Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production.

The in vivo intramolecular recombination of a parental plasmid allows excising prokaryotic backbone from the eukaryotic cassette of interest, leading to the formation of, respectively, a miniplasmid and a minicircle. Here we describe a real-time PCR protocol suitable for the determination of recombination efficiency of parental plasmids with multimer resolution sites (MRS). The protocol was successfully applied to purified DNA samples obtained from E. coli cultures, allowing a more reproducible determination of recombination efficiency than densitometry analysis of agarose gels.

A Real-Time Quantitative PCR Protocol for the Quantification of Plasmid Copy Number in Lactococcus lactis.

The determination of the number of plasmid copies in each cell of Lactococcus lactis is critical for the control and regulation of the production of recombinant proteins and plasmids. This protocol describes a method for the determination of the plasmid copy number per genome of L. lactis, which is based on the detection by real-time quantitative PCR of the number of plasmid molecules and the number of chromosomes and subsequently their ratio after calculating the amplification efficiency.

Bioengineered Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles as an Improved Drug Delivery System: Methods and Applications.

Extracellular vesicles (EVs) are cell-derived nano-sized lipid membranous structures that modulate cell-cell communication by transporting a variety of biologically active cellular components. The potential of EVs in delivering functional cargos to targeted cells, their capacity to cross biological barriers, as well as their high modification flexibility, make them promising drug delivery vehicles for cell-free therapies. Mesenchymal stromal cells (MSCs) are known for their great paracrine trophic activity, which is largely sustained by the secretion of EVs. MSC-derived EVs (MSC-EVs) retain important features of the parental cells and can be bioengineered to improve their therapeutic payload and target specificity, demonstrating increased therapeutic potential in numerous pre-clinical animal models, including in the treatment of cancer and several degenerative diseases. Here, we review the fundamentals of EV biology and the bioengineering strategies currently available to maximize the therapeutic value of EVs, focusing on their cargo and surface manipulation. Then, a comprehensive overview of the methods and applications of bioengineered MSC-EVs is presented, while discussing the technical hurdles yet to be addressed before their clinical translation as therapeutic agents.

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression.

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of Escherichia coli biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h-1 (corresponding to a Reynolds number of 4600 and 2300, respectively). The fluorometric analysis showed that the specific eGFP production was higher in biofilms than in planktonic cells under both hydrodynamic conditions (3-fold higher for 255 L h-1 and 2-fold higher for 128 L h-1). In the biofilm cells, the percentage of eGFP-expressing cells was on average 52% higher at a flow rate of 255 L h-1. Furthermore, a higher plasmid copy number (PCN) was obtained for the highest flow rate for both planktonic (244 PCN/cell versus 118 PCN/cell) and biofilm cells (43 PCN/cell versus 29 PCN/cell). The results suggested that higher flow velocities promoted eGFP expression in E. coli biofilms.

The Effect of Recombinant Protein Production in Lactococcus lactis Transcriptome and Proteome.

Lactococcus lactis is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. The present paper reviews the major findings from L. lactis transcriptome and proteome studies, with an overexpression of native or recombinant proteins. These studies should provide important insights on how to engineer the plasmid vectors and/or the strains in order to achieve high pDNA or recombinant proteins yields, with high quality standards. L. lactis harboring high copy numbers of plasmids for DNA vaccines production showed altered proteome profiles, when compared with a smaller copy number plasmid. For live mucosal vaccination applications, the cell-wall anchored antigens had shown more promising results, when compared with intracellular or secreted antigens. However, previous transcriptome and proteome studies demonstrated that engineering L. lactis to express membrane proteins, mainly with a eukaryotic background, increases the overall cellular burden. Genome engineering strategies could be used to knockout or overexpress the pinpointed genes, so as to increase the profitability of the process. Studies about the effect of protein overexpression on Escherichia coli and Bacillus subtillis transcriptome and proteome are also included.

Mesenchymal Stromal Cells (MSCs): A Promising Tool for Cell-Based Angiogenic Therapy.

