Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.Fil: Tossolini, Ileana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: López Díaz, Fernando. Salk Institute for Biological Studies; Estados UnidosFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Protein design and immunogenic analysis of COVID-19 vaccine candidates based on RBD/trimeric-spike antigens and chimeric VLPs antigens

Spike glycoprotein (S) and its Receptor Binding Domain region (RBD) are the main targets of neutralizing antibodies during an infection with SARS-CoV-2, the etiologic agent of COVID-19 pandemic viral disease. Thus, they are the chosen antigens for the development of diagnostic kits and vaccines candidates. Furthermore, Virus-like Particles (VLPs) constitute potent immunogens that have been engineered to obtain vaccine candidates through expression of SARS-CoV-2 S, M, E and N proteins. Although it might be a challenging platform, as multiple proteins must be expressed in order to assure VLP budding, they are able to induce stronger immune responses than soluble antigens due to their particulate nature and highly repetitive antigen display. Hence, we aimed to generate a serum-free platform to produce soluble SARS-CoV-2 antigens and design a novel chimeric VLP (cVLP) exposing the prefusion stabilized S ectodomain on its surface. Please click Download on the upper right corner to see the full abstract

Crosslinked casein micelles bound paclitaxel as enzyme activated intracellular drug delivery systems for cancer therapy

Nanomedicine for cancer therapy is a successful tool to diminish the side effect of chemotherapeutics such as paclitaxel (PTX). In this regard, Abraxane®, a human serum albumin (HSA)-based nanomedicine system has shown lesser side effects than Taxol®. However, the large-scale production of HSA protein is limited and expensive, which is traduced in a high cost of the treatments in clinical applications. Thus, the use of easily-available alternative nanocarriers could increment the accessibility of patients to nanomedicine for cancer treatments. Casein is a low-cost protein able to self-assemble into micelles which could efficiently encapsulate PTX into their structure. In this work, the synthesis of chemically crosslinked casein micelles (CCM), used to prepare PTX-based nanoformulations, is presented. CCM@PTX nanoformulations showed promising results in vitro to be applied as nanomedicine for cancer therapy. Thus, the obtained nanoformulations are great candidates to be parenterally administered, accumulate in tumor by passive targeting without leakage of PTX in plasma, and release the drug within the tumor microenvironment, in response to overexpressed proteases such as trypsin.Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ronco, Ludmila Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Calderón, Marcelo. Polymat; EspañaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

Design and optimization of a production process of human interferon alpha2b muteins with increased biological activity through glycosylation in mammalian cells

Fil: Gugliotta, Agustina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina.En el presente trabajo de tesis se llevó a cabo la producción de IFN4N empleando células CHO-K1 y HEK293 como huéspedes y purificación. IFN4NCHO e IFN4NHEK fueron caracterizados en términos de su grado de glicosilación, farmacocinética y actividad biológica. El IFN4NCHO presentó una masa molecular aparente superior y N-glicanos altamente ramificados con un mayor contenido de ácido siálico respecto de la molécula obtenida a partir de HEK. Estudios de farmacocinética revelaron la existencia de diferencias significativas sólo en la etapa de absorción. Si bien ensayos de actividad biológica antiviral in vitro demostraron similitud entre ambas proteínas, la actividad antiproliferativa específica in vitro resultó dos veces superior para la variante obtenida en células HEK. El análisis de la actividad biológica antitumoral in vivo permitió confirmar estos resultados, demostrando la mayor potencia antitumoral del IFN4NHEK. Por otra parte se llevó a cabo la generación de nuevas muteínas hiperglicosiladas del hIFN-alfa2b, con el objetivo de optimizar la molécula IFN4N. El estudio de las nuevas moléculas permitió observar que el grado de glicosilación de las mismas se incrementó conforme aumentó el número de sitios potenciales para N-glicosilación, lo cual se tradujo en un incremento del tiempo de vida media plasmática. A su vez, las variantes carentes de la modificación R23N evidenciaron valores de ABE antiproliferativa in vitro superiores respecto del IFN4N. Los resultados de esta tesis revelan la importancia de encontrar el balance apropiado entre la actividad biológica in vitro y las propiedades farmacocinéticas para la obtención de un producto terapéutico innovador y efectivo.In the present thesis work IFN4N was produced in CHO and HEK cells and purified. IFN4NCHO and IFN4NHEK were characterized in terms of glycosylation, pharmacokinetics and biological activity. IFN4NCHO exhibited a higher average molecular mass and more acidic and ramified isoforms compared to IFN4NHEK. Regarding pharmacokinetic studies, IFN4NHEK reached maximum plasma concentration 3-times faster than IFN4NCHO, however their elimination profile did not differ significantly. Also, despite the in vitro antiviral specific biological activity of both proteins was the same, IFN4NHEK was more efficient as an antiproliferative agent in different tumor-derived cell lines. Accordingly, IFN4NHEK showed a higher in vivo antitumor activity in animal models. As part of this thesis work new muteins of rhIFN-alpha2b were designed and constructed in order to improve the glycosylation degree and, consequently, the pharmacokinetic properties, and the in vitro antiproliferative activity of IFN4N. The characterization of the muteins showed that the introduction of new N glycosylation sites resulted in the increment of their glycosylation degree. The in vitro biological characterization showed that variants lacking the mutation R23N presented an increased antiproliferative activity. The results of this thesis demonstrate that searching for an appropriate balance between the in vitro biological activity and the pharmacokinetic properties in the context of an adequate production host would constitute the basis to generate an innovative and effective therapeutic product.Universidad Nacional del LitoralConsejo Nacional de Investigaciones Científicas y Técnica

Epigenetic modulations rendering cell-to-cell variability and phenotypic metastability

Tumor cells display phenotypic plasticity and heterogeneity due to genetic and epigenetic variations which limit the predictability of therapeutic interventions. Chromatin modifications can arise stochastically but can also be a consequence of environmental influences such as the microenvironment of cancer cells. A better understanding of the impact and dynamics of epigenetic modulation at defined chromosomal sites is required to get access to the underlying mechanisms. We investigated the epigenetic modulations leading to cell-to-cell heterogeneity in a tumor cell line model. To this end, we analyzed expression variance in 80 genetically uniform cell populations having a single-copy reporter randomly integrated in the genome. Single-cell analysis showed high intraclonal heterogeneity. Epigenetic characterization revealed that expression heterogeneity was accompanied by differential histone marks whereas contribution of DNA methylation could be excluded. Strikingly, some clones revealed a highly dynamic, stochastically altered chromatin state of the transgene cassette which was accompanied with a metastable expression pattern. In contrast, other clones represented a robust chromatin state of the transgene cassette with a stable expression pattern. Together, these results elucidate locus-specific epigenetic modulation in gene expression that contributes to phenotypic heterogeneity of cells and might account for cellular plasticity.Fil: Spencer, Shawal. Helmholtz Centre For Infection Research; AlemaniaFil: Gugliotta, Agustina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Gödecke, Natascha. Helmholtz Centre For Infection Research; AlemaniaFil: Hauser, Hansjörg. Helmholtz Centre For Infection Research; AlemaniaFil: Wirth, Dagmar. Helmholtz Centre For Infection Research; Alemania. Hannover Medical School; Alemani

Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation

tIntraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenonthat has a strong impact on overall transgene expression levels and that limits the predictability of trans-gene expression in genetically modified cells, thereby hampering single cell based screening approaches.The underlying mechanism(s) leading to this variance are poorly understood. To study the dynam-ics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in morethan 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copyreporter cassettes driven by EF1alpha and CMV promoters. Single cell analysis showed intraclonal vari-ability with heterogeneity in expression in genetically uniform populations. DNA methylation is a wellknown mechanism responsible for silencing of gene expression. Interestingly, loss of expression wasnot associated with DNA methylation of the CMV promoter. However, in most of the clonal popula-tions expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting thatheterogeneity of transgene expression is crucially governed by histone modifications. Further, to deter-mine if the epigenetic status of transgene expression is governed by the chromosomal integration locuswe targeted heterologous expression cassettes into two chromosomal sites using recombinase medi-ated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-establishedwhen the same promoter used. In this way the problem of early stage cell clone instability can bebypassed. However, upon introduction of an unrelated promoter methylation-independent silencingwas observed. Together, these results suggest that histone modifications are the relevant mechanisms bywhich epigenetic modulation of transgene expression cassettes is governed in the early phase of clonegeneration.Fil: Spencer, Shawal. Helmholtz Centre for Infection Research; AlemaniaFil: Gugliotta, Agustina. Helmholtz Centre for Infection Research; Alemania. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Koenitzer, Jennifer. Boehringer Ingelheim Pharma; AlemaniaFil: Hauser, Hansjörg. Helmholtz Centre for Infection Research; AlemaniaFil: Wirth, Dagmar. Helmholtz Centre for Infection Research; Alemania. Hannover Medical School. Department of Experimental Hematology; Alemani

Effect of ANITVNITV peptide fusion on the bioactivity and pharmacokinetics of human IFN-α2b and a hyper-N-glycosylated variant

Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.Fil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Ceaglio, Natalia Analia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Roggero, Marcos Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Strategies to develop therapeutic N- and O-hyperglycosylated proteins

Glycoengineering by N- and/or O-hyperglycosylation represents a procedure to introduce potential sitesfor adding N- and/or O-glycosyl structures to proteins with the aim of producing biotherapeutics withimproved pharmacodynamic and pharmacokinetic properties. In this chapter, a detailed description of thesteps routinely performed to generate new proteins having high content of N- and/or O-glycosyl moietiesis carried out. The rational strategy involves the initial stage of designing N- and/or O-hyperglycosylatedmuteins to be expressed by mammalian cells and includes the upstream and downstream processing stagesnecessary to develop hyperglycosylated versions of the proteins of interest with the purpose of beginningthe long road toward producing biobetters.Fil: Gugliotta, Agustina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Ceaglio, Natalia Analia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Etcheverrigaray, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Roggero, Marcos Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Gelatin and Tannic Acid Based Iongels for Muscle Activity Recording and Stimulation Electrodes

Iongels are soft ionic conducting materials, usually composed of polymer networks swollen with ionic liquids (ILs), which are being investigated for applications ranging from energy to bioelectronics. The employment of iongels in bioelectronic devices such as bioelectrodes or body sensors has been limited by the lack of biocompatibility of the ILs and/or polymer matrices. In this work, we present iongels prepared from solely biocompatible materials: (i) a biobased polymer network containing tannic acid as a cross-linker in a gelatin matrix and (ii) three different biocompatible cholinium carboxylate ionic liquids. The resulting iongels are flexible and elastic with Young's modulus between 11.3 and 28.9 kPa. The morphology of the iongels is based on a dual polymer network system formed by both chemical bonding due to the reaction of the gelatin's amines with the polyphenol units and physical interactions between the tannic acid and the gelatin. These biocompatible iongels presented high ionic conductivity values, from 0.003 and up to 0.015 S·cm-1 at room temperature. Furthermore, they showed excellent performance as a conducting gel in electrodes for electromyography and electrocardiogram recording as well as muscle stimulation.Fil: Aguzin, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Luque, Gisela Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Ronco, Ludmila Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: del Agua, Isabel. No especifíca;Fil: Guzmán González, Gregorio. Universidad del Pais Vasco. Polymat.; EspañaFil: Marchiori, Bastien. No especifíca;Fil: Gugliotta, Agustina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Tomé, Liliana C.. Universidade Nova de Lisboa; PortugalFil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Mecerreyes, David. Universidad del Pais Vasco. Polymat.; EspañaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin