Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Microfluidic isolation of extrachromosomal circular DNA through selective digestion of plasmids and linear DNA using immobilized nucleases.

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.

Characterizing Plasmids in Bacteria Species Relevant to Urinary Health.

The urinary tract has a microbial community (the urinary microbiota or urobiota) that has been associated with human health. Whole genome sequencing of bacteria is a powerful tool, allowing investigation of the genomic content of the urobiota, also called the urinary microbiome (urobiome). Bacterial plasmids are a significant component of the urobiome yet are understudied. Because plasmids can be vectors and reservoirs for clinically relevant traits, they are important for urobiota dynamics and thus may have relevance to urinary health. In this project, we sought plasmids in 11 clinically relevant urinary species: Aerococcus urinae, Corynebacterium amycolatum, Enterococcus faecalis, Escherichia coli, Gardnerella vaginalis, Klebsiella pneumoniae, Lactobacillus gasseri, Lactobacillus jensenii, Staphylococcus epidermidis, Streptococcus anginosus, and Streptococcus mitis. We found evidence of plasmids in E. faecalis, E. coli, K. pneumoniae, S. epidermidis, and S. anginosus but insufficient evidence in other species sequenced thus far. Some identified plasmidic assemblies were predicted to have putative virulence and/or antibiotic resistance genes, although the majority of their annotated coding regions were of unknown predicted function. In this study, we report on plasmids from urinary species as a first step to understanding the role of plasmids in the bacterial urobiota. IMPORTANCE The microbial community of the urinary tract (urobiota) has been associated with human health. Whole genome sequencing of bacteria permits examination of urobiota genomes, including plasmids. Because plasmids are vectors and reservoirs for clinically relevant traits, they are important for urobiota dynamics and thus may have relevance to urinary health. Currently, urobiota plasmids are understudied. Here, we sought plasmids in 11 clinically relevant urinary species. We found evidence of plasmids in E. faecalis, E. coli, K. pneumoniae, S. epidermidis, and S. anginosus but insufficient evidence in the other 6 species. We identified putative virulence and/or antibiotic resistance genes in some of the plasmidic assemblies, but most of their annotated coding regions were of unknown function. This is a first step to understanding the role of plasmids in the bacterial urobiota.

Assessment of the plasmidome of an extremophilic microbial community from the Diamante Lake, Argentina.

Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L-1) and low oxygen pressure. Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.

Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol.

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

Comparative genomic analyses of ß-lactamase (blaCMY-42)-encoding plasmids isolated from wastewater treatment plants in Canada.

Wastewater treatment plants (WWTPs) are useful environments for investigating the occurrence, diversity, and evolution of plasmids encoding clinically relevant antibiotic resistance genes (ARGs). Our objective was to isolate and sequence plasmids encoding meropenem resistance from bacterial hosts within Canadian WWTPs. We used two enrichment culture approaches for primary plasmid isolation, followed by screening for antibiotic resistance, conjugative mobility, and stability in enteric bacteria. Isolated plasmids were sequenced using Illumina MiSeq and Sanger sequencing methods. Bioinformatics analyses resolved a multi-resistance IncF/MOBF12 plasmid, pFEMG (209 357 bp), harbouring resistance genes to ß-lactam (blaCMY-42, blaTEM-1ß, and blaNDM-5), macrolide (mphA-mrx-mphR), tetracycline (tetR-tetB-tetC-tetD), trimethoprim (dfrA12), aminoglycoside (aadA2), and sulfonamide (sul1) antibiotic classes. We also isolated an IncI1/MOBP12 plasmid pPIMR (172 280 bp) carrying similar ß-lactamase and a small multi-drug efflux resistance gene cluster (blaCMY-42-blc-sugE) to pFEMG. The co-occurrence of different ARGs within a single 24 552 bp cluster in pFEMG - interspersed with transposons, insertion sequence elements, and a class 1 integron - may be of significant interest to human and veterinary medicine. Additionally, the presence of conjugative and plasmid maintenance genes in the studied plasmids corresponded to observed high conjugative transfer frequencies and stable maintenance. Extensive investigation is required to further understand the fitness trade-offs of plasmids with different types of conjugative transfer and maintenance modules.

In Vivo Production of Small Recombinant RNAs Embedded in 5S rRNA-Derived Protective Scaffold.

Preparative synthesis of RNA is a challenging task that is usually accomplished by either chemical or enzymatic polymerization of ribonucleotides in vitro. Herein, we describe an alternative approach in which RNAs of interest are expressed as a fusion with a 5S rRNA-derived scaffold. The scaffold provides protection against cellular ribonucleases resulting in cellular accumulations comparable to those of regular ribosomal RNAs. After isolation of the chimeric RNA from the cells, the scaffold can be removed, if necessary, by deoxyribozyme-catalyzed cleavage followed by preparative electrophoretic separation of the reaction products. The protocol is designed for sustained production of high quality RNA on the milligram scale.

Scalable Purification of Plasmid DNA Nanoparticles by Tangential Flow Filtration for Systemic Delivery.

