Minicircle Vector Research Articles

A novel minicircle vector based system for inhibting the replication and gene expression of Enterovirus 71 and Coxsackievirus A16

Enterovirus 71 (EV 71) and Coxsackievirus A16 (CA 16) are two major causative agents of hand, foot and mouth disease (HFMD). They have been associated with severe neurological and cardiological complications worldwide, and have caused significant mortalities during large-scale outbreaks in China. Currently, there are no effective treatments against EV 71 and CA 16 infections. We now describe the development of a novel minicircle vector based RNA interference (RNAi) system as a therapeutic approach to inhibiting EV 71 and CA 16 replication. Small interfering RNA (siRNA) molecules targeting the conserved regions of the 3Cpro and 3Dpol function gene of the EV 71 and CA 16 China strains were designed based on their nucleotide sequences available in GenBank. This RNAi system was found to effectively block the replication and gene expression of these viruses in rhabdomyosarcoma (RD) cells and virus-infected mice model. The inhibitory effects were confirmed by a corresponding decrease in viral RNA, viral protein, and progeny virus production. In addition, no significant adverse off-target silencing or cytotoxic effects were observed. These results demonstrated the potential and feasibility of this novel minicircle vector based RNAi system for antiviral therapy against EV 71 and CA 16 infection.

Efficient Reprogramming of Human Cord Blood CD34+ Cells Into Induced Pluripotent Stem Cells With OCT4 and SOX2 Alone

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure. PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively. We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.

Stabilization of hypoxia-inducible factor-1 via novel minicircle vector improves diabetic wound healing

Stabilization of hypoxia-inducible factor-1 via novel minicircle vector improves diabetic wound healing

Enhanced transgene expression improves immune responses following DNA vaccination (53.1)

Abstract Plasmid DNA (pDNA)-based gene delivery has the potential to advance vaccine development due to advantages such as low cost, ease of production, safety (e.g. pDNA does not cause insertional mutagenesis), and the flexibility to introduce several genes at once. To date, there are more than 300 clinical trials involving pDNA-based gene transfer. Although promising, these trials have revealed that poor transfection efficiency, transient protein production, and low immunogenicity limit therapy efficacy. We are investigating the use of minicircle (MC) DNA as a means of circumventing these limitations. MC vectors contain the expression cassette of full-length plasmids, but lack the bacterial backbone sequences that are targeted for transcriptional repression in mammalian cells. Removal of these plasmid backbone sequences is achieved via site-specific recombination in Escherichia coli prior to episomal (i.e. MC) DNA isolation. The absence of these prokaryotic elements also reduces MC size relative to its parental pDNA, leading to enhanced transfection efficiencies. Data will be presented showing that vaccination with MC DNA results in significantly higher and more prolonged reporter transgene expression. Furthermore, we compare specific responses of adoptively transferred and endogenous CD8+ T cells following vaccination of mice with either parental pDNA or MC-encoded antigen.

Minicircle DNA-based Gene Therapy Coupled With Immune Modulation Permits Long-term Expression of α-L-Iduronidase in Mice With Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease characterized by mutations to the α-L-iduronidase (IDUA) gene resulting in inactivation of the IDUA enzyme. The loss of IDUA protein results in the progressive accumulation of glycosaminoglycans within the lysosomes resulting in severe, multi-organ system pathology. Gene replacement strategies have relied on the use of viral or nonviral gene delivery systems. Drawbacks to these include laborious production procedures, poor efficacy due to plasmid-borne gene silencing, and the risk of insertional mutagenesis. This report demonstrates the efficacy of a nonintegrating, minicircle (MC) DNA vector that is resistant to epigenetic gene silencing in vivo. To achieve sustained expression of the immunogenic IDUA protein we investigated the use of a tissue-specific promoter in conjunction with microRNA target sequences. The inclusion of microRNA target sequences resulted in a slight improvement in long-term expression compared to their absence. However, immune modulation by costimulatory blockade was required and permitted for IDUA expression in MPS I mice that resulted in the biochemical correction of pathology in all of the organs analyzed. MC gene delivery combined with costimulatory pathway blockade maximizes safety, efficacy, and sustained gene expression and is a new approach in the treatment of lysosomal storage disease.

Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver

We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.