The Mesenchymal stromal cells (MSCs) are a diverse subset of adult multipotent precursors, known for their potential therapeutic properties in regenerative medicine mainly sustained by paracrine effects through secretion of a variety of biologically active molecules. MSC secretome includes a wide range of soluble protein factors, composed of growth factors and cytokines, and vesicular components, which transfer proteins and genetic material modulating the host microenvironment. In particular, MSC-derived secretome mediates the different steps of the angiogenic process, inducing endothelial cell functions in vitro and promoting angiogenesis in vivo. As a result, MSCs have been widely explored as a promising cell-based therapy in diseases caused by insufficient angiogenesis. Numerous studies of myocardial infarction, ischemic stroke, and critical limb ischemia in animals have shown that human MSCs can enhance angiogenesis and accelerate tissue regeneration. This extensive preclinical work encouraged the study of these remarkable cells for the treatment of these disorders in human clinical settings. The present review provides a comprehensive overview of the pro-angiogenic potential of MSCs and paracrine effectors of their secretome. In addition, bioengineering strategies, including ex vivo preconditioning and genetic modification approaches, to enhance MSC innate angiogenic properties, and thereby therapeutic potency, will be presented. Finally, an update on completed preclinical and clinical studies with MSCs for the treatment of ischemia-related diseases will be discussed.

Recombination efficiency measurement by real-time PCR: A strategy to evaluate ParA-mediated minicircle production.

Minicircles (MCs) are DNA molecules that are produced in Escherichia coli by replicating a parental plasmid (PP) and inducing its site-specific intramolecular recombination into miniplasmid (MP; containing the prokaryotic backbone) and MC molecules (comprised by the eukaryotic cassette). The determination of the recombination efficiency and the monitoring of PP, MC and MP species during processing and in the final product are critical aspects of MC manufacturing. This work describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The method was evaluated using artificial mixtures of (i) PP and MP, (ii) PP and MC and (iii) MP and MC that were probed for all three DNA molecules. The ratio of molecules of each DNA species in these mixtures were determined with differences lower than 10% relatively to the expected ratio of the species in 90% of the mixtures. Next, the recombination efficiency was successfully estimated by analysing pre-purified DNA samples obtained from cell cultures. A standard deviation < 2% was obtained between replicas and results closely correlated with those obtained by densitometry analysis of agarose gels. Further optimization is required to determine recombination efficiency directly from whole cells.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

BACKGROUND: Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. METHODS: We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. RESULTS: MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. CONCLUSIONS: Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).

Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories.

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.

Plasmid Copy Number of pTRKH3 in Lactococcus lactis is Increased by Modification of the repDE Ribosome-Binding Site.

Plasmids for DNA vaccination are exclusively produced in the Gram-negative Escherichia coli. One important drawback of this system is the presence of lipopolysaccharides. The generally recognized as safe Lactococcus lactis (L. lactis) would constitute a safer alternative for plasmid production. A key requirement for the establishment of a cost-effective L. lactis-based plasmid manufacturing is the availability of high-copy number plasmids. Unfortunately, the highest copy number reported in Gram-positive bacteria for the pAMß1 replicon is around 100 copies. The purpose of this work is to engineer the repDE ribosome-binding site (RBS) of the pTRKH3 plasmid by site-directed mutagenesis in order to increase the plasmid copy number in L. lactis LMG19460 cells. The pTRKH3-b mutant is the most promising candidate, achieving 215 copies of plasmid per chromosome, a 3.5-fold increase when compared to the nonmodified pTRKH3, probably due to a stronger RBS sequence, a messenger RNA secondary structure that promotes the RepDE expression, an ideal intermediate amount of transcriptional repressors and the presence of a duplicated region that added an additional RBS sequence and one new in-frame start codon. pTRKH3-b is a promising high-copy number shuttle plasmid that will contribute to turn lactic acid bacteria into a safer and economically viable alternative as DNA vaccines producers.

Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The VEGF gene was cloned in minicircle and conventional plasmid vectors and used to transfect bone marrow-derived MSC ex vivo. VEGF expression was evaluated both by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The number of VEGF transcripts following MSC transfection with minicircles increased 130-fold relative to the expression in non-transfected MSC, whereas for the plasmid (pVAX1)-based transfection, the increase was 50-fold. Compared to the VEGF basal levels secreted by MSC (11.1 ± 3.4 pg/1,000 cells/day), significantly higher values were detected by enzyme-linked immunosorbent assay after both minicircle and pVAX1 transfection (644.8 ± 82.5 and 508.3 ± 164.0 pg/1,000 cells/day, respectively). The VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays, without affecting the expansion potential ex vivo, as well as multilineage differentiation capacity or immunophenotype of MSC. Although preclinical in vivo studies are required, these results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach for an improved angiogenesis in the context of PAD.

Production and Purification of Supercoiled Minicircles by a Combination of In Vitro Endonuclease Nicking and Hydrophobic Interaction Chromatography.

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled (sc) MC from related miniplasmid (MP) and parental plasmid (PP) impurities. This protocol describes a purification strategy that combines the in vitro enzymatic relaxation of sc MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC, is approximately 50 h. The process delivers sc MCs that are virtually free from MP, PP, RNA, and protein impurities.

Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma.

The Escherichia coli phosphoglucose isomerase (pgi) mutant strain GALG20 was developed previously from wild-type K12 strain MG1655 for increased plasmid yield. To investigate the potential effects of the pgi deletion/higher plasmid levels on recombinant human Interferon Gamma (IFN-γ) production, a detailed network of the central metabolic pathway (100 metabolites, 114 reactions) of GALG20 and MG1655 was constructed. Elementary mode analysis (EMA) was then performed to compare the phenotypic spaces of both the strains and to check the effect of the pgi deletion on flux efficiency of each metabolic reaction. The results suggested that pgi deletion increases amino acid biosynthesis and flux efficiency towards IFN-γ synthesis by 11%. To further confirm the qualitative prediction that the pgi mutation favours recombinant human IFN-γ expression, GALG20 and MG1655 were lysogenised, transformed with a plasmid coding for IFN-γ and tested alongside with BL21(DE3) for their expression capabilities in shake flask experiments using complex media. IFN-γ gene expression was analysed by quantifying plasmid and mRNA copy number per cell and IFN-γ protein production level. Specific IFN-γ yields confirmed the in silico metabolic network predictions, with GALG20(DE3) producing 3.0-fold and 1.5-fold more IFN-γ as compared to MG1655(DE3) and BL21(DE3), respectively. Most of the total IFN-γ was expressed as inclusion bodies across the three strains: 95% in GALG20(DE3), 97% in BL21(DE3) and 72% in MG1655(DE3). The copy number of mRNA coding for IFN-γ was found to be higher in GALG20(DE3) as compared to the other two strains. Overall, these findings show that GALG20(DE3) has the potential to become an excellent protein expression strain.

Draft Genome Sequence of the Plasmid-Free Lactococcus lactis subsp. lactis Strain LMG 19460.

We report here the draft genome sequence of the plasmid-free Lactococcus lactis subsp. lactis strain LMG 19460. This strain has potential application for a cost-effective production of food-grade plasmid DNA to use in DNA vaccines, produce recombinant proteins, and be used as a mucosal delivery vehicle of therapeutic molecules.

Characterizing Slow Photochemical Reaction Kinetics by Enhanced Sampling of Rare Events with Capillary Optical Fibers and Kramers' Theory.

