Publications
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Ibrahim D, Kis Z, Papathanasiou MM, et al., 2024, Strategic Planning of a Joint SARS-CoV-2 and Influenza Vaccination Campaign in the UK, Vaccines, Vol: 12, ISSN: 2076-393X
The simultaneous administration of SARS-CoV-2 and influenza vaccines is being carried out for the first time in the UK and around the globe in order to mitigate the health, economic, and societal impacts of these respiratory tract diseases. However, a systematic approach for planning the vaccine distribution and administration aspects of the vaccination campaigns would be beneficial. This work develops a novel multi-product mixed-integer linear programming (MILP) vaccine supply chain model that can be used to plan and optimise the simultaneous distribution and administration of SARS-CoV-2 and influenza vaccines. The outcomes from this study reveal that the total budget required to successfully accomplish the SARS-CoV-2 and influenza vaccination campaigns is equivalent to USD 7.29 billion, of which the procurement costs of SARS-CoV-2 and influenza vaccines correspond to USD 2.1 billion and USD 0.83 billion, respectively. The logistics cost is equivalent to USD 3.45 billion, and the costs of vaccinating individuals, quality control checks, and vaccine shipper and dry ice correspond to USD 1.66, 0.066, and 0.014, respectively. The analysis of the results shows that the choice of rolling out the SARS-CoV-2 vaccine during the vaccination campaign can have a significant impact not only on the total vaccination cost but also on vaccine wastage rate.
Newall AT, Beutels P, Kis Z, et al., 2023, Placing a value on increased flexible vaccine manufacturing capacity for future pandemics., Vaccine, Vol: 41, Pages: 2317-2319
Geall AJ, Kis Z, Ulmer JB, 2023, Vaccines on demand, part II: future reality., Expert Opin Drug Discov, Vol: 18, Pages: 119-127
INTRODUCTION: Prior to the emergence of SARS-CoV-2, the potential use of mRNA vaccines for a rapid pandemic response had been well described in the scientific literature, however during the SARS-CoV-2 outbreak we witnessed the large-scale deployment of the platform in a real pandemic setting. Of the three RNA platforms evaluated in clinical trials, including 1) conventional, non-amplifying mRNA (mRNA), 2) base-modified, non-amplifying mRNA (bmRNA), which incorporate chemically modified nucleotides, and 3) self-amplifying RNA (saRNA), the bmRNA technology emerged with superior clinical efficacy. AREAS COVERED: This review describes the current state of these mRNA vaccine technologies, evaluates their strengths and limitations, and argues that saRNA may have significant advantages if the limitations of stability and complexities of manufacturing can be overcome. EXPERT OPINION: The success of the SARS-CoV-2 mRNA vaccines has been remarkable. However, several challenges remain to be addressed before this technology can successfully be applied broadly to other disease targets. Innovation in the areas of mRNA engineering, novel delivery systems, antigen design, and high-quality manufacturing will be required to achieve the full potential of this disruptive technology.
Alifia KCH, Kontoravdi C, Kis Z, et al., 2022, Techno-Economic Evaluation of Novel SARS-CoV-2 Vaccine Manufacturing in the Insect Cell Baculovirus Platform, INTERNATIONAL JOURNAL OF TECHNOLOGY, Vol: 13, Pages: 1630-1639, ISSN: 2086-9614
Daniel S, Kis Z, Kontoravdi K, et al., 2022, Quality by design for enabling RNA platform production processes, Trends in Biotechnology, Vol: 40, Pages: 1213-1228, ISSN: 0167-7799
RNA-based products have emerged as one of the most promising and strategic technologies for global vaccination, infectious disease control and future therapy development. The assessment of critical quality attributes, product-process interactions, relevant process analytical technologies, and process modeling capabilities can feed into a robust Quality by Design (QbD) framework for future development, design and control of manufacturing processes. Its implementation will help the RNA technology to reach its full potential and will be central in the development, pre-qualification and regulatory approval of rapid response, disease-agnostic RNA platform production processes.
Palmieri E, Kis Z, Ozanne J, et al., 2022, GMMA as an alternative carrier for a glycoconjugate vaccine against Group A streptococcus, Vaccines, Vol: 10, Pages: 1-17, ISSN: 2076-393X
Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM197; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM197 in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM197, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM197. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.
