pSETT4, an Improved φC31-Based Integrative Vector System for Actinoplanes sp. SE50/110

ABSTRACT The pSETT4 vector integrates into the Actinoplanes sp. SE50/110 chromosome via the bacteriophage φC31 integrase and allows cloning of a gene of interest by Golden Gate assembly (BsaI). T4 terminators surround the expression cassette to isolate the transcriptional unit and to prevent antisense transcription. The system can be used in other Actinomycetales by exchanging the promoter.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.

A Bivalent RecombinantMycobacterium bovisBCG Expressing the S1 Subunit of the Pertussis Toxin Induces a Polyfunctional CD4+T Cell Immune Response

Background. A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment.Objectives. To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice.Methods. Mycobacterial plasmids were constructed by cloning thes1ptgene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins.Findings. S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γwere observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4+IFN-γ+and double-positive CD4+IFN-γ+TNF-α+T cells.Conclusions. rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4+T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer.

Leadership and Leadership Development Tools in a Vuca World

The challenge of the VUCA’s world to us is about the development of not only narrowly professional competencies, but also of the individual as a whole, that is the process of forming “big” minds. What is “leader” and “leadership” for us now? What needs to be changed in our perception of the term “leadership” in order not only to survive, but also to be on the “crest of a wave” in the time we are experiencing VUCA? How to create a community of leaders and a living environment, an incubator environment where leaders can emerge and effectively manifest themselves? The author offers his view on the choice of leadership development tools. The article summarizes the author’s interview with one of the game developers, whose methodology is based on integrative vector modeling.