Transgene Loss and Changes in the Promoter Methylation Status as Determinants for Expression Duration in Nonviral Gene Transfer for Factor IX

Advances in delivery techniques and in expression construct design have renewed interest in nonviral gene transfer. Here, we test plasmid or bacterial backbone free minicircle vectors for factor IX (FIX) expression by hydrodynamic liver-directed delivery. Both constructs are driven by a hepatic control region, the human α(1)-antitrypsin promoter, which results in long-term expression in FIX knockout mice. However, levels of expression were higher and expression loss over time was reduced when using minicircles. Even at the highest expression levels (>700% of normal) FIX was fully functional. Transgene loss was the main determinant for expression loss over time for both vector types. A significant effect of gene silencing was observed only for the plasmid, not for the minicircle vector. To determine the influence of promoter methylation, we performed bisulfite-mediated conversion and sequencing of vector DNA on days 14 and 100 after gene transfer. We determined a higher frequency of methyl-protected cytosines in CpGs and a lower degree of demethylation at bacterial Dcm methylation sequences near transcription factor-binding sites in the α(1)-antitrypsin promoter in plasmid compared with minicircle mice on day 100. Therefore, the methylation status might reflect differences in the levels and durability of expression. Judging from the high levels of functional FIX obtained, small fractions of liver or single liver segments should be sufficient to reach therapeutic efficacy in translating hydrodynamic delivery to humans. However, transgene loss remains to be addressed to further guarantee sustained expression over time.

MicroRNA-210 as a Novel Therapy for Treatment of Ischemic Heart Disease

MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart. Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation. MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.

A nonviral minicircle vector for deriving human iPS cells

Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.

Tightly-wound miniknot vectors for gene therapy: A potential improvement over supercoiled minicircle DNA

Minimized derivatives of bacterial plasmids with removed bacterial backbones are promising vectors for the efficient delivery and for the long-term expression of therapeutic genes. The absence of the bacterial plasmid backbone, a known inducer of innate immune response and a known silencer of transgene expression, provides a partial explanation for the high efficiency of gene transfer using minimized DNA vectors. Supercoiled minicircle DNA is a type of minimized DNA vector obtained via intra-plasmid recombination in bacteria. Minicircle vectors seem to get an additional advantage from their physical compactness, which reduces DNA damage due to the mechanical stress during gene delivery. An independent topological means for DNA compression is knotting, with some knotted DNA isoforms offering superior compactness. I propose that, firstly, knotted DNA can be a suitable compact DNA form for the efficient transfection of a range of human cells with therapeutic genes, and, secondly, that knotted minimized DNA vectors without bacterial backbones ("miniknot" vectors) can surpass supercoiled minicircle DNA vectors in the efficiency of therapeutic gene delivery. Crucially, while the introduction of a single nick to a supercoiled DNA molecule leads to the loss of the compact supercoiled status, the introduction of nicks to knotted DNA does not change knotting. Tight miniknot vectors can be readily produced by the direct action of highly concentrated type II DNA topoisomerase on minicircle DNA or, alternatively, by annealing of the 19-base cohesive ends of the minimized vectors confined within the capsids of Escherichia coli bacteriophage P2 or its satellite bacteriophage P4. After reaching the nucleoplasm of the target cell, the knotted DNA is expected to be unknotted through type II topoisomerase activity and thus to become available for transcription, chromosomal integration or episomal maintenance. The hypothesis can be tested by comparing the gene transfer efficiency achieved with the proposed miniknot vectors, the minicircle vectors described previously, knotted plasmid vectors and standard plasmid vectors. Tightly-wound miniknots can be particularly useful in the gene administration procedures involving considerable forces acting on vector DNA: aerosol inhalation, jet-injection, electroporation, particle bombardment and ultrasound DNA transfer.

Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction

Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel nonviral vector that robustly and persistently expresses the hypoxia-inducible factor-1 alpha (HIF-1alpha) therapeutic gene in the heart, leading to functional benefits after myocardial infarction. We first created minicircles (MC) carrying double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB/N mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging showed stable MC gene expression in the heart for >12 weeks and the activity level was 5.6+/-1.2-fold stronger than regular plasmid at day 4 (P<0.01). Next, we created MC carrying HIF-1alpha (MC-HIF-1alpha) therapeutic gene for treatment of myocardial infarction. Adult FVB/N mice underwent left anterior descending ligation and were injected intramyocardially with: (1) MC-HIF-1alpha; (2) regular plasmid carrying HIF-1alpha (PL-HIF-1alpha) as positive control; and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction in the MC group (51.3%+/-3.6%) compared to regular plasmid group (42.3%+/-4.1%) and saline group (30.5%+/-2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed MC express >50% higher HIF-1alpha level than regular plasmid. Taken together, this is the first study to our knowledge to demonstrate that MC can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel nonviral vector can be of great value for cardiac gene therapy protocols.