Characterization of slow chemical reactions is essential for assessing catalytic efficiency in chemistry and biology. Traditionally, chemical reaction rates are obtained from population relaxation kinetics measurements and the Arrhenius equation. Unfortunately, it is difficult to use this approach to characterize reactions wherein concentrations change slowly. Thus, it is interesting to see whether a dynamical view of chemical reactions may be used to obtain the reaction rates of slow processes. In the present work, we perform Brownian dynamics simulations of an asymmetric double-well potential to investigate how enhanced sampling of barrier crossing at transition states improves the characterization of reaction rate constants. We then present the design of a liquid-filled capillary optical fiber-based fluorescence spectrometer, which, like rare events, is also based on Poissonian statistics. We use the instrument to characterize the slow photochemical degradation kinetics of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) in o-dichlorobenzene. We have employed in situ optical microscopy measurements and electrodynamics simulations to characterize the excitation beam profile inside a liquid-filled capillary fiber. We compare the cuvette and capillary fiber sample holders and show that the MEH-PPV fluorescence line shape is independent of the sample holder, as expected. We characterize the photochemical degradation kinetics of MEH-PPV in o-dichlorobenzene solutions placed in the cuvette versus that in the capillary fiber. We observe small and slow changes in the time-dependent fluorescence spectra when the degradation reaction is performed in the cuvette. On the other hand, we are able to characterize reactant-concentration decay and product-concentration buildup from the time-dependent fluorescence spectra recorded during photochemical degradation of MEH-PPV performed inside the capillary optical fiber. Ultrafast optically heterodyne-detected optical Kerr effect spectroscopy and multimode Brownian oscillator analysis provide further insights into the role of bath oscillator modes of friction in the mechanism of MEH-PPV photochemical degradation. Overall, the work presented herein shows that slow photochemical degradation kinetics of MEH-PPV can be successfully and efficiently assessed in the capillary fiber fluorescence spectrometer.

Design and characterization of plasmids encoding antigenic peptides of Aha1 from Aeromonas hydrophila as prospective fish vaccines.

The current investigation aimed at designing DNA vaccines against Aeromonas hydrophila infections. The DNA vaccine candidates were designed to express two antigenic outer membrane protein (Aha1) peptides and to be delivered by a nanoparticle-based delivery system. Gene sequences of conserved regions of antigenic Aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pVAX-GFP expression vector. The selected DNA vaccine candidates were purified from E. coli DH5α and transfected into Chinese hamster ovary cells. The expression of the antigenic peptides was measured in cells along post-transfection time, through the fluorescence intensity of the reporter GFP. The lipofection efficiency of aha-pVAX-GFP was highest after 24h incubation. Formulated PLGA-chitosan nanoparticle/plasmid DNA complexes were characterized in terms of size, size distribution and zeta potential. Nanocomplexes with average diameters in the range of 150-170nm transfected in a similar fashion into CHO cells confirmed transfection efficiency comparable to that of lipofection. DNA entrapment and further DNase digestion assays demonstrated ability for pDNA protection by the nanoparticles against enzymatic digestion.

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 8.6-14.9 kcal/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 2 h. 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.

DNA Vaccines Against Maedi-Visna Virus.

Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic and progressive disease of sheep with a high prevalence all over the world. Therefore, measures aiming at the control of MVV infection are necessary, and the development of DNA vaccines may be the ideal approach. A DNA vaccine is an antigen-encoding bacterial plasmid designed to mimic infections safely, with ability to generate both humoral and cellular long-lasting immune responses once it is delivered to the host.Here, we describe the development and evaluation of DNA vaccines against ovine maedi-visna virus. The first step is the design of the vaccines, including the choice of the backbone vector and the nucleotide sequences to use as antigen-encoding sequences. Once constructed, the vaccines may be produced with high quality for use in in vitro and in vivo tests. In vitro assays are performed through transfection of animal cells to confirm the expression of the protein, while in vivo tests are carried out by mouse and/or sheep immunization in order to check humoral and cellular responses to the vaccines and conclude about their efficiency. Several approaches may be later performed in order to enhance the effectiveness of the vaccines, such as the introduction of targeting sequences, the use of a prime-boost strategy, the administration of a combined vaccine, and the use of liposomes as delivery vehicle.

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.

Towards effective non-viral gene delivery vector.

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids' performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.