Class 2016
PhD Grant: PD/BD/116842/2016
PhD thesis
Escherichia coli Genome Engineering For Improved Minicircle Production
Supervisor
• Gabriel A. Monteiro, Professor @ IST/ULisboa, PT
Co-Supervisor
• Duarte Miguel Prazeres, Professor @ IST/ULisboa, PT
Collaborators
• Pedro M. Paulo, Research Scientist @ IST/ULisboa, PT
• Christopher Reisch, Professor @ University of Florida, Gainesville, FL, US
PhD degree complete: Discussion on May 14 2021, Pass with distinction
THESIS ABSTRACT
In the last 20 years, a high interest has been registered in non-viral vectors for gene therapy and DNA vaccination applications. In this context, plasmid DNA has been used advantageously although presenting low transfection efficiency and transgene expression. Also, some minor concerns regarding their safety have been raised. Therefore, efforts were made to develop safer and more efficient non-viral delivery vectors, which led to the creation of minicircle vectors. Minicircles are plasmid-derived DNA molecules devoid of bacterial sequences, which can safely deliver a gene of interest to cells, allowing for a higher expression when compared to conventional plasmids. Minicircles are produced in Escherichia coli by replicating a parental plasmid and inducing its intramolecular recombination into miniplasmid and minicircle molecules. Despite the advantages of minicircles, current manufacturing processes present low yields. This highlights the need for E. coli strains with improved capability to produce the amounts of minicircle required for gene therapy and DNA vaccination pre-clinical and clinical trials.
The main goal of this work is to improve minicircle production in E. coli. In pursuit of this objective, the 5’-untranslated region of the ParA resolvase gene was optimized, generating a sequence (PBAD/araC-par3A) that improves recombination efficiency by ≈2-fold over a previously optimized cassette. To improve production yield, CRISPR/Cas9-mediated genome engineering was further used to modify the expression of genes in the central carbon metabolism as a strategy to achieve a high parental plasmid producing E. coli strain. This resulted in the construction of five new strains with knockouts in the pykA, pykF and pgi genes. While promising results were obtained for the pgi knockout, current production yield studies are still inconclusive. A strain harboring the above referred cassette with the new ParA 5’- UTR in the genome was also constructed. Finally, a real-time PCR based method for molecule-specific detection of recombination products was developed and successfully used to determine recombination efficiencies in pure DNA samples.
RESUMO DA TESE
Nos últimos 20 anos tem sido registado um elevado interesse em vectores não-virais para aplicações em terapia génica e vacinas de DNA. Os plasmídeos têm sido usados neste contexto, apesar da sua baixa eficiência de transfecção e expressão génica. A incerteza relacionada com a segurança destes vectores levou ao desenvolvimento de vectores não-virais mais seguros e eficientes, incluindo os minicírculos. Estas moléculas circulares de DNA não contêm sequências procarióticas, sendo por isso associadas a uma entrega de genes mais segura e a uma maior expressão quando comparadas com plasmídeos convencionais. A produção de minicírculos é efectuada em Escherichia coli a partir de um plasmídeo parental que, após recombinação intramolecular, origina miniplasmídeos contendo a informação procariótica e minicírculos com a informação terapêutica. Apesar das suas vantagens, os processos de produção actuais apresentam baixa produtividade, sendo evidente a necessidade de desenvolver estirpes de E. coli melhoradas e com capacidade de produzir minicírculos em quantidade suficiente para ensaios pre-clínicos e clínicos.
Com o objectivo de melhorar a produção de minicírculos em E. coli, optimizou-se a sequência não codificante da resolvase ParA, envolvida na recombinação do plasmídeo parental, resultando numa recombinação com o dobro da eficiência relativamente à sequência optimizada anteriormente. Para melhorar o rendimento de produção, foi utilizado um método de engenharia genómica baseado na tecnologia CRISPR/Cas9. Neste contexto, foram construídas cinco estirpes por eliminação dos genes pykA, pykF e pgi envolvidos no metabolismo de carbono, com o intuito de aumentar a produção de plasmídeo em E. coli. Apesar de serem obtidos resultados promissores com a delecção do gene pgi, os resultados de produção são ainda inconclusivos. Foi também contruída uma estirpe contendo uma cassete com a nova
sequência para optimização da recombinação (PBAD/araC-par3A). Finalmente, foi desenvolvido um método de qPCR para detecção específica de plasmídeo parental, miniplasmídeo e minicírculo, aplicável na determinação da eficiência de recombinação em amostras puras de DNA.
