-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.


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

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.


To test knockdown of the MiniVector-encoded shRNA gene targets in vitro, cells will be plated and transfected with Lipofectamine™3000 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.


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.


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.


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.


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.


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.).


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.


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.


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.


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.


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

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

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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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, EF1α, or another promoter for mammalian RNA polymerase II.









TABLE 1







Therapeutic sequences to be encoded on MiniVector











SEQ


Dharmacon



ID NO
Gene
Description
Cat. No.
Mature Antisense





   1.
AKT2
RAC-beta serine/threonine-
V2LHS_237948
AAATTCATCATCGAAGTAC




protein kinase (gene AKT2)






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
Retinal dehydrogenase 1
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
Aurora kinase B Q96GD4
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
Basal Cell Adhesion Molecule
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
Baculoviral IAP repeat-
V2LHS_94585
ATCAAATCCATCATCTTAC




containing protein 5 O15392




  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-specific cyclin-B1
V3LHS_369356
TTACCATGACTACATTCTT




P14635




  39.


V3LHS_369358
TGCTTGCAATAAACATGGC


  40.


V3LHS_369355
TAATTTTCGAGTTCCTGGT


  41.


V3LHS_369360
AAAGCTCTTAGAATCTTCA


  42.


V3LHS_369359
AGAATCTTCATTTCCATCT





  43.
CD133
Prominin-1 O43490
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
CD44 antigen, Receptor for
V2LHS_111680
TATATTCAAATCGATCTGC




hyaluronic acid (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
Cell division cycle protein 20
V2LHS_112883
TTCCAGATGCGAATGTGTC




homolog Q12834




  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-dependent kinase 4
V2LHS_262156
AATAAATAGAATCCATTTC




inhibitor D P55273




  67.


V3LHS_401207
ATGAATAACTCATAACTCA


  68.


V3LHS_310385
CCACTAGGACCTTCAGGGT


  69.


V3LHS_310386
CGGGATGCACCAGCTCGCG


  70.


V3LHS_310389
AGGACCTTCAGGGTGTCCA


  71.


V3LHS_310387
GAACTGCCAGATGGATTGG





  72.
CDKN3
Cyclin-dependent kinase
V2LHS_262397
TATAGTAGGAGACAAGCAG




inhibitor 3 Q16667




  73.


V2LHS_201585
TGCTTGATGGTCTGTATTG


  74.


V3LHS_386043
TGATTGTGAATCTCTTGAT


  75.


V3LHS_386040
ATCTTGATACAGATCTTGA


  76.


V3LHS_386041
TGATACAGATCTTGATTGT





  77.
CENPA
Histone H3-like centromeric
V2LHS_150535
ATATGATGGAAATGCCCAG




protein A P49450




  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 (gene KIAA1524)
V2LHS_206422
TACTCAATGTCTTTATGTG




Q8TCG1




  86.


V3LHS_308568
TGAATGTGATCTATCAGGA


  87.


V3LHS_308569
TGTTCTCTATTATCTGACG


  88.


V3LHS_308565
TTCATTTCATATACATCCA


  89.


V3LHS_308566
TGAACAGAAAGATTGTGCC





  90.
FOXM1
Forkhead box protein M1
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




kinase PLK1 P53350




  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
PR domain zinc finger protein
V2LHS_215636
TAAAGCCTCAGAATCTAAG




16 Q9HAZ2




104


V2LHS_251390
TAAATTACGACTCTGACAC


105


V3LHS_300082
ATTATTTACAACGTCACCG


106


V3LHS_300078
TTCTCGTCTAAAAGTGCGT


107


V3LHS_300081
AAAAGTGCGTGGTTGTCCG





108
SALL4
Sal-like protein 4 Q9UJQ4
V3LHS_363661
TAGCTGACCGCAATCTTGT


109


V3LHS_363659
TAGTGAACTTCTTCTGGCA


110


V3LHS_363662
TCGGCTTGACTATTGGCCG


111


V3LHS_363664
TTCTGAGACTCTTTTTCCG





112
SLC25A6
ADP/ATP translocase 3 (gene
V3LHS_314256
TGTACTTATCCTTGAAGGC




SLC25A6) P12236




113


V3LHS_314257
TGCCCGCAAAGTACCTCCA





114
WDFY2
WD repeat and FYVE domain-
V2LHS_118254
TATCCCACAACTTAATAAC




containing protein 2 Q96P53




115


V2LHS_118249
TAACCAAACACGAACTGTC


116


V3LHS_405758
ATTGTATGAACAAGTTGGA


117


V3LHS_341295
TTCACAGGAGTCATCTTGT


118


V3LHS_405756
TATATTGTATGAACAAGTT





119
CBCP1
cyclin Y
V2LHS_243158
ATACTTGGCATAGACACTG


120


V3LHS_314369
TACTGAGGAATATTGTGCT


121


V3LHS_314371
TAATGAAGAGACTCTTGCG
















TABLE 2







MiniVector elements


Table 2. MiniVector elements










Module
Element
Description
Use





A
λ-attL
attL from the λ-integrase system
Recombination



λ-attR
attR from the λ-integrase system
sites (product of



λ-attB
attB from the λ-integrase system
site-specific



λ-attP
attP from the λ-integrase system
recombination



loxP
loxP site for Cre recombinase
used to generate



γδ-res
res site for the γδ (Tn1000) resolvase
MiniVector).



