Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

DNA origami is an emerging technology that can be used as a nanoscale platform in numerous applications ranging from drug delivery systems to biosensors. The DNA nanostructures are assembled from large single-stranded DNA (ssDNA) scaffolds, ranging from hundreds to thousands of nucleotides and from short staple strands. Scaffolds are usually obtained by asymmetric PCR (aPCR) or Escherichia coli infection/transformation with phages or phagemids. Scaffold quantification is typically based on agarose gel electrophoresis densitometry for molecules obtained by aPCR, or by UV absorbance, in the case of scaffolds obtained by infection or transformation. Although these methods are well-established and easy-to-apply, the results obtained are often inaccurate due to the lack of selectivity and sensitivity in the presence of impurities. Herein, we present an HPLC method based on ion-pair reversed-phase (IP-RP) chromatography to quantify DNA scaffolds. Using IP-RP chromatography, ssDNA products (449 and 1000 nt) prepared by aPCR were separated from impurities and from the double stranded (ds) DNA byproduct. Additionally, both ss and dsDNA were quantified with high accuracy. The method was used to guide the optimization of the production of ssDNA by aPCR, which targeted the maximization of the ratio of ssDNA to dsDNA obtained. Moreover, ssDNA produced from phage infection of E. coli cells was also quantified by IP-RP using commercial ssDNA from the M13mp18 phage as a standard.

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.

Manufacturing of non-viral protein nanocages for biotechnological and biomedical applications.

Protein nanocages are highly ordered nanometer scale architectures, which are typically formed by homo- or hetero-self-assembly of multiple monomers into symmetric structures of different size and shape. The intrinsic characteristics of protein nanocages make them very attractive and promising as a biological nanomaterial. These include, among others, a high surface/volume ratio, multi-functionality, ease to modify or manipulate genetically or chemically, high stability, mono-dispersity, and biocompatibility. Since the beginning of the investigation into protein nanocages, several applications were conceived in a variety of areas such as drug delivery, vaccine development, bioimaging, biomineralization, nanomaterial synthesis and biocatalysis. The ability to generate large amounts of pure and well-folded protein assemblies is one of the keys to transform nanocages into clinically valuable products and move biomedical applications forward. This calls for the development of more efficient biomanufacturing processes and for the setting up of analytical techniques adequate for the quality control and characterization of the biological function and structure of nanocages. This review concisely covers and overviews the progress made since the emergence of protein nanocages as a new, next-generation class of biologics. A brief outline of non-viral protein nanocages is followed by a presentation of their main applications in the areas of bioengineering, biotechnology, and biomedicine. Afterwards, we focus on a description of the current processes used in the manufacturing of protein nanocages with particular emphasis on the most relevant aspects of production and purification. The state-of-the-art on current characterization techniques is then described and future alternative or complementary approaches in development are also discussed. Finally, a critical analysis of the limitations and drawbacks of the current manufacturing strategies is presented, alongside with the identification of the major challenges and bottlenecks.

Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules.

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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.

Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microfluidic Assays.

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).

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).

Manufacturing of bacteriophages for therapeutic applications.

Bacteriophages, or simply phages, are the most abundant biological entities on Earth. One of the most interesting characteristics of these viruses, which infect and use bacteria as their host organisms, is their high level of specificity. Since their discovery, phages became a tool for the comprehension of basic molecular biology and originated applications in a variety of areas such as agriculture, biotechnology, food safety, veterinary, pollution remediation and wastewater treatment. In particular, phages offer a solution to one of the major problems in public health nowadays, i.e. the emergence of multidrug-resistant bacteria. In these situations, the use of virulent phages as therapeutic agents offers an alternative to the classic, antibiotic-based strategies. The development of phage therapies should be accompanied by the improvement of phage biomanufacturing processes, both at laboratory and industrial scales. In this review, we first present some historical and general aspects related with the discovery, usage and biology of phages and provide a brief overview of the most relevant phage therapy applications. Then, we showcase current processes used for the production and purification of phages and future alternatives in development. On the production side, key factors such as the bacterial physiological state, the conditions of phage infection and the operation parameters are described alongside with the different operation modes, from batch to semi-continuous and continuous. Traditional purification methods used in the initial phage isolation steps are then described followed by the presentation of current state-of-the-art purification approaches. Continuous purification of phages is finally presented as a future biomanufacturing trend.

A Cellulose Paper-Based Fluorescent Lateral Flow Immunoassay for the Quantitative Detection of Cardiac Troponin I.

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the cardiac biomarker troponin I (cTnI) that features an analytical strip made of cellulose filter paper. The results show that the wicking and test time are comparable to those obtained with conventional nitrocellulose (NC)-based LFAs. Further, the cellulose paper provides an excellent background with no auto-fluorescence that is very adequate in detecting fluorescent lines. While fluorescence that was generated with cellulose strips was lower when compared to that generated in NC strips, signals could be improved by layering carbon nanofibers (CNF) on the cellulose. A nonlinear behavior of the concentration-response relationship was observed for the LFA architectures with NC, cellulose, and cellulose-CNF in the 0 to 200 ng/mL cTnI concentration range. The measurements were consistent and characterized by coefficients of variation lower than 2.5%. Detection and quantitation limits that were in the range 1.28-1.40 ng/mL and 2.10-2.75 ng/mL were obtained for LFA with cellulose and cellulose CNF strips that are equivalent to the limits obtained with the standard NC LFA. Overall, we showed that commercially available filter paper can be used in the analytical strip of LFA.

Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation.

A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (>80%).

Purification of Plasmid DNA by Multimodal Chromatography.

Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.

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.

Separation of plasmid DNA topoisomers by multimodal chromatography.

The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.

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.

G protein-coupled receptors: an overview of signaling mechanisms and screening assays.

The existence of cellular receptors, a group of specialized biomolecules to which endogenous and exogenous compounds bind and exert an effect, is one of the most exciting aspects of cell biology. Among the different receptor types recognized today, G-protein-coupled receptors (GPCRs) constitute, undoubtedly, one of the most important classes, in part due to their versatility, but particularly, due to their central role in a multitude of physiological states. The unveiling of GPCR function and mode of action is a challenging task that prevails until our days, as the full potential of these receptors is far from being established. Such an undertaking calls for a joint effort of multidisciplinary teams that must combine state-of-the-art technologies with in-depth knowledge of cell biology to probe such specialized molecules. This review provides a concise coverage of the scientific progress that has been made in GPCR research to provide researchers with an updated overview of the field. A brief outline of the historical breakthroughs is followed by a discussion of GPCR signaling mechanisms and by a description of the role played by assay technologies.

Use of ImageJ to recover information from individual cells in a G protein-coupled receptor assay.

Live-cell assays used in GPCR research often rely on fluorescence techniques that generate large amounts of raw image data. Consequently, the capacity to accurately and timely extract useful information from image and video data has become more and more important. Image J is an open-source program that provides powerful tools with a simple interface designed to fit the needs of image analysis of most researchers. In this chapter, Image J routines to extract information from individual cells in a calcium GPCR assay are described. In these routines, individual cells in the same image/video data can be separated using either a progressive threshold or a local threshold method. Both methods can be optimized to either a maximum number of selection or maximum area selected resulting in conceptually distinct selections.

Plasmid Biopharmaceuticals.

Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

Impact of plasmid quality on lipoplex-mediated transfection.

This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery.

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.