Plasmid DNA (pDNA) nanoparticles synthesized by complexation with linear polyethylenimine (lPEI) are one of the most effective non-viral gene delivery vehicles. However, the lack of scalable and reproducible production methods and the high toxicity have hindered their clinical translation. Previously, we have developed a scalable flash nanocomplexation (FNC) technique to formulate pDNA/lPEI nanoparticles using a continuous flow process. Here, we report a tangential flow filtration (TFF)-based scalable purification method to reduce the uncomplexed lPEI concentration in the nanoparticle formulation and improve its biocompatibility. The optimized procedures achieved a 60% reduction of the uncomplexed lPEI with preservation of the nanoparticle size and morphology. Both in vitro and in vivo studies showed that the purified nanoparticles significantly reduced toxicity while maintaining transfection efficiency. TFF also allows for gradual exchange of solvents to isotonic solutions and further concentrating the nanoparticles for injection. Combining FNC production and TFF purification, we validated the purified pDNA/lPEI nanoparticles for future clinical translation of this gene nanomedicine.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

A novel plasmid from Aerococcus urinaeequi of porcine origin co-harboring the tetracycline resistance genes tet(58) and tet(61).

Tetracyclines are the broad-spectrum agents used in veterinary medicine and food animal production. Known mechanisms of tetracycline resistance include ribosome protection, active efflux and enzymatic inactivation. However, the presence of two different tet genes conferring different resistance mechanisms on the same plasmid has rarely been reported. In this study, we identified the tandem tetracycline resistance genes tet(61)-tet(58) on the novel plasmid pT4303. These tet genes were identified for the first time in Aerococcus urinaeequi. Reduced susceptibility to doxycycline was observed in S. aureus RN4220 harboring tet(61) when an extra tet(58) was expressed. Plasmid pT4303 was electrotransformed into S. aureus RN4220, E. faecalis JH2-2, S. suis BAA and E. coli DH5α and conferred tetracycline resistance (MIC ≥ 16) in both Gram-positive and Gram-negative bacteria, assuming that it might serve as a vehicle for the dissemination of the tetracycline resistance genes tet(61) and tet(58).

Isolation of Chromosomal and Plasmid DNA from Bifidobacteria.

Rapid and efficient protocols aimed at the isolation and purification of DNA for the purpose of downstream applications, such as cloning, PCR, Southern blotting, or sequencing, are essential for genetic, biochemical, and molecular biological analyses of a given bacterium. The protocols herein presented provide a robust and efficient method for the isolation of chromosomal and plasmid DNA from Bifidobacterium strains by organic extraction. The methods are simple, and the yield, purity, and quality of the DNA are adequate to perform various downstream applications including next-generation sequencing.

Enrichment of plasmid pool from E. coli for mass sequencing to detect untargeted mutagenic events.

Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations in vivo is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The concept of this protocol could be applied into any species. For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).

Dilemma on plasmid DNA purification: binding capacity vs selectivity.

Plasmid DNA chromatography is a powerful field in constant development and evolution. The use of this technique is considered mandatory in the production of an efficient and safe formulation to be applied for plasmid-mediated gene therapy. Concerning this, the search for an ideal chromatographic support/ligand combination motivated scientist to pursue a continuous improvement on the plasmid chromatography performance, looking for a progression on the ligands and supports used. The present review explores the different approaches used over time to purify plasmid DNA, ambitioning both high recovery and high purity levels. Overall, it is presented a critical discussion relying on the relevance of the binding capacity versus selectivity of the supports.

Scale-Up of Plasmid DNA Downstream Process Based on Chromatographic Monoliths.

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.

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.

Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment.

Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.

Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators.

CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs.

Occurrence and characteristics of Escherichia coli mcr-1-like in rabbits in Shandong, China.

Polymyxin is regarded as the last retort to fight against multidrug-resistant (MDR) Enterobacteriaceae. The emergency and spread of polymyxin-associated resistance gene mcr-1 evoked great panic of no medicine to cure the bacterial infection in society. mcr-1 is widespread in domestic and wild animals. Therefore, continuous monitoring of its prevalence and characteristics is required. In this study, we used a polymerase chain reaction (PCR)-based method to detect the mcr-1 of Escherichia coli isolated from rabbits of Tai'an, China, and determined the characteristics of mcr-1-bearing plasmids. A total of 55 non-duplicated E. coli was recovered from the swabs of rabbit faeces. Plasmid profiling, plasmid and chromosome PCR, complete genome sequencing, a conjugation experiment, lactose fermentation experiment, multilocus sequence typing and polymyxin resistance tests were performed to determine the characteristics of mcr-1-bearing plasmids. 14.6% (8/55) of the specimens were mcr-1 positive. The mcr-1-positive E. coli harboured more drug-resistant genes compared with the mcr-1-negative specimens, and results showed four sequence types. Overall, these findings suggested the possible threat of the transmission of mcr-1 from rabbits to humans, especially since the gene is located on transferable plasmids making horizontal transfer relatively easy. Since food-producing animals are necessary for our daily diet, worldwide cooperation is needed in fighting the spread of this drug resistance gene to avoid human infections with MDR pathogenic bacteria.