910. Gene Transfer to Mouse Heart – A Study of Mini-Circle Vector Efficiency

910. Gene Transfer to Mouse Heart – A Study of Mini-Circle Vector Efficiency

749. Development of S/MAR Minicircle Vectors for Persistent Expression In Vivo

749. Development of S/MAR Minicircle Vectors for Persistent Expression In Vivo

Gene Transfer to Mouse Heart and Skeletal Muscles Using a Minicircle Expressing Human Vascular Endothelial Growth Factor

: Gene transfer to heart muscle is a promising modality to treat ischemic heart disease. However, current vectors are inefficient and need to be improved. A novel vector system that shows great promise is the minicircle (MC) vector being smaller than conventional plasmid vectors and devoid of bacterial sequences. : To study gene transfer of MC DNA, expressing the human vascular endothelial growth factor (hVEGF), to mouse heart and skeletal muscles and to compare it with one of the efficient plasmids used in cardiovascular trials, the phVEGF165 containing the same expression cassette as the MC. : The MC and the phVEGF165 plasmid show similar expression patterns both in vitro and in mouse heart and skeletal muscle studies in vivo on molar basis (equal expression in heart 24 hours, 0.9 fold lower expression from MC in heart and 1.9 fold higher in skeletal muscle at 7 days), whereas on weight basis the MC construct was more efficient in skeletal muscle (5.6 fold higher expression, P < 0.05), and at least as efficient in heart (1.6 fold higher expression). : The gene expression is similar in the 2 vector systems, so the smaller size and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene therapy.

Minicircle-IFNγ Induces Antiproliferative and Antitumoral Effects in Human Nasopharyngeal Carcinoma

The aims of this work were to investigate the antitumor effect of IFNgamma gene transfer on human nasopharyngeal carcinoma (NPC) and to assess the potential of minicircle vector for antitumor gene therapy. We developed a recombinant minicircle vector carrying the human IFNgamma gene and evaluated the effects of minicircle-mediated IFNgamma gene transfer on NPC cell lines in vitro and on xenografts in vivo. Relative to p2PhiC31-IFNgamma, minicircle-mediated IFNgamma gene transfer in vitro resulted in 19- to 102-fold greater IFNgamma expression levels in transfected cells (293, NIH 3T3, CNE-1, CNE-2, and C666-1) and inhibited the growth of CNE-1, CNE-2, and C666-1 cells more efficiently, reducing relative growth rates to 7.1 +/- 1.6%, 2.7 +/- 1.0%, and 6.1 +/- 1.6%, respectively. Flow cytometry and caspase-3 activity assays suggested that the antiproliferative effects of IFNgamma gene transfer on NPC cell lines could be attributed to G(0)-G(1) arrest and apoptosis. Minicircle-mediated intratumoral IFNgamma expression in vivo was 11 to 14 times higher than p2PhiC31-IFNgamma in CNE-2- and C666-1-xenografted mice and lasted for 21 days. Compared with p2PhiC31-IFNgamma treatment, minicircle-IFNgamma treatment significantly increased survival and achieved inhibition rates of 77.5% and 83%, respectively. Our data indicate that IFNgamma gene transfer exerts antiproliferative effects on NPC cells in vitro and leads to a profound antitumor effect in vivo. Minicircle-IFNgamma is more efficient than corresponding conventional plasmids due to its capability of mediating long-lasting high levels of IFNgamma gene expression. Therefore, minicircle-mediated IFNgamma gene transfer is a promising novel approach in the treatment of NPC.

RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia colistrain for Cre mediated production of minicircle DNA

BackgroundMinicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions.ResultsWe describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination.ConclusionUsing transient RecET-driven recombination we inserted a single copy of the araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtained recA- minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors.