FRT
FRT site for Flp recombinase
Sequences listed



hixL
hixL site for Hin recombinase
in Table 3.



hixR
hixR site for Hin recombinase




Tn3 res
res site for Tn3 resolvase




Tn21 res
res site for Tn21 resolvase




cer
cer site for XerCD system




psi
psi site for XerCD






B

Tissue-specific promoter of alcohol
Initiation of




dehydrogenase 1 (ALDH1)
transciption.



AMY1C
Tissue-specific promoter of human amylase
Includes




alpha 1C (AMY1C)
promoters for



β-actin
Promoter from the (human) beta actin gene
RNA polymerase



CaMKIIα
Ca2+/calmolulin-dependent protein kinase II
II and RNA




alpha promoter
polymerase III.



CMV
Promoter from the human cytobegalovirus (CMV)
Full sequences



Mini CMV
Minimized version of CMV
of selected



CAG
CMV early enhancer/chicken β actin promoter (CAG).
promoters




Synthetic hybrid promoter made from a) the CMV
provided in




early enhancer element, b) the promoter, the first
Table 4.




exon and the first intron of chicken beta-actin





gene, and c) the splice acceptor of the rabbit





beta-globin gene




Cyto-
Cell-specific promoters of the human keratin 18




keratin 18
and 19 genes




and 19





EF1α
Strong expression promoter from human elongation





factor 1 alpha




GFAP
Tissue-specific promoter of the glial fibrillary





acidic protein (GFAP)





Promoter from the human polymerase III RNA





promoter




Kallikrein
Tissue-specific promoter of the kallikrein gene.




NFK-β
Nuclear factor kappa-light-chain-enhancer of





activated B cells (NF-Kβ)




PGK1
Promoter from human or mouse phosphoglycerate





kinase gene (PGK)




RSV
Long terminal repeat (LTR) of the rous sarcoma





virus (RSV)




SV40
Mammalian expression promoter from the simian





vacuolating virus 40




UBC
Promoter of the human ubiquitin C gene (UBC)




U6
Promoter from the human U6 small nuclear promoter






C
shRNA
(DNA) sequence encoding short hairpin RNA (shRNA)
Knockdown of




transcript. Sequences for use in target validation
gene expression




are listed in Table 1. Potential therapeutic
through RNA




sequences will be designed de novo and optimized
interference




for knockdown efficiency.




miRNA
(DNA) sequence encoding micro-RNA (miRNA)





transcript.




lhRNA
(DNA) sequence encoding long hairpin RNA (lhRNA)





transcript




lncRNA
(DNA) sequence encoding long non-coding RNA





(lncRNA) transcript




piRNA
(DNA) sequence encoding piwi-interacting (piRNA)





RNA transcript






D

Transcriptional terminator sequence






E
S/MAR
Scaffold/matrix attached region from eukaryotic
Episomal




chromosomes (Sequences in Table 5)
replication





F/G
β-globin
Intron of the human β globin gene (130 bp)




intron





HGH
Intron of the human growth hormone gene (262 bp)
Gene expression



intron

enhancer





H
SV40
Simian virus 40 early promoter (351 bp)
Nuclear



early

localization



promoter





NF-Kβ
Binding site of nuclear factor kappa-light-chain-





enhancer of activated B cells (55 bp (5 repeats





of GGGGACTTTCC SEQ ID NO 122))




p53 NLS
Binding site of tumor protein 53 (p53):





AGACTGGGCATGTCTGGGCA SEQ ID NO 123




p53 NLS
Binding site of tumor protein 53 (p53):





GAACATGTCCCAACATGTTG SEQ ID NO 124




Adeno-
GGGGCTATAAAAGGG SEQ ID NO 125




virus





major late





promoter
















TABLE 3







Complete sequences for element A (recombination sites)









SEQ ID NO
Site
Sequence (5′-3′)





126.
λ-attL
TCCGTTGAAGCCTGCTTTcustom-character TAAGTTGGCATTATAAAAAAGCATTGC




TTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTA




TT





127.
λ-attR
AGATGCCTCAGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCA




TAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGC




AAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAG




CTTTcustom-character TAACTTGAGCGAAACG





128.
λ-attB
TCCGTTGAAGCCTGCTTTcustom-character TAACTTGAGCGAAACG





129.
λ-attP
AGATGCCTCAGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCA




TAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGC




AAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAG




CTTTcustom-character TAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTG




CAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATT





130.
loxP
ATAACTTCGTATAGCATACATTATACGAAGTTAT





131.
γδ-res
ATTTTGCAACCGTCCGAAATATTATAAATTATCGCACACATAAAAACAGTGC




TGTTAATGTGTCTATTAAATCGATTTTTTGTTATAACAGACACTGCTTGTCC




GATATTTGATTTAGGATACATTTTTA





132.
FRT
GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC





133.
hixL
TTCTTGAAAACCAAGGTTTTTGATAA





134.
hixR
TTTTCCTTTTGGAAGGTTTTTGATAA





135.
Tn3 res
CAACCGTTCGAAATATTATAAATTATCAGACATAGTAAAACGGCTTCGTTTG




AGTGTCCATTAAATCGTCATTTTGGCATAATAGACACATCGTGTCTGATATT




CGATTTAAGGTACATTT





136.
Tn21 res
GCCGCCGTCAGGTTGAGGCATACCCTAACCTGATGTCAGATGCCATGTGTAA




ATTGCGTCAGGATAGGATTGAATTTTGAATTTATTGACATATCTCGTTGAAG




GTCATAGAGTCTTCCCTGACAT





137.

GGTGCGTACAATTAAGGGATTATGGTAAAT





138.

GGTGCGCGCAAGATCCATTATGTTAAAC
















TABLE 4







Complete sequences for element B (promoters)









SEQ ID NO
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



β-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-β
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 5







Complete sequences for elements E, F and G (accessory sequences)









SEQ ID NO
Element
Sequence (5′-3′)





152.
250 bp S/MAR
TCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAG




AATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTT




AGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATAT




TTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATAT




ATTTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAAT




ATATTTAGAA





153.
439 bp S/MAR
TCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAG




AATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTT




AGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATAT




TTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATAT




ATTTAGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAAT




ATATTTAGAA





154.
(45 bp) Type A
GGTGCATCGATGCAGCATCGAGGCAGGTGCATCGATACAGGGGGG



Cpg motif






155.
(24 bp) Type B
TCGTCGTTTTGTCGTTTTGTCGTT



Cpg motif






156.
(21 bp) Type C
TCGTCGAACGTTCGAGATGAT



CpG motif






157.
β-globin intron
GTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTG




GGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACT




CTCTCTGCCTATTGGTCTATTTTCCCACCCTTAG





158.
Human growth
TTCGAACAGGTAAGCGCCCCTAAAATCCCTTTGGGCACAATGTGTCCT



hormone intron
GAGGGGAGAGGCAGCGACCTGTAGATGGGACGGGGGCACTAACCCTCA




GGTTTGGGGCTTCTGAATGTGAGTATCGCCATGTAAGCCCAGTATTTG




GCCAATCTCAGAAAGCTCCTGGTCCCTGGAGGGATGGAGAGAGAAAAA




CAAACAGCTCCTGGAGCAGGGAGAGTGCTGGCCTCTTGCTCTCCGGCT




CCCTCTGTTGCCCTCTGGTTTC
















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 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 8







Other Ovarian Cancer Targets (some including target sequences)









SEQ ID NO
Target
Detail/Targeting Sequence






4EBP1
shRNA against human 4E-BP1: (hshBP1)(sigma: TRCN0000040203):





159.

CCGGGCCAGGCCTTATGAAAGTGATCTCGAGATCACTTTCATAAGGCCTGGCTTTTTG





160.
AKT
AKT1 TRCN0000010174 GGACTACCTGCACTCGGAGAA





161.

AKT2 TRCN0000000564 CTTCGACTATCTCAAACTCCT





162.

AKT3 TRCN0000010187 CTGCCTTGGACTATCTACATT












ANG1/ANG2 and Tie, ATM, AXL, BCL-2, BET, bFGF, BRAF, BRIPI, CDC42, c-KIT



CD40, CD184, CDCP1, CHK1, TNNB1, CXCL1 aka Fractalkine, CX3CR1 aka



Fractalkine, CXCR4












163.
DXL1
The targeting sequence for Dlx1 was 5′-AACCGGAGGTTCCAACAAACT-3′


164.

