Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.

Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells.

This article describes a novel method merging the cloning of viral vector producer cells with vector titer screening, allowing for screening 200-500 clones in 2 weeks. It makes use of a GFP separated into two fragments, S10 and S11 (Split GFP), fluorescing only upon transcomplementation. Producer cells carrying a S11 viral transgene are cloned in 96-well plates and co-cultured with target cells stably expressing S10. During the period of clone expansion, S11 viruses infect S10 target cells reconstituting the GFP signal. Transcomplemented fluorescence data provide direct estimation of the clone's productivity and can be analyzed in terms of density distribution, offering valuable information on the average productivity of the cell population and allowing the identification of high-producing clones. The method was validated by establishing a retrovirus producer from a nude cell line, in <3 months, inserting three vector constructs without clone selection or screening in between. Clones producing up to 10(8) infectious particles per ml were obtained, delivering optimal ratios of infectious-to-total particles (1 to 5). The method was additionally used to evaluate the production performance of HEK 293 and HEK 293T cell lines demonstrating that the latter sustains increased titers. Finally, it was used to study genetic manipulation of glutathione metabolism in retrovirus production showing that changing cell metabolism steers higher vector expression with titer increases of more than one order of magnitude.This method is a valuable tool not only for cell line development but also for genetic manipulation of viral vector and/or producer cells contributing to advancing the field of viral gene therapy.

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

PINCHAR HACE DAÑO Representaciones del dolor en el niño, en edad escolar,sometido a punción venosa / No disponible

Introducción: El dolor forma parte de la condición humana y es inalienable de su existencia. Es entendido como una experiencia universal con inicio precoz en cada individuo y gana heterogeneidad de configuraciones sociales y variabilidad de grados de intensidad. Como experiencia intransmisible, marcará, de diversas formas, la construcción psicológica y social de la persona. En el niño es una experiencia común y perturbadora, muchas veces subestimada y subintervencionada en contextos de salud. Metodología: Con este estudio exploratorio de naturaleza mixta, basado en la teoría de las representaciones sociales, pretendemos dar voz a los niños sometidos a punción venosa. Los objetivos de este trabajo son identificar las representaciones asociadas a la experiencia de punción venosa y evaluar el grado de dolor asociado a esta experiencia, en niños en edad escolar. Se aplicó la asociación libre de palabras con dos cuestiones estímulo: «El dolor me hece pensar en…», «El pinchazo de la aguja me hace sentir…» y la escala numérica de evaluación del dolor, a 43 niños con edades comprendidas entre 6 y 12 años ingresados en un hospital de la Sub-región de Salud de Lisboa. Los datos se analizaron con recurso del software SPAD-T (análisis factorial de correspondencias simples) y SPSS (estadística descrpitiva). Resultados: Los datos obtenidos permiten decir que los niños que participaron en este estudio consideraron que el dolor funciona como factor desencadenante de sufrimiento y está asociado a manifestaciones físicas como el llanto y a sentimentos expresados ante el dolor, como el miedo y la ansiedad. El grado de dolor está asociado a la punción venosa y presenta variabilidad con factores como la edad y las experiencias anteriores. Conclusión: La punción venosa es un procedimiento doloroso y, según los niños que participaron en el estudio, provoca miedo y ansiedad. Sin embargo, muchos evitaron tener comportamientos sugestivos de dolor por vergüenza de ser considerados más flojos o por entender que es un dolor necesario. De ahí el hecho de que algunos niños, asociaran al dolor la valentía que se necesita tener para soportarla (AU)

Introduction: Pain is a part ofhuman nature and is an unalienable part of our existence. It is understood as a universal experience, with an early beginning, in each person, and it gans heterogeneity from social configurations and varying intensity. As an untransferrableexperience, it marks the construction of the psychological and social person in various ways. For children, pain is a common and disturbing experience that is sometimes underestimated in health contexts. Methodology: This exploratory study is of a mixed nature, which was based on the theory of social representations, aims to dentify the representation associated to pain, in school age children submitted to venous puncture and to the degree ofpain associated to venous puncture. Therefore, the free association of words technique was applied using two stimulus questions: «Pain makes me think about…», the needle prick makes me feel…», and the numerical scale of pain evaluation, to forty three children with ages between six and twelve years old, who were hospitalized in Lisbon’s Health Sub-region. The data have been analyzed using SPAD-T (factorial analysis of simple correspondence) and SPSS (descriptive statistics) software. The Results show that for those children pain is a factor which arouses suffering and is associated, by children, to physical manifestations, such as crying and to feelings expressed in the presence of pain, like fear. This procedure provokes fear and anxiety in hospitalized children. The degree of pain associated to venous puncture changes with factors like age and mainly with previous experience. In Conclusion, venipuncture is a painful procedure and according to the children who participated in the study, it causes fear and anxiety. However, many avoided suggestive feelings of pain, because they were ashamed to be considered weaker or deem it a necessary pain; hence the fact that some children associate pain and the need to have the necessary courage to support it (AU)

Down-regulation of CD81 tetraspanin in human cells producing retroviral-based particles: tailoring vector composition.

Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by reducing the amounts of the pseudotyped envelope and/or by incorporating protein capable of inducing immune responses when non-human VPC are used. Manipulating the repertoire of VPC proteins integrated onto the vector structure is an underexplored territory and should provide valuable insights on potential targets to improve vector pharmacokinetic and pharmacodynamic properties. In this work, human HEK 293 cells producing retrovirus-like particles (rVLPs) and infectious RV vectors were used to prove the concept of customizing RV composition by manipulating cellular protein content. The tetraspanin CD81 was chosen since it is significantly incorporated in the RV membrane, conferring to the vector significant immunogenicity when used in mice. RNA interference-mediated by shRNA lentiviral vector transduction was efficiently used to silence CD81 expression (up to 99%) and the rVLPs produced by knocked-down cells lack CD81. Silenced clones were analyzed for cell proliferation, morphological changes, susceptibility to oxidative stress conditions, and rVLP productivities. The results showed that the down-regulation of VPC proteins requires close monitoring for possible side effects on cellular production performance. Yet, they confirm that it is possible to change the composition of host-derived immunogens in RV by altering cellular protein content with no detriment for vector productivity and titers. This constitutes an important manipulation tool in vaccinology--by exploiting the potential adjuvant effect of VPC proteins or using them as fusion agents to other proteins of interest to be exposed on the vector membrane--and in gene therapy, by reducing the immunogenicity of RV-based vector and enhancing in vivo half-life. Such tools can also be applied to lentiviral or other enveloped viral vectors.