516. Development of a Minicircle Vector Free of Plasmid Bacterial DNA Sequences and Capable of øC31-Mediated Site-Specific Integration

Top of pageAbstract It has been well documented that plasmid bacterial DNA sequences (plasmid BB) can silence the expression of an episomal or integrated transgene. Current site-specific integrating technologies are hampered in part by the fact that the integrated transgene expression cassette is covalently linked with the plasmid BB. As a result, a substantial proportion of integrated transgenes are expressed at reduced levels or silenced. We have previously reported the development of a robust method for producing episomal minicircle vectors that are devoid of plasmid BB and capable of persistent and high level transgene expression in quiescent tissues in vivo. The minicircle was generated from a parental plasmid, which contained an expression cassette flanked with the short form recombination sites, sattB and sattP, two copies of the recombinase |[oslash]|C31 and one copy of the endonuclease I-Sce I genes under the control of the inducible promoter BAD. An I-Sce I site was engineered into the plasmid BB. Minicircle containing the therapeutic transgene was generated by the intramolecular recombination between the sattB and sattP sites mediated by the |[oslash]|C31 recombinase after the addition of the inducer L-Arabinose to the bacterial culture. A second DNA circle composed of plasmid BB sequences together with the I-Sce I site was also formed but was degraded after linearization by the I-Sce I endonuclease. This resulted in milligram yields of minicircle DNA from a liter of culture that was then purified by simple column chromatography. To extend the application of the minicircle to rapidly replicating cells, we elected to make a site-selective integrating form of this vector. To do this, we devised a strategy to include a functional sattB site into the minicircle. We inserted an additional sattB site between the sattP and the expression cassette, and replaced the original upstream sattB with a full length attB (LattB), resulting in the efficient recombination between the LattB and sattP, and a high yield of sattB-containing minicircle (sattB.MC). An in vitro study demonstrated that the sattB site is capable of mediating efficient integration. To test for in vivo integration, 10 |[mu]|g of the integrating sattB.MC expressing human factor IX (hFIX) was infused into mouse liver together with 20 |[mu]|g of a plasmid expressing either wild type or mutant |[oslash]|C31. Seven weeks later a surgical two-thirds partial hepatectomy (PH) was performed. The procedure induces liver regeneration resulting in the loss of about 95% of the episomal DNA. Nine weeks after PH, the mice receiving wild type and mutant |[oslash]|C31 expressed 40 and 15% of serum hFIX, respectively as compared to corresponding non-PH group. Southern blot analysis confirmed integration of attB.MC in the genome of mice receiving wild type |[oslash]|C31, but not mice treated with mutant |[oslash]|C31. These results suggest that the sattB.MC is capable of integration in vivo. Further studies are underway to establish the advantages of the integrating minicircle vector.

141. Development of a One-Step Column Purification of Minicircle Vector DNA Devoid of Bacterial Sequences That Results in High Level Persistent Transgene Expression In Vivo

We have shown that episomal circular DNA vectors free of plasmid bacterial DNA sequences are capable of indefinite high level transgene expression in vivo. The minicircle vector is generated in bacteria from a parental plasmid containing an inducible phiC31 integrase gene, and att P and att B recognition sites flanking the therapeutic expression cassette. Recombination results in the formation of two circular DNA molecules, one containing the plasmid bacterial backbone and the other the eukaryotic expression cassette. Previously, the minicircle was purified away from the plasmid bacterial DNA by cesium chloride banding in an ultracentrifuge (Chen et al., Mol Ther. 8:495, 2003). We have now devised a one-step purification method that results in a high yield of minicircle vector. To do this, two copies of the phiC31 integrase gene under the control of an inducible promoter (BAD), together with the transgene expression cassette flanked by the attB and attP sites, were built into a minicircle-producing plasmid, p2phiC31. An intron encoded restriction enzyme I-Sce I gene, also under the control of the BAD promoter, together with an I-Sce I restriction site, were also built into p2phiC31. Top 10 E. coli was used to amplify p2phiC31 using standard plasmid preparation. To induce the phiC31 integrase-mediated minicircle formation, an over night culture was re-suspended 4 to 1 (vol/vol) in fresh LB broth containing the pBAD inducer 1% L-arabinose and incubated at 32°C with constant shaking for one hour. The temperature of the incubation was increased to 37°C, to optimize I-Sce I enzymatic activity, for an additional 2 hours. Linearization of the plasmid bacterial DNA circle by the restriction enzyme resulted in its rapid degradation by exonucleases in bacteria. The mini-circle expression cassette becomes the only episomal circular DNA and is easily isolated by an affinity purification column. Using this technology to prepare a minicircle encoding a 4 kb human factor IX expression cassette, > 1 mg of minicircle of 97 percent purity can be prepared from 1 liter of over night bacterial growth. When injected into the livers of animals by hydrodynamic infusion, about 10 μg/ml of serum hFIX was expressed for up to 7 weeks (length of study). The high yield, simple purification, and robust and persistence of transgene expression makes these vectors viable for in vivo gene therapy applications.

Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo.

We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.