TREATMENT OF LIVER CANCER OR LIVER FIBROSIS

Abstract

Compositions containing MiniVectors and gene therapy uses, including long term repeated gene therapy uses, to treat liver fibrosis or liver cancer.

Claims

1) A composition comprising an MiniVector plus a pharmaceutically acceptable carrier, said MiniVector being a double-stranded, supercoiled circular DNA lacking a bacterial origin of replication or an antibiotic selection gene, having a length of about 250-600 base pairs exclusive of expressible payload and having <1% contamination by a parent plasmid DNA, said expressible payload being a sequence selected from one or more that upregulates one or more of P53 or relaxin, or downregulates one or more of FOXM1, CAD11, MDM2, MDM4, or STATS.

2) The ultrapure MiniVector of claim 1, wherein contamination by parent DNA is <0.1%.

3) The ultrapure MiniVector of claim 1, wherein contamination by parent DNA is <0.02% and is assessed by gel electrophoresis and staining at a sensitivity of ≤0.1 ng, or preferably ≤0.01 ng.

4) The ultrapure MiniVector of claim 1, wherein contamination by parent DNA is <0.02% and is assessed by gel electrophoresis and staining with SYBR Gold staining at a sensitivity of ≤0.1 ng.

5) The ultrapure MiniVector of claim 1, wherein said MiniVector is separated from said parent plasmid and recombination side-products on the basis of size, and does not use sequence-specific endonuclease cleavage in vivo for preparation of said MiniVector.

6) The ultrapure MiniVector of claim 1, wherein contamination by parent DNA is <0.02% and wherein said MiniVector is separated from said parent plasmid and recombination side-products on the basis of size, and does not use sequence-specific endonuclease cleavage in vivo for preparation of said MiniVector.

7) The ultrapure MiniVector of claim 1, wherein said MiniVector is purified by cross flow filtration or by PEG precipitation of large DNA or by at least two passes through multiple gel-filtration columns using progressively smaller size range size exclusion resins or by a combination thereof.

8) The ultrapure MiniVector of claim 1, wherein said MiniVector is purified by PEG precipitation of larger DNA species followed by anion exchange chromatography to remove RNA and non-nucleic acid components, followed by at least two passes through multiple gel-filtration columns using progressively smaller size range size exclusion resins.

9) The ultrapure MiniVector of claim 1, wherein said MiniVector is purified by PEG precipitation, anion exchange chromatography, and at least two passes through multiple gel-filtration columns using progressively smaller size range size exclusion resins, and one or more alcohol precipitations.

10) The ultrapure MiniVector of claim 1, comprising a promoter operably connected to said payload operably connected to a terminator.

11) The MiniVector of claim 1, wherein said MiniVector is ≤500 bp in length, excluding said payload.

12) A method of treating liver fibrosis or liver cancer, said method comprising administering the composition of any of claim 1 to a patient having liver fibrosis or liver cancer in an amount sufficient to upregulate one or more of P53 or relaxin by at least 10% as assessed by protein activity, or in an amount sufficient to downregulate one or more of FOXM1, CAD11, MDM2, MDM4, or STAT3 by at least 10% as assessed by protein activity.

13) The method of claim 12, wherein said administration is by injection into or onto a liver of said patient.

14) The method of claim 12, wherein said administration is by surgical entry into a liver of said patient.

15) The method of claim 12, wherein said administration is by IV delivery, lavage of a liver, or surface coating of the liver.

16) The method of claim 12, wherein said administration occurs a plurality of times.

17) The method of claim 12, wherein said administration occurs a plurality of times 1-4 weeks apart.

18) A composition comprising an MiniVector plus a pharmaceutically acceptable carrier, said MiniVector being a double-stranded, supercoiled circular DNA lacking a bacterial origin of replication or an antibiotic selection gene, having a length of about 250-600 base pairs exclusive of expressible payload and having <1% contamination by a parent plasmid DNA, said expressible payload being a sequence selected from one or more that upregulates one or more of P53 or relaxin, or downregulates one or more of FOXM1, CAD11, MDM2, MDM4, STAT3, STAT6 or TGFB1.

19) A method of treating liver fibrosis or liver cancer, said method comprising administering the composition of any of claim 18 to a patient having liver fibrosis or liver cancer in an amount sufficient to upregulate one or more of P53 or relaxin by at least 10% as assessed by protein activity, or in an amount sufficient to downregulate one or more of FOXM1, CAD11, MDM2, MDM4, STAT3, STAT6 or TGFB1 by at least 10% as assessed by protein activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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.

[0049] 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., Table 1), and D) a transcriptional terminator. 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. Potential sequences for these various components are listed in Tables 2-7.

DETAILED DESCRIPTION

[0050] The disclosure provides ultrapure MiniVectors that are sufficiently pure for use in gene therapy to treat liver cirrhosis or liver cancer.

Minivector Synthesis

[0051] MiniVector DNA is generated in bacterial cells using in vivo site-specific recombination as described previously in U.S. Pat. No. 7,622,252. In more detail, parent plasmids contain attB and attP (recognition sites for λ-integrase) oriented in the same direction. Site-specific recombination between the attB and attP sites exchanges the sequences between the two sites results in two product circles that are linked together (catenated) because of supercoiling in the parent plasmid. One of these product circles is the minivector. The other circle is the “miniplasmid”, which contains the unwanted bacterial sequences. The recombination reaction also results in two new integrase sites, attL and attR, which are hybrid sites each containing sequences from attB and attP. If attB comes first on the parent plasmid, followed by attP, with the minivector sequence in between, the larger (˜180 bp) attR site will end up on the minivector and the smaller (˜100 bp) attL site is on the miniplasmid. Conversely, if attP comes first, followed by attB, then the attL site will be on the minivector. The intervening sequence between the attB and attP sites becomes incorporated into the minivector. Therefore, any sequence can be engineered into a minivector by simply cloning between the integrase sites on the parent plasmid. The system was first tested with pMC339 with attB preceding attP on the parent plasmid, which generates a 339 bp minivector containing an attR site and otherwise random sequence.

[0052] MiniVectors are generated using engineered Escherichia coli strains (examples include but are not limited to LZ31 and LZ54. These strains express, k-integrase (λ-Int) under the tight control of the temperature-sensitive cI857 repressor. The Escherichia coli strain is transformed with the relevant parent plasmid. When cells are grown at 30° C., no λ-Int is expressed because of the tight control afforded by the cI857 repressor. This prevents premature recombination which would result in excision of the minivector sequence from the parent plasmid. An aliquot of the transformed strain is grown up at 30° C. in shaker flasks and used to inoculate a fermenter containing modified terrific broth medium.

[0053] Cells are grown at 30° C., maintaining the pH at 7 and the dissolved oxygen concentration above 60%, to ensure the cells remain in exponential phase. Once cells have reached mid-exponential phase, λ-Int expression is induced by shifting the culture to 43° C. for ˜30 minutes to induce λ-Int expression. The increased temperature leads to denaturation of the cI857 repressor, which prevents λ-Int expression at lower temperatures. λ-Int is not active at the higher 43° C. temperature, therefore the culture is subsequently shifted down to 30° C. to allow recombination to proceed for about an hour (1-4 hrs). Prior to the temperature shift back to the lower temperature, norfloxacin is added to the fermenter prevent decatenation of the recombination products by topoisomerase IV.

Minivector Purification

[0054] Step 1: The first step in purification is to harvest the cells containing MiniVector by centrifugation.

[0055] Step 2: Cells are first incubated with lysozyme to break down the bacterial cell walls and then lysed using a standard alkaline lysis procedure.

[0056] Step 3: The nucleic acid (DNA and RNA) in the lysate is precipitated with isopropanol then resuspended to reduce the volume per usual procedures. Nucleic acid solution is then incubated with RNaseA to degrade the RNA, followed by incubation with proteinase K to degrade any residual proteins.

[0057] Step 4: Nucleic acid solution is incubated with polyethylene glycol (PEG) and NaCl and incubated on ice for ˜15 minutes. By carefully controlling the concentration of PEG, larger DNA species are selectively precipitated while the smaller minivector DNA stays in solution. For the 339 bp MiniVector exemplified herein a solution containing an equal volume of 10% PEG-8000, 1.6 M NaCl was added to the nucleic acid solution (final concentrations: 5% PEG-8000, 0.8M NaCl). For larger MiniVectors lower concentrations of PEG are used. The precipitated larger DNA species is thus pelleted by centrifugation. The smaller nucleic acid (DNA and RNA) species in the supernatant are subsequently precipitated with ethanol to remove the PEG.

[0058] PEG precipitation is quick and has high capacity but has low resolution and can only separate DNA species significantly different in size (two-fold or more). It is used to remove a majority of the unwanted large circle (miniplasmid) recombination byproduct and any unrecombined parent plasmid. Reducing the mass of contaminating large DNA species makes subsequent downstream purification steps much more efficient.

