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).

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.

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.

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.

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.

Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters.

Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition, which, despite much progress towards its elucidation, still remains incompletely understood. We report here the results of a meta-analysis of IS2 target sites from genomic, phage, and plasmid DNA and find that newly acquired IS2 elements are consistently inserted around abrupt DNA compositional shifts, particularly in the form of switch sites of GC skew. The results presented in this study not only corroborate our previous observations that both the insertion sequence (IS) minicircle junction and target region adopt intrinsically bent conformations in IS2, but most interestingly, extend this requirement to other families of IS elements. Using this information, we were able to pinpoint regions with high propensity for transposition and to predict and detect, de novo, a novel IS2 insertion event in the 3' region of the gfp gene of a reporter plasmid. We also found that during amplification of this plasmid, process parameters such as scale, culture growth phase, and medium composition exacerbate IS2 transposition, leading to contamination levels with potentially detrimental clinical effects. Overall, our findings provide new insights into the role of target DNA structure in the mechanism of transposition of IS elements and extend our understanding of how culture conditions are a relevant factor in the induction of genetic instability.

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production.

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.

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.

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.

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.

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.

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.

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.

Evidence for the in vivo expression of a distant downstream gene under the control of ColE1 replication origin.

ColE1-like plasmids are widely used as expression vectors and as gene delivery vehicles. We have recently described a naturally occurring plasmid deletion phenomenon in the ColE1-type plasmid, pCI-neo, which leads to the detectable expression of an apparently promotorless kanamycin resistance gene. In the current work, we found that the expression of that aminoglycoside phosphotransferase (aph) gene is regulated by an RNAII preprimer promoter located within the plasmid ColE1 replication origin, as a consequence of the extension of the RNA II species for at least 1.5 kb, up to the aph gene. This mechanism is dependent on the nonformation and/or dissociation of the hybrid between plasmid DNA and RNA II preprimer transcript. This is the first in vivo description of RNA II transcription beyond ori in wild-type Escherichia coli strains and nonmutated RNAII plasmid sequences resulting in productive transcription of distant downstream genes. Our results raise questions about unwanted expression of genes from expression or cloning vectors of ColE1 type and highlight the importance of a more careful design of ColE1-derived plasmid vectors.

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events.

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.

Stabilization of naked and condensed plasmid DNA against degradation induced by ultrasounds and high-shear vortices.

Micrometre-sized aggregates of a 6050-bp plasmid obtained by the addition of 1.5-3.0 mM CaCl2 and 20% (v/v) t-butanol or 0.3-1.0% (v/v) APG (aluminium phosphate gel) were subjected to degradation induced by sonication or vortex flows. Dynamic light scattering revealed that the plasmid hydrodynamic radius increases from 116 nm to >1300 nm and approx. 1000 nm, when formulated with CaCl2/t-butanol and APG respectively. CD showed that addition of CaCl2/t-butanol leads to transition in plasmid structure from B-DNA to a psi-DNA negative form, whereas no detectable transitions were observed for APG formulations. The ability of the condensing agents to stabilize supercoiled plasmid isoforms subjected to sonication or turbulent Taylor vortices was assessed by agarose-gel electrophoresis. Although naked plasmid was completely fragmented after 5 s of sonication, condensing agents increased the plasmid stability dramatically [e.g. up to 80% after 30 s with 1.5 mM CaCl2+20% (v/v) t-butanol]. In the case of the vorticular flow system, the extent of degradation correlated well with the shear stress associated with flow of the solutions being processed. Overall, the results from the present study demonstrate that condensing agents such as CaCl2/t-butanol and APG can effectively stabilize plasmids against shear-induced degradation; the extent of protection, however, depends on both the condensing agents and the shear-inducing system used.

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.

Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

High frequency plasmid recombination mediated by 28 bp direct repeats.

The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative candidate DNA vaccine (pGPV-PV) is described. These multicopy pMB1-type plasmids were found to recombine in several recA E. coli strains due to the presence of two 28 bp direct repeats flanking intervening sequences of 1.6 kb (pCIneo) and 3.2 kb (pGPV-PV). In this recombination event, one of the direct repeats and the intervening sequence were deleted or duplicated, originating monomeric or/and hetero-dimeric plasmid forms, respectively. Additionally, the plasmid rearrangement led to the acquisition of a kanamycin resistance phenotype. Recombination frequencies between 7.8 x 10(-7) and 3.1 x 10(-5) were determined for DH5alpha and JM109(DE3) strains, respectively. Higher recombination frequencies were found in cells previously grown up to stationary growth phase being the monomeric plasmid form the prevalent one. Real-time PCR quantification revealed the presence of approximately 1.5 x 10(4) recombined molecules per 2 x 10(5 )cells pre-kanamycin exposure. Under selective pressure of this antibiotic, the number of recombined molecules increased approximately 2,000-fold in a 48-h period replacing the original plasmid form. The high frequency, at which deletion-duplication occurred in the absence of kanamycin selective pressure, should be regarded as a safety concern. This work highlights the impact of mutational hot spots on expression and cloning plasmid vectors and the need to carefully design plasmid vectors.