Ly HH, Daniel S, Soriano SKV, et al., 2022, Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach., Mol Pharm, Vol: 19, Pages: 1892-1905
Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.
Rostami F, Kis Z, Koppelaar R, et al., 2022, Comparative sustainability study of energy storage technologies using data envelopment analysis, ENERGY STORAGE MATERIALS, Vol: 48, Pages: 412-438, ISSN: 2405-8297
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Kis Z, Tak K, Ibrahim D, et al., 2022, Pandemic-response adenoviral vector and RNA vaccine manufacturing, npj Vaccines, Vol: 7, ISSN: 2059-0105
Rapid global COVID-19 pandemic response by mass vaccination is currently limited by the rate of vaccine manufacturing. This study presents a techno-economic feasibility assessment and comparison of three vaccine production platform technologies deployed during the COVID-19 pandemic: (1) adenovirus-vectored (AVV) vaccines, (2) messenger RNA (mRNA) vaccines, and (3) the newer self-amplifying RNA (saRNA) vaccines. Besides assessing the baseline performance of the production process, impact of key design and operational uncertainties on the productivity and cost performance of these vaccine platforms is evaluated using variance-based global sensitivity analysis. Cost and resource requirement projections are computed for manufacturing multi-billion vaccine doses for covering the current global demand shortage and for providing annual booster immunisations. The model-based assessment provides key insights to policymakers and vaccine manufacturers for risk analysis, asset utilisation, directions for future technology improvements and future pidemic/pandemic preparedness, given the disease-agnostic nature of these vaccine production platforms.
Kis Z, 2022, Stability modelling of mRNA vaccine quality based on temperature monitoring throughout the distribution chain, Pharmaceutics, Vol: 14, ISSN: 1999-4923
The vaccine distribution chains in several low- and middle-income countries are not adequate to facilitate the rapid delivery of high volumes of thermosensitive COVID-19 mRNA vaccines at the required low and ultra-low temperatures. COVID-19 mRNA vaccines are currently distributed along with temperature monitoring devices to track and identify deviations from predefined conditions throughout the distribution chain. These temperature readings can feed into computational models to quantify mRNA vaccine critical quality attributes (CQAs) and the remaining vaccine shelf life more accurately. Here, a kinetic modelling approach is proposed to quantify the stability-related CQAs and the remaining shelf life of mRNA vaccines. The CQA and shelf-life values can be computed based on the conditions under which the vaccines have been distributed from the manufacturing facilities via the distribution network to the vaccination centres. This approach helps to quantify the degree to which temperature excursions impact vaccine quality and can also reduce vaccine wastage. In addition, vaccine stock management can be improved due to the information obtained on the remaining shelf life of mRNA vaccines. This model-based quantification of mRNA vaccine quality and remaining shelf life can improve the deployment of COVID-19 mRNA vaccines to low- and middle-income countries.
Kis Z, Tak K, Ibrahim D, et al., 2022, Quality by design and techno-economic modelling of RNA vaccine production for pandemic-response, Computer Aided Chemical Engineering, Pages: 2167-2172
Vaccine production platform technologies have played a crucial role in rapidly developing and manufacturing vaccines during the COVID-19 pandemic. The role of disease agnostic platform technologies, such as the adenovirus-vectored (AVV), messenger RNA (mRNA), and the newer self-amplifying RNA (saRNA) vaccine platforms is expected to further increase in the future. Here we present modelling tools that can be used to aid the rapid development and mass-production of vaccines produced with these platform technologies. The impact of key design and operational uncertainties on the productivity and cost performance of these vaccine platforms is evaluated using techno-economic modelling and variance-based global sensitivity analysis. Furthermore, the use of the quality by digital design framework and techno-economic modelling for supporting the rapid development and improving the performance of these vaccine production technologies is also illustrated.