[0059] Step 5: DNA is then further purified using anion-exchange chromatography, although other methods are available. We use Qiagen plasmid purification kits for this (e.g., Maxiprep kit or Gigaprep Kit) but columns from other manufacturers may be used. The major purpose of this step is to remove the (degraded) RNA and other (non-nucleic acid) contaminants, and it does not differentiate between different sized DNA species. Following anion-exchange the DNA is again precipitated with isopropanol and resuspended in a small volume for gel-filtration chromatography.

[0060] Step 6: Gel-filtration. This step completely removes any remaining large DNA contaminants, separating DNA according to size (larger DNA species eluting first). Although described in the original U.S. Pat. No. 7,622,252 patent, we have made several modifications since that patent was filed.

[0061] The contaminating DNA species are not able to enter the beads in the gel filtration matrix and are typically eluted in the “void volume,” while the MiniVector DNA elutes later. Here, instead of using a single gel filtration column, two or three columns are connected in series such that when DNA is eluted from one column it enters the next column in the series. This significantly increases the separation of DNA species. Using multiple columns in series also allows different combinations of gel filtration resin to be used, thus optimizing size separation. For example, Sephacryl S-500 is best for separating MiniVector DNA from the parent plasmid. Sephacryl S-400 provides better separation of monomeric MiniVector from any multimeric length byproducts.

[0062] Using different columns in decreasing size separation range sequentially like this allows ultrapure monomeric MiniVector to be isolated and the recovery efficiency of DNA from gel-filtration is very high. Essentially all the DNA loaded onto the columns is eluted (provided that the DNA stays in solution). Therefore, there is no penalty in terms of yield for running the same DNA through the gel-filtration columns multiple times. To further remove any remaining contaminants, the DNA may simply be loaded again through the series of gel filtration columns.

[0063] Other purification methods can also be used in various combination(s), including high density centrifugation, dead end filtration, cross flow filtration, ultrafiltration, precipitations, binding to various resins, phenol-chloroform extraction; proteinase K digestion, electrophoresis, ion exchange chromatography, affinity chromatography, and the like, as well as techniques to be developed in the future.

Minivector Payloads

[0064] Target sequences currently under development for gene therapy uses to treat liver cirrhosis and liver cancer include P53.sup.+, Relaxin.sup.+, FOXM1.sup.−, CAD11.sup.−, MDM2.sup.−, MDM4− and STAT3−. Herein, the + sign means the protein/gene is upregulated in the therapy, usually by adding a copy of the gene under a strong promoter, or by upregulating the endogenous promoter, and the − sign meaning the protein/gene is downregulated, usually by adding an RNAi or by downregulating the endogenous promoter.

[0065] Additional targets that will be tested in the near future include transforming growth factor beta 1 (TGFB) (OMIM 190180) and signal transducer and activator of transcription 6 (STAT6) (OMIM 601512), which can be downregulated using an RNAi approach, possibly both in conjunction with other targets.

[0066] TGFB is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGFB acts synergistically with TGFA (OMIM 190170) in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGFB activation and signaling may result in apoptosis. Many cells synthesize TGFB and almost all of them have specific receptors for this peptide. TGFB1, TGFB2 (190220), and TGFB3 (190230) all function through the same receptor signaling systems. TGFB is known to be important in wound healing and fibrosis is associated with increased expression of TGFB, making it a logical target, and the other members of the pathway may also prove useful.

[0067] Lipid metabolism, especially fatty acid oxidation (FAO) dysfunction, is a major driver of renal fibrosis; however, until recently the mechanisms remained unclear. Recently, scientists demonstrated an association between STAT6 and tubular lipid metabolism in fibrotic kidneys. Specifically, STAT6 was activated along with the accumulation of lipids via the downregulation of FAO-related genes when mice were subjected to unilateral ureteral obstruction or high-fat diet challenge. Tubular-specific depletion, or pharmacologic inhibitor of STAT6 in mice, and STAT6 knockdown in cultured tubular cells attenuated lipid accumulation and renal fibrosis by enhancing FAO. Mechanistically, STAT6 transcriptionally inhibited the expression of PPARα (OMIM 170998) and its FAO-related target genes through a sis-inducible element located in the promoter region of the protein.

[0068] Although we focused initially on the above targets, there are many known liver specific targets that have known associations with liver disease, such as HGF (OMIM 604375), ATP7B (OMIM 606882), ABCB4 (OMIM 171060), ALDOB (OMIM 612724, GBE1 (OMIM 607839), FAH (OMIM 613871), ASL (OMIM 608310), SLC25A13 (OMIM 603859), LIPA (OMIM 613497), SERPINA1 (OMIM 107400), CFTR (602421), HFE (OMIM 613609), KRT18 (OMIM 215600), possibly MTDPS4A (OMIM 215600), CASP8 (OMIM 601763), CTNNB1 (OMIM 116806), PIK3CA (OMIM 171834), APC (OMIM 611731), IGF2R (OMIM 147280), MET (OMIM 164860), PDGFRL (OMIM 604584), AXIN (OMIM 603816), TP53 (OMIM 191170), FOCAD (OMIM 614606), MU (OMIM 613282), TRIM37 (OMIM 605073), MARS1 (OMIM 156560), PBC1 (OMIM 109720), LBR (OMIM 600024), KIF12 (OMIM 611278), PBC3 (OMIM 613008), PCB4 (OMIM 614220), PCB5 (OMIM 614221), to name a few. There is insufficient time and space to describe each of these liver cirrhosis/cancer targets in detail, but each OMIM entry cited (and their links) are incorporated by reference in their entireties for all purposes. Further, the OMIM entries are hyperlinked to the relevant DNA and protein sequences, for use as targets in the MiniVectors described herein. Various inhibitory RNA sequences to these targets can be found in the many RNAi databases, such as RNACentral, GenomeRNAi.org, the RNAi Consortium's SHRNA library (stocks distributed by SigmaAldrich), the RNAiAtlas, DocleraWiki, NCBI, the RNAi Codex, to name just a few.

[0069] One payload for which we already have significant efficacy data is the FOXM1 shRNA (targeting 5′-ATAATTAGAGGATAATTTG-3′). Additional shRNA payloads of strong interest are CDH11, and MDM2 and 4. Other payloads encode genes that promote apoptosis (e.g., p53).

[0070] Any shRNAs or other RNAi sequences can be designed using freely available, open access, algorithms (e.g., siRNA Wizard™ Software, siDESIGN Center, etc.) and then screened for off-target effects using NCBI-BLAST. Alternatively, there are significant numbers of commercially available sequences that can be used for initial proof of concept work. SigmaAldrich, for example has more than 200,000 individual sequences available, including more than 20,000 prescreened human specific sequences from the TRC1.5 and TRC2 collections.

[0071] 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. Tables 1-6 show exemplary payload and MiniVector sequences. Additional sequences and various disease targets are discussed in U.S. Ser. No. 16/180,046, incorporated by reference in its entirety for all purposes.

TABLE-US-00001 TABLE 1 Payload therapeutic sequences that may be encoded on an ultrapure MiniVector Dharmacon SEQ ID NO Gene Description Cat. No. Mature Antisense 1. FOXM1 Forkhead V2LHS_283849 ATAATTAGAGGATAATTTG box protein M1 Q08050 2. FOXM1 V3LHS_396939 ATTGTTGATAGTGCAGCCT 3. FOXM1 V3LHS_396937 TGAATCACAAGCATTTCCG 4. FOXM1 V3LHS_396941 TGATGGTCATGTTCCGGCG 5. FOXM1 V3LHS_396940 AATAATCTTGATCCCAGCT 6. FOXM1 V3LHS_314369 TACTGAGGAATATTGTGCT 7. MDM4 O15151 V2LHS_11941 TATGTACTGACCTAAATAG 8. MDM4 V2LHS_151660 ATCTGAATACCAATCCTTC 9. MDM4 V3LHS_356802: TGAACACTGAGCAGAGGTG 10. MDM4 V3LHS_356797: AACAGTGAACATTTCACCT

TABLE-US-00002 TABLE 2 MiniVector elements Table 2. MiniVector elements Module Element Description Use A λ-attL attL from the λ-integrase system Recombination sites (product of site- specific recombination used to generate MiniVector). Sequences listed in Table 3. λ-atR attR from the λ-integrase system λ-atB attB from the λ-integrase system λ-attP attP from the λ-integrase system loxP loxP site for Cre recombinase γδ-res res site for the γδ (Tn1000) resolvase FRT FRT site for Flp recombinase hixL hixL site for Hin recombinase 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 dehydrogenase 1 Initiation of (ALDH1) transcription. Includes promoters for RNA polymerase II and RNA polymerase III. Full sequences of selected promoters provided in Table 4. AMY1C Tissue-specific promoter of human amylase alpha 1C (AMY1C) β-actin Promoter from the (human) beta actin gene CaMKIIα Ca2+/calmodulin-dependent protein kinase II alpha promoter CMV Promoter from the human cytomegalovirus (CMV) Mini CMV Minimized version of CMV CAG CMV early enhancer/chicken p actin promoter (CAG). Synthetic hybrid promoter made from a) the CMV early enhancer element, b) the promoter, the first 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 and 19 keratin 18 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 gene transcript. Sequences for use in target validation are expression through listed in Table 1. Potential therapeutic sequences will be RNA interference designed de novo and optimized for knockdown efficiency. miRNA (DNA) sequence encoding micro-RNA (miRNA) transcript IhRNA (DNA) sequence encoding long hairpin RNA (IhRNA) transcript IncRNA (DNA) sequence encoding long non-coding RNA Knockdown of gene (IncRNA) transcript expression (not RNAi) piRNA (DNA) sequence encoding piwi-interacting (piRNA) RNA transcript D Transcriptional terminator sequence (Any can be used) E S/MAR Scaffold/matrix attached region from eukaryotic Episomal replication chromosomes (Sequences in Table 5) CpG Unmethylated deoxycytidyl-deoxyguanosine (CpG) Immunostimulatory motifs dinucleotides: (Sequences in Table 5) activity F/G β-globin Intron of the human β globin gene (130 bp) Gene expression intron enhancer HGH Intron of the human growth hormone gene (262 bp) intron H SV40 Simian virus 40 early promoter (351 bp) Nuclear localization early promoter NF-κβ Binding site of nuclear factor kappa-light-chain-enhancer of activated B cells (55 bp (5 repeats of GGGGACTTTCC SEQ ID NO 11)) p53 NLS Binding site of tumor protein 53 (p53): AGACTGGGCATGTCTGGGCA SEQ ID NO 12 p53 NLS Binding site of tumor protein 53 (p53): GAACATGTCCCAACATGTTG SEQ ID NO 13 Adeno- GGGGCTATAAAAGGG SEQ ID NO 14 virus major late promoter

TABLE-US-00003 TABLE 3 Complete sequences for element A (underline = recombination sites) SEQ ID NO Site Sequence (5′-3′) 15. λ-attL TCCGTTGAAGCCTGCTTT TAAGTTGGCATTATAAAAAAGCATTG CTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCAT TATT 16. λ-attR AGATGCCTCAGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTC ATAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTAT GCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTT CAGCTTT TAACTTGAGCGAAACG 17. λ-attB TCCGTTGAAGCCTGCTTT TAACTTGAGCGAAACG 18. λ-attP AGATGCCTCAGCTCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTC ATAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTAT GCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTT CAGCTTT TAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTT GTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATT 19. loxP ATAACTTCGTATAGCATACATTATACGAAGTTAT 20. γδ-res ATTTTGCAACCGTCCGAAATATTATAAATTATCGCACACATAAAAACAGTG CTGTTAATGTGTCTATTAAATCGATTTTTTGTTATAACAGACACTGCTTGT CCGATATTTGATTTAGGATACATTTTTA 21. FRT GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC 22. hixL TTCTTGAAAACCAAGGTTTTTGATAA 23. hixR TTTTCCTTTTGGAAGGTTTTTGATAA 24. Tn3 res CAACCGTTCGAAATATTATAAATTATCAGACATAGTAAAACGGCTTCGTTT GAGTGTCCATTAAATCGTCATTTTGGCATAATAGACACATCGTGTCTGATA TTCGATTTAAGGTACATTT 25. Tn21 res GCCGCCGTCAGGTTGAGGCATACCCTAACCTGATGTCAGATGCCATGTGTA AATTGCGTCAGGATAGGATTGAATTTTGAATTTATTGACATATCTCGTTGA AGGTCATAGAGTCTTCCCTGACAT 26. GGTGCGTACAATTAAGGGATTATGGTAAAT 27. GGTGCGCGCAAGATCCATTATGTTAAAC

TABLE-US-00004 TABLE 4 Complete sequences for element B (promoters) SEQ ID NO Promoter Sequence (5′-3′) 28. CMV GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTA GTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT 29. mini-CMV CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGA CGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT 30. RSV GGTGCACACCAATGTGGTGAATGGTCAAATGGCGTTTATTGTATCGAGCT AGGCACTTAAATACAATATCTCTGCAATGCGGAATTCAGTGGTTCGTCCA ATCCATGTCAGACCCGTCTGTTGCCTTCCTAATAAGGCACGATCGTACCA CCTTACTTCCACCAATCGGCATGCACGGTGCTTTTTCTCTCCTTGTAAGG CATGTTGCTAACTCATCGTTACCATGTTGCAAGACTACAAGAGTATTGCA TAAGACTACATT 31. CAG GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAAT AGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCC CCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGC GGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAAT CAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCG GCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG 32. EF1a GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAG AAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCG CGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT TTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTC CCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTA CTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGT TGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGC CTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGA ATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGC CATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATA GTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGG GCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAG GCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAG CTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGC GCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTT CCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTT GGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAG ACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGC CCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTC AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA 33. EFS ATCGATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAG TCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGA AGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT TTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGA ACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGACGC G 34. Human GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACG β-actin GCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTC CGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAG AACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCT AGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTC CCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGAT GATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCG GGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGT GAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCT CGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTC CGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATG GCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGTGA GGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTC TTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTG GGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACT CGGTTTGTCGTCTGTTGCGGGGGCGGCAGTTATGGCGGTGCCGTTGGGCA GTGCACCCGTACCTTTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCAC CCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTG CGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGC TCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGA GGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTG AAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGA AGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATT TTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCT TTTTTGTTAGAC 35. NFK-β GCTAGCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCAGAT CTGCCGCCCCGACTGCATCTGCGTGTTCGAATTCGCCAATGACAAGACGC TGGGCGGGGTTTGTGTCATCATAGAACTAAAGACATGCAAATATATTTCT TCCGGGGACACCGCCAGCAAACGCGAGCAACGGGCCACGGGGATGAAGCA GAAGCTTGGCA 36. Ubiquitin-C GTCTAACAAAAAAGCCAAAAACGGCCAGAATTTAGCGGACAATTTACTAG TCTAACACTGAAAATTACATATTGACCCAAATGATTACATTTCAAAAGGT GCCTAAAAAACTTCACAAAACACACTCGCCAACCCCGAGCGCATAGTTCA AAACCGGAGCTTCAGCTACTTAAGAAGATAGGTACATAAAACCGACCAAA GAAACTGACGCCTCACTTATCCCTCCCCTCACCAGAGGTCCGGCGCCTGT CGATTCAGGAGAGCCTACCCTAGGCCCGAACCCTGCGTCCTGCGACGGAG AAAAGCCTACCGCACACCTACCGGCAGGTGGCCCCACCCTGCATTATAAG CCAACAGAACGGGTGACGTCACGACACGACGAGGGCGCGCGCTCCCAAAG GTACGGGTGCACTGCCCAACGGCACCGCCATAACTGCCGCCCCCGCAACA GACGACAAACCGAGTTCTCCAGTCAGTGACAAACTTCACGTCAGGGTCCC CAGATGGTGCCCCAGCCCATCTCACCCGAATAAGAGCTTTCCCGCATTAG CGAAGGCCTCAAGACCTTGGGTTCTTGCCGCCCACCATGCCCCCCACCTT GTTTCAACGACCTCACAGCCCGCCTCACAAGCGTCTTCCATTCAAGACTC GGGAACAGCCGCCATTTiGCTGCGCTCCCCCCAACCCCCAGTTCAGGGCA ACCTTGCTCGCGGACCCAGACTACAGCCCTTGGCGGTCTCTCCACACGCT TCCGTCCCACCGAGCGGCCCGGCGGCCACGAAAGCCCCGGCCAGCCCAGC AGCCCGCTACTCACCAAGTGACGATCACAGCGATCCACAAACAAGAACCG CGACCCAAATCCCGGCTGCGACGGAACTAGCTGTGCCACACCCGGCGCGT CCTTATATAATCATCGGCGTTCACCGCCCCACGGAGATCCCTCCGCAGAA TCGCCGAGAAGGGACTACTTTTCCTCGCCTGTTCCGCTCTCTGGAAAGAA AACCAGTGCCCTAGAGTCACCCAAGTCCCGTCCTAAAATGTCCTTCTGCT GATACTGGGGTTCTAAGGCCGAGTCTTATGAGCAGCGGGCCGCTGTCCTG AGCGTCCGGGCGGAAGGATCAGGACGCTCGCTGCGCCCTTCGTCTGACGT GGCAGCGCTCGCCGTGAGGAGGGGGGCGCCCGCGGGAGGCGCCAAAACCC GGCGCGGAGGC 37. SV40 GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATG CATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGC AGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCC CGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCAT TCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGC CGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAG GCCTAGGCTTTTGCAAA 38. PGK CCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCT TCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTC GTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGA TGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGC CAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGG TGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCC GAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCT GCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCC 39. H1 AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAAT GTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACAGATCCC 40. U6 GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTT GTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATAT TTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGT TCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAG AGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTC ACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGG AGAAAAGCCTTGTT