(sense strand: 5′-CCGGAGGTTCCAA-CAAACTTT-3′;


165.

antisense strand: 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
shRNA 1 for MARCH7(NM_022826) AAGTGCTAGGATGATGTCTGGAA





170.

shRNA 2 for MARCH7(NM_022826) AAGAACAGATTCCTCTATTAGTA





171.

shRNA 3 for MARCH7(NM_022826) AAGATCTAGTCAGGATTCCTTGA





172.

shRNA 4 for MARCH7(NM_022826) AAGAGATGAATCTTCAAGGATAC












MAST1, MDM2, MEK, MET, MMP9












173.
mTOR
V2LHS_262100 Mature Antisense: TAGGAGGCAGCAGTAAATG; many others




listed from company












NOX1, OX40, PAK1, PARP, PDGF, PD-1, PDL1, PICT- aka GLTSCR2












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
The shRNA sequence of PRAS40 was GCTGAGTTCTAAGCTCTAA (sense)












PRMT5, Raf, RAS, RB, Ror1, Ror2, Shp2, SLIT3, STAT, SYK, TGF alpha, TLR4,



TNF-alpha, TNF, TOP1, TOP2, TP53, TRAIL, TYK2












179.
USP7
USP7 (GCGATTACAAGAAGAGAAA) through Dhamracon





180.
USP15
shRNA-1




5′-CCGGCCGTAATCAATGTGGGCCTATCTCAGATAGGCCCACATTGATTACGGTTTTT-3′;












VEGF, WDR77, WDR79, WEE1, XIAP, Y1349
















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










The following references are incorporated by reference in their entirety for all purposes.

  • ATCC Sarcoma Cell Lines. atcc. org/˜/media/PDF s/Cancer %20and %20Normal %20cell %20lines %20tables/Sarcoma %20cel 1%20lines.ashx
  • Catanese, D. J., et al., Supercoiled MiniVector DNA resists shear forces associated with gene therapy delivery, Gene Ther. 19(1): 94-100 (2012).
  • 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).
  • Darquet A. M., et al., Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer, Gene Ther., 6: 209-218 (1999).
  • Douard R., et al., Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer, Surgery. 139(5): 665-670 (2006).
  • ES4: Cellosaurus, web.expasy.org/cellosaurus/CVCL_1200.
  • Fogg, J. M., et al., Exploring writhe in supercoiled minicircle DNA. J. Phys.-Condes. Matter, 18: S145-S159 (2006).
  • Hardee, C. L., Advances in Non-Viral DNA Vectors for Gene Therapy, Genes 8, 65 (2017)
  • Hornstein, B. D., et al., Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells, PLoS One. 11(12): e0167537 (2016).
  • Jaiswal N., et al., Biology of FOXM1 and its emerging role in cancer therapy, J. Proteins Proteomics 5(1): 1-24 (2014).
  • 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).
  • 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).
  • Kannan K., CDKN2D-WDFY2 is a cancer-specific fusion gene recurrent in high-grade serous ovarian carcinoma, PLoS Genet. 10(3):e1004216 (2014).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • Lokody, I., Signalling: FOXM1 and CENPF: co-pilots driving prostate cancer, Nature Reviews Cancer 14: 450-451 (2014).
  • 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).
  • 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
  • Teh M. T., et al., FOXM1 is a downstream target of Gli1 in basal cell carcinomas, Cancer Res., 62(16): 4773-4780 (2002).
  • US20150376645, US20140056868, 61/653,279, filed May 30, 2012, Supercoiled MiniVectors as a tool for DNA repair, alteration and replacement
  • 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).
  • WE68: Cellosaurus, web.expasy.org/cellosaurus/CVCL_9717.
  • Wei, Ping et al., FOXM1 promotes lung adenocarcinoma invasion and metastisis by upregulating snail, International Journal of Biological Sciences 11(2): 186-198 (2015).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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
  • U.S. Pat. No. 7,622,252, US20070020659, 60/689,298, filed Jun. 10, 2005, Generation of minicircle DNA with physiological supercoiling
  • US20060211117 Methods of making minicircles
  • WO1994009127 Supercoiled minicircle DNA as a unitary promoter vector
  • WO2002083889 Methods for the production of minicircles
  • 62/470,997, Mar. 14, 2017, TARGETING MINIVECTORS TO SPECIFIC TISSUE USING SHAPE
  • 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
  • Ramamoorth M., & Narvekar, A., Non Viral Vectors in Gene Therapy—An Overview, J. Clinical & Diagnostic Res. 2015 January, Vol-9(1): GE01-GEO6.
  • Hidai C., & Kitano, H., Nonviral Gene Therapy for Cancer: A Review, Diseases 2018, 6, 57.
  • 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.

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; andd) 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; andd) 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%.
PRIOR RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 62/581,614, filed Nov. 3, 2017, and 62/680,588, filed Jun. 5, 2018, each incorporated by reference in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH STATEMENT

This invention was made with government support under grants R01GM115501, R56AI054830, R01AI054830 and R01CA060651 awarded by the National Institutes of Health. The government has certain rights in the invention.

Provisional Applications (2)
Number Date Country
62581614 Nov 2017 US
62680588 Jun 2018 US