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Institute for Bioengineering and Biosciences
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Determination of Recombination Efficiency in Minicircle Production by Real-time PCR

Determination of Recombination Efficiency in Minicircle Production by Real-time PCR | iBB | Scoop.it

The determination of the recombination efficiency and the monitoring of parental plasmids (PP), minicircles (MC) and miniplasmid (MP) species during processing and in the final product are critical aspects of minicircle manufacturing. A paper published in Analytical Biochemistry by BERG-iBB researchers Cláudia Alves, Miguel Prazeres and Gabriel Monteiro 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 results presented indicate that the method can be applied for the determination of recombination efficiency in pure DNA samples.

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Minicircle‐based Expression of VEGF in Mesenchymal Stromal Cells from Human Tissues

Minicircle‐based Expression of VEGF in Mesenchymal Stromal Cells from Human Tissues | iBB | Scoop.it

Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. A recent study published in the Journal of Gene Medicine by BERG and SCERG researchers compares the angiogenic potential of MSC obtained from bone marrow (BM), adipose tissue (AT) and umbilical cord matrix (UCM) that were genetically modified with VEGF‐encoding minicircle vectors. Transfected cells displayed higher in vitro angiogenic potential than non‐transfected controls, as demonstrated by functional in vitro assays, but no significant differences were observed among cells from different sources.

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Featured Photo: CRISPR/Cas9 Engineering of E. coli for Minicircle Production

Featured Photo: CRISPR/Cas9 Engineering of E. coli for Minicircle Production | iBB | Scoop.it

Description: plate for screening CRISPR modifications, featured photo by Cláudia Alves, Copyright BERG-iBB and University of Florida 2019.

 

Context: BIOTecnico PhD student Cláudia Alves, under the supervision of BERG-iBB researchers Gabriel Monteiro and Miguel Prazeres and in collaboration with Christopher Reisch from the University of Florida, is using CRISPR/Cas9 to create mutations in E. coli that improve minicircle production.

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Engineering Human MSC with VEGF-encoding Minicircles for Angiogenic Gene Therapy

Engineering Human MSC with VEGF-encoding Minicircles for Angiogenic Gene Therapy | iBB | Scoop.it

Peripheral artery disease (PAD) is a debilitating condition characterized by the blockage of arteries, which leads to limb amputation in more severe cases. Researchers from SCERG- and BERG-iBB propose the use of human bone marrow (BM) MSC transiently transfected with minicircles encoding for VEGF as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The data shows that VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays.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 to improve angiogenesis in the context of PAD. The work was published in Human Gene Therapy.

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Purification of Plasmid DNA by Multimodal Chromatography

Purification of Plasmid DNA by Multimodal Chromatography | iBB | Scoop.it
Production of plasmid DNA is important in the context of gene therapy and DNA vaccination. In a paper just published in the journal Separation and Purification Technology, Ana Rita Santos, Cláudia Alves, Ana Azevedo and Miguel Prazeres from BERG-iBB, describe a process based on tandem precipiatation and multimodal chromatography that yields pure supercoiled plasmid DNA. The chromatography step, which uses a cationic multimodal ligand (Capto™ adhere) and an NaCl step gradient, provides baseline separation of isoforms and yields supercoiled-rich fractions (>90%) that are virtually free from RNA and have levels of gDNA and protein impurities within specifications. Click on title to learn more.
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Separation of Plasmid DNA Topoisomers by Multimodal Chromatography

Separation of Plasmid DNA Topoisomers by Multimodal Chromatography | iBB | Scoop.it
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. In a recent paper in Analytical Biochemistry, BERG-iBB researchers led by Miguel Prazeres and Ana Azevedo 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. Click on title to learn more.
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Cláudia Alves to Defend PhD Thesis in Biotechnology and Biosciences

Cláudia Alves to Defend PhD Thesis in Biotechnology and Biosciences | iBB | Scoop.it

Cláudia Patrícia Almeida Alves will be defending her PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Friday the 14th of May 2021, at 14:00 am (https://videoconf-colibri.zoom.us/j/82471896554). During the last years, and under the supervision of Gabriel Monteiro and Miguel Prazeres from iBB, Cláudia engineered Escherichia coli strains for the production of minicircle vectors by resolvase-catalyzed in vivo recombination. The title of her thesis is “Escherichia coli Genome Engineering For Improved Minicircle Production". Yo can check more about Cláudia's work here.

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Minicircle Biopharmaceuticals: An Overview of Purification Strategies

Minicircle Biopharmaceuticals: An Overview of Purification Strategies | iBB | Scoop.it

Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates the minicircle of interest and an unwanted miniplasmid. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles. The characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. In a review published in Frontiers in Chemical Engineering, Cláudia Alves, Miguel Prazeres and Gabriel Monteiro from BERG-IST summarize the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.

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Multimodal Chromatography of Supercoiled Minicircles

Multimodal Chromatography of Supercoiled Minicircles | iBB | Scoop.it

The ability to purify supercoiled minicircles (sc MC) from related miniplasmid (MP) and parental plasmid (PP) impurities is critical for a wider application of MC vectors in gene therapy research. BERG-iBB researchers developed a process that relies on multimodal chromatography with a 2-step NaCl elution to separate sc MC from open circular impurities. A detailed study of the DNA-ligand interactions underlying the separation shows that the order of elution reflects the increasing degree of base exposure of the species. Data further suggests that nucleic acid bases most likely engage in cation-π, π-π stacking and H-bonding with ligand. The work was published in Separation and Purification Technology.

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Production and Purification of Supercoiled Minicircles

Production and Purification of Supercoiled Minicircles | iBB | Scoop.it

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled MC from related miniplasmid (MP) and parental plasmid (PP) impurities. BERG-iBB researchers published a protocol that describes a purification strategy that combines the in vitro enzymatic relaxation of supercoiled 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 hr. The process delivers supercoiled MCs that are virtually free from MP, PP, RNA and protein impurities. The work was published in Human Gene Therapy Methods.

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Improvement of DNA Minicircle Production

Improvement of DNA Minicircle Production | iBB | Scoop.it
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, a team led by Gabriel Monteiro and Miguel Prazeres from iBB-BERG and Kristala Prather from MIT, optimized the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA) using a predictive thermodynamic model. The optimized parA gene was then inserted in the chromosome of an E. coli strain with improved arabinose uptake. overall the 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.The research was developed under the framework of the MIT-Portugal program and is a direct result of Michaela Simcikova's Ph D. thesis in Bioengineering. Click on title to read the publication in Applied Microbiology and Biotechnology.
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Purification of Minicircles by a Nicking Endonuclease-Assisted Method

Purification of Minicircles by a Nicking Endonuclease-Assisted Method | iBB | Scoop.it

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. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from miniplasmid (MP) impurities. In a proof-of-concept study published in Journal of Chromatography A, a team led by Miguel Prazeres from iBB-BERG describe a reproducible process to improve the purification of supercoiled (sc) MCs. The study shows that sc MC can be obtained completely free from nucleic acid impurities by modifying the topology of MP impurities with a nicking endonuclease and subsequently exploring the ability of hydrophonic interaction chromatography to fractionate topoisomers.

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