TABLE-US-00005 TABLE 5 Complete sequences for elements E, F and G (accessory sequences) SEQ ID NO Element Sequence (5′-3′) 41. 250 bp S/MAR TCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAGA ATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAG AATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTA GAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTT AGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATT TAGAA 42. 439 bp S/MAR TCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAGA ATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTAG AATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTTA GAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATTT AGAATCTTTAATTTCTAATATATTTAGAATCTTTAATTTCTAATATATT TAGAA 43. (45 bp) Type A GGTGCATCGATGCAGCATCGAGGCAGGTGCATCGATACAGGGGGG Cpg motif 44. (24 bp) Type B TCGTCGTTTTGTCGTTTTGTCGTT Cpg motif 45. (21 bp) Type C TCGTCGAACGTTCGAGATGAT CpG motif 46. β-globin GTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGG intron GCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCT CTCTGCCTATTGGTCTATTTTCCCACCCTTAG 47. Human growth TTCGAACAGGTAAGCGCCCCTAAAATCCCTTTGGGCACAATGTGTCCTG hormone intron AGGGGAGAGGCAGCGACCTGTAGATGGGACGGGGGCACTAACCCTCAGG TTTGGGGCTTCTGAATGTGAGTATCGCCATGTAAGCCCAGTATTTGGCC AATCTCAGAAAGCTCCTGGTCCCTGGAGGGATGGAGAGAGAAAAACAAA CAGCTCCTGGAGCAGGGAGAGTGCTGGCCTCTTGCTCTCCGGCTCCCTC TGTTGCCCTCTGGTTTC

TABLE-US-00006 TABLE 6 Relaxin and P53 sequences SEQ ID No cDNA Sequence (5′ to 3′) RLN2 48. ATGCCTCGCCTGTTTTTTTTCCACCTGCTAGGAGTCTGTTTACTACTGA (human) ACCAATTTTCCAGAGCAGTCGCGGACTCATGGATGGAGGAAGTTATTAA ATTATGCGGCCGCGAATTAGTTCGCGCGCAGATTGCCATTTGCGGCATG AGCACCTGGAGCAAAAGGTCTCTGAGCCAGGAAGATGCTCCTCAGACAC CTAGACCAGTGGCAGAAATTGTGCCATCCTTCATCAACAAAGATACAGA AACCATAAATATGATGTCAGAATTTGTTGCTAATTTGCCACAGGAGCTG AAGTTAACCCTGTCTGAGATGCAGCCAGCATTACCACAGCTACAACAAC ATGTACCTGTATTAAAAGATTCCAGTCTTCTCTTTGAAGAATTTAAGAA ACTTATTCGCAATAGACAAAGTGAAGCCGCAGACAGCAGTCCTTCAGAA TTAAAATACTTAGGCTTGGATACTCATTCTCGAAAAAAGAGACAACTCT ACAGTGCATTGGCTAATAAATGTTGCCATGTTGGTTGTACCAAAAGATC TCTTGCTAGATTTTGCTGA RLN1 49. ATGTCCAGCAGATTTTTGCTCCAGCTCCTGGGGTTCTGGCTATTGCTGA (mouse) GCCAGCCTTGCAGGACGCGAGTCTCGGAGGAGTGGATGGACGGATTCAT TCGGATGTGCGGCCGTGAATATGCCCGTGAATTGATCAAAATCTGCGGG GCCTCCGTGGGAAGATTGGCTTTGAGCCAGGAGGAGCCAGCTCTGCTTG CCAGGCAAGCCACTGAAGTTGTGCCATCCTTCATCAACAAAGATGCAGA GCCTTTCGATACGACGCTGAAATGCCTTCCAAATTTGTCTGAAGAGCTC AAGGCAGTACTGTCTGAGGCTCAGGCCTCGCTCCCAGAGCTACAACACG CACCTGTGTTGAGCGATTCTGTTGTTAGCTTGGAAGGCTTTAAGAAAAC TCTCCATGATAAACTGGGTGAAGCAGAAGACGGCAGTCCTCCAGGGCTT AAATACTTGCAATCAGATACCCATTCACGGAAAAAGAGGGAGTCTGGTG GATTGATGAGCCAGCAATGTTGCCACGTCGGTTGTAGCAGAAGATCTAT TGCTAAACTCTATTGC Amino Acid Sequence (5′ to 3′) P53 50. MEEPQSDPSV EPPLSQETFS DLWKLLPENN VLSPLPSQAM isoform DDLMLSPDDI EQWFTEDPGP DEAPRMPEAA PPVAPAPAAP a TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS SCMGGMNRRP ILTIITLEDS SGNLLGRNSF EVRVCACPGR DRRTEEENLR KKGEPHHELP PGSTKRALPN NTSSSPQPKK KPLDGEYFTL QIRGRERFEM FRELNEALEL KDAQAGKEPG GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD 51. MFCQLAKTCP VQLWVDSTPP PGTRVRAMAI YKQSQHMTEV VRRCPHHERC SDSDGLAPPQ HLIRVEGNLR VEYLDDRNTF RHSVVVPYEP PEVGSDCTTI HYNYMCNSSC MGGMNRRPIL TIITLEDSSG NLLGRNSFEV RVCACPGRDR RTEEENLRKK GEPHHELPPG STKRALPNNT SSSPQPKKKP LDGEYFTLQI RGRERFEMFR ELNEALELKD AQAGKEPGGS RAHSSHLKSK KGQSTSRHKK LMFKTEGPDS D