Ibrahim D, Kis Z, Tak K, et al., 2022, Optimal design and planning of supply chains for viral vectors and RNA vaccines, Computer Aided Chemical Engineering, Pages: 1633-1638
This work develops a multi-product MILP vaccine supply chain model that supports planning, distribution, and administration of viral vectors and RNA-based vaccines. The capability of the proposed vaccine supply chain model is illustrated using a real-world case study on vaccination against SARS-CoV-2 in the UK that concerns both viral vectors (e.g., AZD1222 developed by Oxford-AstraZeneca) and RNA-based vaccine (e.g., BNT162b2 developed by Pfizer-BioNTech). A comparison is made between the resources required and logistics costs when viral vectors and RNA vaccines are used during the SARS-CoV-2 vaccination campaign. Analysis of results shows that the logistics cost of RNA vaccines is 85% greater than that of viral vectors, and that transportation cost dominates logistics cost of RNA vaccines compared to viral vectors.
Ibrahim D, Kis Z, Tak K, et al., 2021, Model-based planning and delivery of mass vaccination campaigns against infectious disease: application to the COVID-19 pandemic in the UK, Vaccines, Vol: 9, Pages: 1-19, ISSN: 2076-393X
Vaccination plays a key role in reducing morbidity and mortality caused by infectious diseases, including the recent COVID-19 pandemic. However, a comprehensive approach that allows the planning of vaccination campaigns and the estimation of the resources required to deliver and administer COVID-19 vaccines is lacking. This work implements a new framework that supports the planning and delivery of vaccination campaigns. Firstly, the framework segments and priorities target populations, then estimates vaccination timeframe and workforce requirements, and lastly predicts logistics costs and facilitates the distribution of vaccines from manufacturing plants to vaccination centres. The outcomes from this study reveal the necessary resources required and their associated costs ahead of a vaccination campaign. Analysis of results shows that by integrating demand stratification, administration, and the supply chain, the synergy amongst these activities can be exploited to allow planning and cost-effective delivery of a vaccination campaign against COVID-19 and demonstrates how to sustain high rates of vaccination in a resource-efficient fashion.
van de Berg D, Kis Z, Behmer CF, et al., 2021, Quality by design modelling to support rapid RNA vaccine production against emerging infectious diseases, npj Vaccines, Vol: 6, ISSN: 2059-0105
Rapid-response vaccine production platform technologies, including RNA vaccines, are being developed to combat viral epidemics and pandemics. A key enabler of rapid response is having quality-oriented disease-agnostic manufacturing protocols ready ahead of outbreaks. We are the first to apply the Quality by Design (QbD) framework to enhance rapid-response RNA vaccine manufacturing against known and future viral pathogens. This QbD framework aims to support the development and consistent production of safe and efficacious RNA vaccines, integrating a novel qualitative methodology and a quantitative bioprocess model. The qualitative methodology identifies and assesses the direction, magnitude and shape of the impact of critical process parameters (CPPs) on critical quality attributes (CQAs). The mechanistic bioprocess model quantifies and maps the effect of four CPPs on the CQA of effective yield of RNA drug substance. Consequently, the first design space of an RNA vaccine synthesis bioreactor is obtained. The cost-yield optimization together with the probabilistic design space contribute towards automation of rapid-response, high-quality RNA vaccine production.
Antonakoudis A, Kis Z, Kontoravdi K, et al., 2021, Accelerating product and process development through a model centric approach, Quality by Design - An Indispensable Approach to Accelerate Biopharmaceutical Product Development, Editors: Campa, Khan, Publisher: Parenteral Drug Association, Inc., Pages: 285-338, ISBN: 978-1-945584-22-0
Kis Z, Papathanasiou M, Kotidis P, et al., 2021, Stability modelling for biopharmaceutical process intermediates, Quality by Design - An Indispensable Approach to Accelerate Biopharmaceutical Product Development, Editors: Campa, Khan, Publisher: Parenteral Drug Association, Inc, Pages: 200-225, ISBN: 978-1-945584-22-0
Kis Z, Kontoravdi C, Shattock R, et al., 2021, Correction: Kis, Z. et al. Resources, Production Scales and Time Required for Producing RNA Vaccines for the Global Pandemic Demand. Vaccines 2021, 9, 3, Vaccines, Vol: 9, Pages: 1-1, ISSN: 2076-393X
Kis Z, 2021, Enhancing Vaccine Platforms: Computational Models Accelerate Development, Manufacturing, and Distribution, BioProcess International
Kis Z, Kontoravdi C, Shattock R, et al., 2020, Resources, production scales and time required for producing RNA vaccines for the global pandemic demand., Vaccines (Basel), Vol: 9, Pages: 1-14, ISSN: 2076-393X
To overcome pandemics, such as COVID-19, vaccines are urgently needed at very high volumes. Here we assess the techno-economic feasibility of producing RNA vaccines for the demand associated with a global vaccination campaign. Production process performance is assessed for three messenger RNA (mRNA) and one self-amplifying RNA (saRNA) vaccines, all currently under clinical development, as well as for a hypothetical next-generation saRNA vaccine. The impact of key process design and operation uncertainties on the performance of the production process was assessed. The RNA vaccine drug substance (DS) production rates, volumes and costs are mostly impacted by the RNA amount per vaccine dose and to a lesser extent by the scale and titre in the production process. The resources, production scale and speed required to meet global demand vary substantially in function of the RNA amount per dose. For lower dose saRNA vaccines, global demand can be met using a production process at a scale of below 10 L bioreactor working volume. Consequently, these small-scale processes require a low amount of resources to set up and operate. RNA DS production can be faster than fill-to-finish into multidose vials; hence the latter may constitute a bottleneck.
Kis Z, Kontoravdi K, Dey AK, et al., 2020, Rapid development and deployment of high-volumevaccines for pandemic response, Journal of Advanced Manufacturing and Processing, Vol: 2, Pages: 1-10, ISSN: 2637-403X
Overcoming pandemics, such as the current Covid‐19 outbreak, requires the manufacture of several billion doses of vaccines within months. This is an extremely challenging task given the constraints in small‐scale manufacturing for clinical trials, clinical testing timelines involving multiple phases and large‐scale drug substance and drug product manufacturing. To tackle these challenges, regulatory processes are fast‐tracked, and rapid‐response manufacturing platform technologies are used. Here, we evaluate the current progress, challenges ahead and potential solutions for providing vaccines for pandemic response at an unprecedented scale and rate. Emerging rapid‐response vaccine platform technologies, especially RNA platforms, offer a high productivity estimated at over 1 billion doses per year with a small manufacturing footprint and low capital cost facilities. The self‐amplifying RNA (saRNA) drug product cost is estimated at below 1 USD/dose. These manufacturing processes and facilities can be decentralized to facilitate production, distribution, but also raw material supply. The RNA platform technology can be complemented by an a priori Quality by Design analysis aided by computational modeling in order to assure product quality and further speed up the regulatory approval processes when these platforms are used for epidemic or pandemic response in the future.
Kis Z, Shattock R, Shah N, et al., 2019, Correction: Emerging technologies for low‐cost, rapid vaccine manufacture, Biotechnology Journal, Vol: 14, Pages: 1-2, ISSN: 1860-6768
Kis Z, Papathanasiou M, CalvoSerrano R, et al., 2019, A model‐based quantification of the impact of new manufacturing technologies on developing country vaccine supply chain performance: A Kenyan case study, Journal of Advanced Manufacturing and Processing, Vol: 1, ISSN: 2637-403X
Kis Z, Shattock R, Shah N, et al., 2019, Emerging technologies for low-cost, rapid vaccine manufacture, Biotechnology Journal, Vol: 14, ISSN: 1860-6768
To stop the spread of future epidemics and meet infant vaccination demands in low‐ and middle‐income countries, flexible, rapid and low‐cost vaccine development and manufacturing technologies are required. Vaccine development platform technologies that can produce a wide range of vaccines are emerging, including: a) humanized, high‐yield yeast recombinant protein vaccines; b) insect cell‐baculovirus ADDomer vaccines; c) Generalized Modules for Membrane Antigens (GMMA) vaccines; d) RNA vaccines. Herein, existing and future platforms are assessed in terms of addressing challenges of scale, cost, and responsiveness. To assess the risk and feasibility of the four emerging platforms, the following six metrics are applied: 1) technology readiness; 2) technological complexity; 3) ease of scale‐up; 4) flexibility for the manufacturing of a wide range of vaccines; 5) thermostability of the vaccine product at tropical ambient temperatures; and 6) speed of response from threat identification to vaccine deployment. The assessment indicated that technologies in the order of increasing feasibility and decreasing risk are the yeast platform, ADDomer platform, followed by RNA and GMMA platforms. The comparative strengths and weaknesses of each technology are discussed in detail, illustrating the associated development and manufacturing needs and priorities.
Pereira GC, Malik S, Kis Z, et al., 2019, Computationally Designed Recombinant-DNA-Based Compounds Production Driven in Plants During Secondary Metabolism and Their Implication in Antimalarial Therapies, NATURAL BIO-ACTIVE COMPOUNDS, VOL 2: CHEMISTRY, PHARMACOLOGY AND HEALTH CARE PRACTICES, Editors: Swamy, Akhtar, Publisher: SPRINGER-VERLAG SINGAPORE PTE LTD, Pages: 127-146, ISBN: 978-981-13-7204-9
Kis Z, Koppelaar RHEM, Sule MN, et al., 2018, Framework for WASH sector data improvements in data-poor environments, applied to Accra, Ghana, Water, Vol: 10, ISSN: 2073-4441
Improvements in water, sanitation and hygiene (WASH) service provision are hampered by limited open data availability. This paper presents a data integration framework, collects the data and develops a material flow model, which aids data-based policy and infrastructure development for the WASH sector. This model provides a robust quantitative mapping of the complete anthropogenic WASH flow-cycle: from raw water intake to water use, wastewater and excreta generation, discharge and treatment. This approach integrates various available sources using a process-chain bottom-up engineering approach to improve the quality of WASH planning. The data integration framework and the modelling methodology are applied to the Greater Accra Metropolitan Area (GAMA), Ghana. The highest level of understanding of the GAMA WASH sector is achieved, promoting scenario testing for future WASH developments. The results show 96% of the population had access to improved safe water in 2010 if sachet and bottled water was included, but only 67% if excluded. Additionally, 66% of 338,000 m3 per day of generated wastewater is unsafely disposed locally, with 23% entering open drains, and 11% sewage pipes, indicating poor sanitation coverage. Total treated wastewater is <0.5% in 2014, with only 18% of 43,000 m3 per day treatment capacity operational. The combined data sets are made available to support research and sustainable development activities.
Kis Z, Pandya N, Koppelaar RHEM, 2018, Electricity generation technologies: Comparison of materials use, energy return on investment, jobs creation and CO2 emissions reduction, Energy Policy, Vol: 120, Pages: 144-157, ISSN: 0301-4215
Abstract Shifting to a low-carbon electricity future requires up-to-date information on the energetic, environmental and socio-economic performance of technologies. Here, we present a novel comprehensive bottom-up process chain framework that is applied to 19 electricity generation technologies, consistently incorporating 12 life-cycle phases from extraction to decommissioning. For each life-cycle phase of each technology the following 4 key metrics were assessed: material consumption, energy return ratios, job requirements and greenhouse gas emissions. We also calculate a novel global electricity to grid average for these metrics and present a metric variability analysis by altering transport distance, load factors, efficiency, and fuel density per technology. This work quantitatively supports model-to-policy frameworks that drive technology selection and investment based on energetic-economic viability, job creation and carbon emission reduction of technologies. The results suggest energetic-economic infeasibility of electricity generation networks with substantial shares of: i) liquefied natural gas transport, ii) long distance transport based hard and brown coal and pipeline natural gas, and iii) low-load factor solar-photovoltaic, concentrated solar power, onshore and offshore wind. Direct sector jobs can be expected to double in renewable-majority scenarios. All combustion-powered technologies without natural (biomass) or artificial carbon capture (fossil fuels) are not compatible with a low carbon electricity generation future.
Lacroix J, Kis Z, Josupeit R, et al., 2018, Preclinical testing of an Oncolytic Parvovirus in Ewing Sarcoma: Protoparvovirus H-1 induces Apoptosis and Lytic infection In Vitro but fails to improve survival In Vivo, Viruses, Vol: 10, ISSN: 1999-4915
About 70% of all Ewing sarcoma (EWS) patients are diagnosed under the age of 20 years. Over the last decades little progress has been made towards finding effective treatment approaches for primarily metastasized or refractory Ewing sarcoma in young patients. Here, in the context of the search for novel therapeutic options, the potential of oncolytic protoparvovirus H-1 (H-1PV) to treat Ewing sarcoma was evaluated, its safety having been proven previously tested in adult cancer patients and its oncolytic efficacy demonstrated on osteosarcoma cell cultures. The effects of viral infection were tested in vitro on four human Ewing sarcoma cell lines. Notably evaluated were effects of the virus on the cell cycle and its replication efficiency. Within 24 h after infection, the synthesis of viral proteins was induced. Efficient H-1PV replication was confirmed in all four Ewing sarcoma cell lines. The cytotoxicity of the virus was determined on the basis of cytopathic effects, cell viability, and cell lysis. These in vitro experiments revealed efficient killing of Ewing sarcoma cells by H-1PV at a multiplicity of infection between 0.1 and 5 plaque forming units (PFU)/cell. In two of the four tested cell lines, significant induction of apoptosis by H-1PV was observed. H-1PV thus meets all the in vitro criteria for a virus to be oncolytic towards Ewing sarcoma. In the first xenograft experiments, however, although an antiproliferative effect of intratumoral H-1PV injection was observed, no significant improvement of animal survival was noted. Future projects aiming to validate parvovirotherapy for the treatment of pediatric Ewing sarcoma should focus on combinatorial treatments and will require the use of patient-derived xenografts and immunocompetent syngeneic animal models.
Geiss C, Kis Z, Leuchs B, et al., 2017, Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro, Viruses, Vol: 9, Pages: 301-301, ISSN: 1999-4915
Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.
Kis Z, Rodin T, Zafar A, et al., 2016, Development of a synthetic gene network to module gene expression by mechanical forces, Scientific Reports, Vol: 6, ISSN: 2045-2322
The majority of (mammalian) cells in our body are sensitive to mechanical forces, but little work hasbeen done to develop assays to monitor mechanosensor activity. Furthermore, it is currently impossibleto use mechanosensor activity to drive gene expression. To address these needs, we developed thefrst mammalian mechanosensitive synthetic gene network to monitor endothelial cell shear stresslevels and directly modulate expression of an atheroprotective transcription factor by shear stress. Thetechnique is highly modular, easily scalable and allows graded control of gene expression by mechanicalstimuli in hard-to-transfect mammalian cells. We call this new approach mechanosyngenetics. To insertthe gene network into a high proportion of cells, a hybrid transfection procedure was developed thatinvolves electroporation, plasmids replication in mammalian cells, mammalian antibiotic selection,a second electroporation and gene network activation. This procedure takes 1 week and yielded over60% of cells with a functional gene network. To test gene network functionality, we developed a fowsetup that exposes cells to linearly increasing shear stress along the length of the fow channel foor.Activation of the gene network varied logarithmically as a function of shear stress magnitude.
Krams R, Towhidi L, Khodadadi D, et al., 2016, Comparison between direct and reverse electroporation of cells in situ: a simulation study, Physiological Reports, Vol: 4, ISSN: 2051-817X
The discovery of the human genome has unveiled new fields of genomics,transcriptomics, and proteomics, which has produced paradigm shifts on howto study disease mechanisms, wherein a current central focus is the under-standing of how gene signatures and gene networks interact within cells. Thesegene function studies require manipulating genes either through activation orinhibition, which can be achieved by temporarily permeabilizing the cellmembrane through transfection to deliver cDNA or RNAi. An efficient trans-fection technique is electroporation, which applies an optimized electric pulseto permeabilize the cells of interest. When the molecules are applied on top ofseeded cells, it is called “direct” transfection and when the nucleic acids areprinted on the substrate and the cells are seeded on top of them, it is termed“reverse” transfection. Direct transfection has been successfully applied in pre-vious studies, whereas reverse transfection has recently gained more attentionin the context of high-throughput experiments. Despite the emerging impor-tance, studies comparing the efficiency of the two methods are lacking. In thisstudy, a model for electroporation of cells in situ is developed to address thisdeficiency. The results indicate that reverse transfection is less efficient thandirect transfection. However, the model also predicts that by increasing theconcentration of deliverable molecules by a factor of 2 or increasing theapplied voltage by 20%, reverse transfection can be approximately as efficientas direct transfection.
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