Minicircle DNA Research Articles

Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016.

First finding of Trypanosoma cruzi II in vampire bats from a district free of domestic vector-borne transmission in Northeastern Argentina.

Establishing the putative links between sylvatic and domestic transmission cycles of Trypanosoma cruzi, the etiological agent of Chagas disease, is of public health relevance. We conducted three surveys to assess T. cruzi infection in wild mammals from a rural and a preserved area in Misiones Province, Northeastern Argentina, which had recently been declared free of vector- and blood-borne transmission of human T. cruzi infection. A total of 200 wild mammals were examined by xenodiagnosis (XD) and/or polymerase chain reaction (PCR) amplification of the hyper-variable region of kinetoplast DNA minicircles of T. cruzi (kDNA-PCR). The overall prevalence of T. cruzi infection was 8%. Nine (16%) of 57 Didelphis albiventris opossums and two (7%) of 29 Desmodus rotundus vampire bats were positive by both XD and kDNA-PCR. Additionally, one D. rotundus positive for T. cruzi by kDNA-PCR tested positive by satellite-DNA-PCR (SAT-DNA-PCR). The T. cruzi-infected bats were captured indoors and in the yard of a vacant dwelling. All D. albiventris were infected with TcI and both XD-positive D. rotundus by TcII. Fifty-five opossum cubs within the marsupium were negative by XD. The mean infectiousness to the vector was 62% in D. albiventris and 50% in D. rotundus. Mice experimentally infected with a parasite isolate from a vampire bat displayed lesions typically caused by T. cruzi. Our study documents the presence of the genotype TcII in a sylvatic host for the first time in Argentina, and the occurrence of two transmission cycles of T. cruzi in a district free of domestic vector-borne transmission.

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05–0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.

Vertical transmission of Anaplasma platys and Leishmania infantum in dogs during the first half of gestation.

BackgroundLeishmania infantum is a canine zoonotic vector-borne protozoan pathogen transmitted by phlebotomine sand flies, whereas Anaplasma platys is a bacterium most likely transmitted by ticks. While vertical transmission of L. infantum from pregnant bitches to their offspring has been documented, thus far no studies have explored the possibility of vertical transmission of A. platys in dogs. This study investigated the occurrence of vertical transmission of L. infantum and A. platys in sheltered dogs during the first half of gestation, in an area of southern Italy characterised by a high incidence of infection by both pathogens.MethodsThe study population included 20 bitches (n = 10 pregnant, at 25–35 days of pregnancy; n = 10 non-pregnant), all subjected to ovariohysterectomy, which were examined for the presence of L. infantum and A. platys via cytological screening of bone marrow and whole blood samples. Infection by L. infantum and A. platys was also tested by immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR) targeting both pathogens. Selected tissue samples (n = 210) collected during surgical procedures from bitches and foetuses (n = 20) were assessed for the presence of L. infantum and A. platys by qPCR targeting a fragment of the kinetoplast minicircle DNA (kDNA) and the 16S rRNA gene, respectively.ResultsLeishmania infantum DNA was not amplified from either uteri or ovaries from pregnant bitches or foetal tissue samples, whereas a subset of ovarian (n = 2) and uterine (n = 4) tissue samples from non-pregnant bitches were infected, with parasite loads of up to 3.09 × 10 and 7.51 parasite/PCR reaction, respectively. Conversely, uterine (n = 10) and ovarian (n = 8) tissues from both pregnant and non-pregnant bitches, together with a subset (n = 5) of foetal tissue samples were qPCR positive for A. platys. Leishmania infantum and A. platys nucleic acids were amplified from two uteri from non-pregnant bitches, with parasite loads of up to 2.32 × 10-3 and 2.05 parasite/per PCR reaction, respectively.ConclusionsResults from this study suggest that, in contrast to L. infantum, A. platys can be transmitted from pregnant dogs to their offspring during the first half of gestation. This hypothesis remains to be verified, for instance via direct observation of parasites in postpartum foetal tissues.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8±0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.

Augmenter of Liver Regeneration Gene Therapy Using a Novel Minicircle DNA Vector Alleviates Liver Fibrosis in Rats.

Liver fibrosis results in cirrhosis, liver cancer, and liver failure, which is a major cause of mortality worldwide. Gene therapy is a relatively new paradigm in medicine, with enormous therapeutic potential. The development of an efficient and safe delivery system is essential for clinical gene therapy. In the present study, we evaluated augmenter of liver regeneration/growth factor ERV1-like (ALR/GFER) gene therapeutic effect mediated by a novel minicircle vector (MC-hALR). The results in liver fibrotic rats that received MC-hALR through hydrodynamics-based transfection (HBT) for 8 weeks indicated that the minicircle DNA vector produced a more effective gene therapy effect than traditional plasmids (pcDNA3.1-hALR). Even when we reduced the treatment dose of MC-hALR to 30% (w/w) and the treatment frequency from weekly to biweekly, the in vitro and in vivo results still demonstrated that higher ALR gene expression significantly blocked increases in transforming growth factor-β1 (TGF-β1), platelet derived growth factor-BB (PDGF-BB), and α-smooth muscle aorta (α-SMA) levels; effectively suppressed the production of collagens, especially collagen I; and effectively alleviated liver injury and fibrosis in rats, thereby improving the survival rate of liver fibrotic rats. It is preliminarily concluded that the relative overexpression of MC-hALR inhibits the activation of hepatic stellate cells (HSCs), thereby alleviating liver fibrosis in rats.

73. Enhanced Engineering of Chimeric Antigen Receptor (CAR)-Modified T Cells Using Non-Viral Sleeping Beauty Transposition from Minicircle Vectors

Adoptive therapy with T cells that were modified with gamma-retroviral and lentiviral (LV) vectors to express a CD19-specific chimeric antigen receptor (CAR) has shown remarkable efficacy in pilot clinical trials. However, there are persistent concerns with the use of viral vectors with regard to safety, as well as the cost and scale of vector production required for making CAR T-cell therapy available to patients on a global level. In this study, we have refined non-viral Sleeping Beauty (SB) transposition to provide an effective and broadly applicable gene-transfer strategy with superior safety profile. We demonstrate that SB transposition of CAR transgenes from minicircle (MC) DNA vectors enables dramatically improved transposition rates compared to conventional plasmids. MC-derived CD19-CAR transposons display a highly favorable integration profile and confer stable CAR expression that permits potent anti-tumor functions and rapid manufacture of CAR T-cell products.We prepared MC transposon donor vectors encoding a CD19-CAR or eGFP from corresponding parental pT2 plasmids through site specific recombination, and a MC encoding SB100X transposase. Each MC contained only the promotor and gene of interest, and was devoid of antibiotic resistance gene and bacterial origin of replication. We then performed transfections into CD8+ and CD4+ T cells of healthy donors (n=7) and analyzed transposition rate and stability that could be accomplished when CD19-CAR transposon and SB100X transposase were encoded by MCs vs. equimolar amounts of corresponding conventional plasmids. In each donor, we achieved a significantly higher transposition rate with MCs (mean 49.8% on day 14 post transfection) compared to plasmids (mean 12.8%; 4.4-fold difference; p<0,001). Expression of the CD19-CAR was stable over multiple rounds of expansion and several weeks in culture. Importantly, MC transfection was not only more effective, but also substantially less toxic compared to plasmids and on average, a 6-fold higher yield of CAR T cells could be obtained within 14 days of culture without the need for feeder cell expansion. In functional experiments, CD19-CAR T cells modified with our enhanced MC-based SB transposition strategy were equally effective as LV transduced CD19-CAR T cells, and exerted high levels of specific cytolytic activity, cytokine production including IFN-g and IL-2, and productive proliferation after stimulation with CD19+ lymphoma. Moreover, a single administration of SB-modified CD19-CAR T cells leads to complete eradication of systemic lymphoma in a murine xenograft model (NSG/Raji-ffLuc), and was at least equivalently potent as LV transduced CD19-CAR T cells prepared from the same donor. To address safety, we determined the gene copy number (n=5, range 3-8 in CD8+ T cells) and performed a comprehensive genomic insertion site analysis. The data show a close-to-random integration profile of MC-derived CD19-CAR transposons, without preference for highly expressed or cancer related genes. Intriguingly, a significantly higher proportion of SB integrations had occurred in genomic safe harbors compared to LV integrations (7-fold difference; p<0,001), close to the perfect score expected for random integration. In conclusion, we demonstrate the potential to manufacture CAR T cells using virus-free SB-mediated transposition from MC DNA vectors. The superior safety profile, high level stable transposition rate and ease-of-handling MC vectors position our novel approach to become a preferred gene-transfer strategy in advanced cellular and gene-therapy.

355. Minicircles Show Improved Hepatic Expression of Their Transgene from a Natural Endogenous Promoter and Are Lost Upon Partial Hepatectomy Due to the Episomal Nature of the Vector

We have previously showed the successful treatment of phenylketonuria (PKU) in the PKU mouse model (C57Bl/6-Pahenu2) by using non-viral naked DNA minicircle (MC) vectors, which are devoid of any viral or bacterial sequences, upon liver-directed phenylalanine hydroxylase (Pah) gene transfer via hydrodynamic vein injection (Viecelli et al., Hepatology 2014). Here we improved the efficacy of transgene expression and investigated the fate of MC vectors in mouse liver. We found that the therapeutic doses of MCs could be significantly lowered by using a codon-optimized murine Pah cDNA in combination with a truncated 5’-intron. Moreover, when using the natural or endogenous 3.6 kb murine Pah-promoter to drive the Pah transgene, vector doses could again be lowered compared to two other “minimal” liver-specific promoters, a synthetic hybrid enhancer/promoter (P3) or the classical CBA (modified cytomegalovirus enhancer/chicken β-actin) promoter. Following 70% partial hepatectomy, MC-vector-treated PKU mice showed normal liver regeneration and blood L-Phe concentration increased to pretreatment levels, indicating that treated PKU mice had lost therapeutic MC vectors during liver regeneration. These results corroborate previous observations that MC-DNA do not integrate - or only at a very low frequency that cannot be detected by our assays - and thus express their transgene as episomal vectors. In conclusion, MC-vectors which do not have a defined size-limitation, offer a favorable safety profile due to their non-integrating behavior in combination with a (large) natural or endogenous promoter, and at the same time have the potential for long-term gene-therapy of liver defects.

306. Production of Safe and Efficient AAV Vectors Using Minicircle DNA

Especially in gene therapy applications, certain sequence motifs contained in plasmid DNA have to be avoided wherever possible. Such sequences are e.g. the bacterial ori or selection markers, only used for controlling the bacterial replication of the plasmid or to select for the plasmid during cloning or during production. Such sequence motifs are redundant in the intended therapeutic application and are completely removed in minicircles, i.e. circular and ccc-supercoiled expression cassettes.Since Adeno-Associated viral (AAV) vectors are produced by co-transfection of HEK293 producer cells, such bacterial sequence motifs may be an issue of an AAV vector-mediated gene transfer as well. Here, as a result of so-called reverse packaging events, an AAV vector-mediated transfer of not only the therapeutic gene but also of the antibiotic resistance gene into the target cells has been reported. Hence, this appears to be a potential risk of plasmid derived AAV vectors which can be overcome by using minicircle DNA for AAV production.Here we present first results showing that both constructs, the Helper & Packaging plasmid as well as the Transfer plasmid can be produced as minicircles although certain sequence motifs such as the ITRs have been identified to be an issue in minicircle production which has been overcome. These minicircles have been used for efficient AAV vector productions. However, only by replacing both, the Helper & Packaging plasmid as well as the transfer plasmid, encapsidation of the antibiotics resistance gene can be avoided.

The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.

Construction of minicircle DNA vectors capable of correcting familial hypercholesterolemia phenotype in a LDLR-deficient mouse model.

Familial hypercholesterolemia (FH) caused by defect in low-density lipoprotein receptor (LDLR) is a life-threatening disease with poor response to conventional treatments. Earlier gene therapy studies have generated promising results, but further development is hampered because the cells harboring the viral vectors were eliminated by host immune system soon after delivery, whereas the nonviral vectors were too bulky to be delivered to target cells. To overcome these problems, we constructed multiple minicircle (MC) DNA vectors to express the therapeutic LDLR. MC is an optimized nonviral vector that is capable of expressing high level of transgene product persistently. We found that among the seven MCs tested, the best is MC5 with multiple advanced features. First, the LDLr gene was placed under the control of sterol regulatory element (SRE) using LDLr gene promoter or apoprotein E (ApoE) promoter, allowing the transcription of the LDLr gene to be regulated by serum low-density lipoprotein (LDL) cholesterol as its functional gene counterpart. Second, a hepatic control region (HCR) was placed upstream of the promoter that serves as a controller to ensure liver-specific expression. Third, the modified Kozak sequence was placed in front of the LDLr gene start codon to enhance its translation efficiency. MC5 was 5.23 kb in size, and was capable of tight physiological control in intracellular LDL cholesterol level even when challenged with high dose of sterols in vitro. Importantly, it was able to correct the phenotype of LDLR-deficient mice C57BL/6 LDLR(-/-) for more than 105 days without detectable toxicity. Therefore, this MC has the clinical application potential for treating FH.

Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.

BackgroundNon-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses.MethodsRat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction.ResultsMinicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRα and FGFR2, respectively.ConclusionsA novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

Development of a nicking endonuclease-assisted method for the purification of minicircles

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled (sc) MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by hydrophobic interaction chromatography (HIC) step. At the early stage of vector design, a site for the nicking endonuclease Nb.BbvCI was strategically placed in the MP part of the PP backbone. A process was then established that involves E. coli culture and recombination of PPs into target MC, cell harvesting and alkaline lysis, precipitation with isopropanol and ammonium sulfate and diafiltration/concentration by microfiltration. Next, an in vitro digestion step was carried out with Nb.BbvCI to nick of one of the strands of the MPs and of non-recombined PPs by Nb.BbvCI. As a result, sc MPs and non-recombined PPs were converted into the corresponding open circular (oc) forms whereas sc MCs remain unaffected. Finally, sc MC was isolated from oc DNA molecules (oc MPs, oc MC) and RNA by performing HIC with a phenyl-Sepharose column using a series of elution steps with decreasing ammonium sulfate concentrations. On the basis of agarose gel electrophoresis analysis, the sc MC-containing fractions were determined to be virtually free from nucleic acid impurities.

Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer.

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.

Host-Feeding Sources and Infection With Trypanosoma cruzi of Triatoma infestans and Triatoma eratyrusiformis (Hemiptera: Reduviidae) From the Calchaqui Valleys in Northwestern Argentina.

We assessed the prevalence of infection with Trypanosoma cruzi, parasite genotypes (discrete typing units, DTUs), and the host-feeding sources of domestic and peridomestic Triatoma infestans Klug and Triatoma eratyrusiformis Del Ponte in eight rural communities of the subandean Calchaqui valleys in northwestern Argentina. We sought to analyze their epidemiological role in the context of routine vector surveillance and control actions. Infection with T. cruzi was determined by optic microscopy or polymerase chain reaction (PCR) amplification of the hypervariable region of kinetoplast DNA minicircles. Parasite genotypes were identified through a multi PCR-based strategy. Bloodmeal contents were tested with a direct ELISA assay against nine antisera. Human sleeping quarters (domiciles) and peridomestic dry-shrub fences concentrated most of the T. infestans and T. eratyrusiformis infected with T. cruzi, respectively. The most frequent host-feeding sources of T. infestans were chickens (73.1%) in peridomiciles and humans (73.3%) in domiciles, whereas T. eratyrusiformis fed more often on cavid rodents (92.6%), which thrived in the dry-shrub fences. The main T. cruzi DTU identified in both vectors was T. cruzi I (TcI). Triatoma eratyrusiformis was implicated in the local circulation of TcI among cavies and perhaps mice, but infection with other typically domestic DTUs (TcVI and TcII/TcV/TcVI) indicated overlap between (peri)domestic transmission cycles in both vector species. Because dry-shrub fences were not targeted for routine insecticide spraying, they may act as sources of (peri)domestic reinfestation. Triatoma eratyrusiformis is an emergent secondary vector of T. cruzi and plays a significant role in the local transmission of T. cruzi.

Molecular Diversity of Trypanosoma cruzi Detected in the Vector Triatoma protracta from California, USA.

BackgroundTrypanosoma cruzi, causative agent of Chagas disease in humans and dogs, is a vector-borne zoonotic protozoan parasite that can cause fatal cardiac disease. While recognized as the most economically important parasitic infection in Latin America, the incidence of Chagas disease in the United States of America (US) may be underreported and even increasing. The extensive genetic diversity of T. cruzi in Latin America is well-documented and likely influences disease progression, severity and treatment efficacy; however, little is known regarding T. cruzi strains endemic to the US. It is therefore important to expand our knowledge on US T. cruzi strains, to improve upon the recognition of and response to locally acquired infections.Methodology/Principle FindingsWe conducted a study of T. cruzi molecular diversity in California, augmenting sparse genetic data from southern California and for the first time investigating genetic sequences from northern California. The vector Triatoma protracta was collected from southern (Escondido and Los Angeles) and northern (Vallecito) California regions. Samples were initially screened via sensitive nuclear repetitive DNA and kinetoplast minicircle DNA PCR assays, yielding an overall prevalence of approximately 28% and 55% for southern and northern California regions, respectively. Positive samples were further processed to identify discrete typing units (DTUs), revealing both TcI and TcIV lineages in southern California, but only TcI in northern California. Phylogenetic analyses (targeting COII-ND1, TR and RB19 genes) were performed on a subset of positive samples to compare Californian T. cruzi samples to strains from other US regions and Latin America. Results indicated that within the TcI DTU, California sequences were similar to those from the southeastern US, as well as to several isolates from Latin America responsible for causing Chagas disease in humans.Conclusions/SignificanceTriatoma protracta populations in California are frequently infected with T. cruzi. Our data extend the northern limits of the range of TcI and identify a novel genetic exchange event between TcI and TcIV. High similarity between sequences from California and specific Latin American strains indicates US strains may be equally capable of causing human disease. Additional genetic characterization of Californian and other US T. cruzi strains is recommended.

Atomistic Molecular Dynamics Simulations of DNA Minicircle Topoisomers: A Practical Guide to Setup, Performance, and Analysis.

While DNA supercoiling is ubiquitous in vivo, the structure of supercoiled DNA is more challenging to study experimentally than simple linear sequences because the DNA must have a closed topology in order to sustain superhelical stress. DNA minicircles, which are closed circular double-stranded DNA sequences typically containing between 60 and 500 base pairs, have proven to be useful biochemical tools for the study of supercoiled DNA mechanics. We present detailed protocols for constructing models of DNA minicircles in silico, for performing atomistic molecular dynamics (MD) simulations of supercoiled minicircle DNA, and for analyzing the results of the calculations. These simulations are computationally challenging due to the large system sizes. However, improvements in parallel computing software and hardware promise access to improve conformational sampling and simulation timescales. Given the concurrent improvements in the resolution of experimental techniques such as atomic force microscopy (AFM) and cryo-electron microscopy, the study of DNA minicircles will provide a more complete understanding of both the structure and the mechanics of supercoiled DNA.

Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

Studies of G-quadruplexes formed within self-assembled DNA mini-circles.

We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.

DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations.

Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.