TABLE-US-00007 TABLE 7 Additional expression sequences for STAT6 and TGFB1 targets The bold underlined TTCAAGAGA is the 9-mer loop SEQ ID of the stemloop of the shRNA structure. Gene NO This sequence is from pSUPER. Mouse 52. AGACCTGTCCATTCGCTCA TGAGCGAATGGACAGGTCT STAT6 Mouse 53. GCATCTTGCCGCACATCAG STAT6 Mouse 54. GAGTAAGGGAGACCCGGCT STAT6 Mouse 55. CCTGATGCTTCCATGCAAC STAT6 Human 56. CAATTCCTGGCGATACCTC GAGGTATCGCCAGGAATTG TGFB1 Human 57. CCAACATGATCGTGCGCTC TGFB1 Human 58. AGAACTGCTGCGTGCGGCA TGFB1 Human 59. TCGCCAGAGTGGTTATCTT TGFB1 Mouse 60. ATACGTCAGACATTCGGGA TGFB1 Mouse 61. ACGCCTGAGTGGCTGTCTT TGFB1 Mouse 62. GAAACGGAAGCGCATCGAA TGFB1 Mouse 63. CCAAGGGCTACCATGCCAA TGFB1

[0072] The following references are incorporated by reference in their entirety for all purposes. [0073] Catanese, D. J., et al., Supercoiled MiniVector DNA resists shear forces associated with gene therapy delivery, Gene Ther. 19(1): 94-100 (2012). [0074] Darquet A. M., et al., Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer, Gene Ther., 6: 209-218 (1999). [0075] Fogg, J. M., et al., Exploring writhe in supercoiled minicircle DNA. J. Phys.—Condes. Matter, 18: S145-S159 (2006). [0076] Hardee, C. L., Advances in non-viral DNA vectors for gene therapy, Genes 8, 65 (2017) [0077] Hornstein, B. D., et al., Effects of circular DNA length on transfection efficiency by electroporation into HeLa cells, PLoS One. 11(12): e0167537 (2016). [0078] Lis and Schleif, Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. Nucleic Acids Research. 2, 383-389 (1975). [0079] US20150376645, US20140056868, 61/653,279, filed May 30, 2012, Supercoiled MiniVectors as a tool for DNA repair, alteration and replacement [0080] 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 [0081] U.S. Pat. No. 7,622,252, US20070020659, 60/689,298, filed Jun. 10, 2005, Generation of minicircle DNA with physiological supercoiling [0082] 63/243,087 Ultrapure minivectors for gene therapy [0083] US20060211117 Methods of making minicircles [0084] WO1994009127 Supercoiled minicircle DNA as a unitary promoter vector [0085] WO2002083889 Methods for the production of minicircles [0086] Ramamoorth M., & Narvekar, A., Non viral vectors in gene therapy—An overview, J. Clinical & Diagnostic Res. 2015 January, Vol-9(1): GE01-GE06. [0087] Hidai C., & Kitano, H., Nonviral gene therapy for cancer: A review, Diseases 2018, 6, 57.

[0088] The following UniProt cites include all sequences taught/linked therein:

[0089] P04637, P04090, Q08050, P55287, Q00987, O15151, P40763.