PUBLICATIONS
Papers
Alves CPA, Prazeres DMF and Monteiro GA, Recombination efficiency measurement by real-time PCR: A strategy to evaluate ParA-mediated minicircle production. Anal. Biochem. 628:114285, 2021. https://doi.org/10.1016/j.ab.2021.114285
Alves CPA, Prazeres DMF and Monteiro GA, Minicircle Biopharmaceuticals–An Overview of Purification Strategies. Front. Chem. Eng. 2:612594, 2021. https://doi.org/10.3389/fceng.2020.612594
Serra J, Alves CPA, Cabral JMS, Monteiro GA, da Silva CL, Prazeres DMF, Minicircle‐based expression of vascular endothelial growth factor (VEGF) in mesenchymal stromal cells (MSC) from diverse human tissues, J. Gene Med. 23(7):e3342, 2021. doi: 10.1002/jgm.3342
Silva-Santos AR, Alves CPA, Monteiro GA, Azevedo AM, Prazeres DMF, “Multimodal chromatography of supercoiled minicircles: a closer look into DNA-ligand interactions”, Separation and Purification Technology, 212, 161-170, 2019
Serra J, Alves CPA, Brito L, Monteiro GA, Cabral JMS, Prazeres DMF, da Silva CL, “Engineering of human mesenchymal stem/stromal cells (MSC) with VEGF-encoding minicircles for angiogenic ex vivo gene therapy”, Human Gene Therapy 30(3):316-329, 2018
Alves CPA, Simčíková M, Brito L, Monteiro GA, Prazeres DMF, “Production and Purification of Supercoiled Minicircles by a Combination of In Vitro Endonuclease Nicking and Hydrophobic Interaction Chromatography”, Human Gene Therapy Methods 29(4):157-168, 2018.
Oral Communications
Alves C, Prazeres DM, Reisch C, Monteiro G, “Improving minicircle DNA production in E. coli – in vivo analysis of recombination efficiency after re-design of ParA resolvase 5’-UTR”, MicroBiotec2019 December 5-7, 2019, Coimbra, Portugal
Poster Communications
Cláudia P.A. Alves; Duarte Miguel F. Prazeres; Gabriel A. Monteiro. Development of a real-time PCR method to evaluate ParA-mediated recombination efficiency in minicircle production. Virtual ESBES2021
Cláudia P.A. Alves; Duarte Miguel F. Prazeres; Gabriel A. Monteiro. Determination of recombination efficiency by real-time PCR – a strategy to evaluate minicircle production. Microbiotec2019, Coimbra, Portugal
Alves CPA, Šimčíková M, Prazeres DMF, Monteiro GA, ”Optimization of ParA resolvase untranslated region – a strategy to boost minicircle DNA production”, MicroBiotec2017 December 7-8, 2017, Porto, Portugal
Alves CPA, Prazeres DMF, Monteiro GA, ”Escherichia coli Genome Engineering for Improved Minicircle Production”, PhD open days 2018Instituto Superior Técnico, Lisboa, Portugal
Cláudia P.A. Alves, Duarte Miguel F. Prazeres, Gabriel Monteiro, ”Improving minicircle DNA production by in silico re-design of the ParA resolvase untranslated region », 12th ESBES – European Symposium on Biochemical Engineering Sciences, September 2018, Lisboa, Portugal
A.Rita Silva-Santos, Cláudia P.A. Alves, Michaela Simčíková, Gabriel Monteiro, Ana M Azevedo, Duarte Miguel F. Prazeres, “Multimodal chromatography of supercoiled minicircles: a closer look into DNA-ligand interactions, 12th ESBES – European Symposium on Biochemical Engineering Sciences, September 2018, Lisboa, Portugal
DOCTORAL PROGRAM (36 ECTS)
Curricular units:
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Gene Therapy (6 ECTS)
• Steam cell engineering(6 ECTS)
Advanced courses:
• Animal Cell Technology course, organized by the European Society of Animal Cell Technology in Llafranc, Girona, Spain, from 24th to 28th September 2017 (30 hours)
• Cell Culture-based Viral Vaccines course – 3rd edition, organized by the European Society for Animal Cell Technology in Llafranc, Girona, Spain from 18-22 September 2018 (25 hours)
Current position
06/2022 – 11/2022: Researcher @ Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
11/2021 – 03/2022: Researcher @ Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal