Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism

Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host rang

Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism

Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range

One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle

Genetically modified cattle are important for developing new biomedical models and for an improved understanding of the pathophysiology of zoonotic diseases. However, genome editing and genetic engineering based on somatic cell nuclear transfer suffer from a low overall efficiency. Here, we established a highly efficient one-step multiplex gene transfer system into the bovine genome.Fil: Garrels, Wiebke. Institut für Nutztiergenetik; AlemaniaFil: Talluri, Thirumala R.. Institut für Nutztiergenetik; AlemaniaFil: Apfelbaum, Ronja. Institut für Nutztiergenetik; AlemaniaFil: Carratalá, Yanet P.. Institut für Nutztiergenetik; AlemaniaFil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Pötzsch, Kerstin. Paul Ehrlich Institute; AlemaniaFil: Grueso, Esther. Paul Ehrlich Institute; AlemaniaFil: Ivics, Zoltan. Paul Ehrlich Institute; AlemaniaFil: Kues, Wilfred. Institut für Nutztiergenetik; Alemani

Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation

Assessing Tn5 and sleeping beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.Instituto de BiotecnologíaFil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Gibbons, Alejandro Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Texeira, D.I.A. Universidade Estadual do Ceará. Faculdade de Veterinária; BrasilFil: Lange, F. Universidad Maimónides. Laboratorio de Clonación y Transgenesis; ArgentinaFil: Vans Landschoot, Geraldina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fil: Lange, F. Universidad Maimónides. Laboratorio de Clonación y Transgenesis; ArgentinaFil: Savy, Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Briski, Olinda. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Grueso, Esther. Paul-Ehrlich-Institute; AlemaniaFil: Ivics, Zoltán. Paul-Ehrlich-Institute; AlemaniaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Kues, Wilfried A. Friedrich-Loeffler-Institut; AlemaniaFil: Ferraris, S.R. Universidad Maimónides. Laboratorio de Clonación y Transgenesis; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Millora de la xarxa sense fils municipal Wi-Fi MESH de Viladecans

El creixement en els darrers anys de les xarxes d’accés sense fils ha generat la necessitat d’algunes ciutats d’implantar xarxes ciutadanes amb la finalitat d’apropar a l’usuari a l’ús d’Internet, moltes d’elles de tecnologia Wi-Fi Mesh. L’Ajuntament de Viladecans és el propietari i ens gestor d’una xarxa Wi-Fi Mesh desplegada sobre una infraestructura d’accés troncal amb fibra òptica. Els dispositius instal·lats al carrer que donen servei d’accés sense fils són equips Tropos 6320 del fabricant Tropos Networks. Aquesta xarxa es va implantar per a millorar l’accés de la ciutadania a la Societat de la Informació i a l’Administració Electrònica i, d’aquest mode, situar a la ciutat en l’avantguarda tecnològica. Aquest treball fi de carrera té com a objectius: estudiar aspectes de seguretat en tecnologies Wi-Fi, estudiar la xarxa d’accés sense fils mallada existent per tal de detectar les mancances i els problemes que pugui presentar a nivell de rendiment, eficiència, seguretat i fiabilitat. Així com proposar, justificar i dur a terme actuacions de millora de la mateixa, avaluar aquestes millores i aconseguir que el propietari integri nous serveis. D’aquesta manera, es realitzaria una correcta explotació de la infraestructura i dels recursos de la xarxa.English: In recent years, the growth of wireless networks has created the needed for cities to implement citizen networks in order to bring the Internet access to the users, many of these networks are Wi-Fi Mesh technologies. The Viladecans Cityhall is the owner and manager of a Wi-Fi Mesh access infrastructure deployed on a fiber optic backbone. The devices installed on the street providing wireless access are Tropos 6320 equipment of the manufacturer Tropos Networks. This network was set up to improve public access to Information Society and e-Government, thus, the city is located at the forefront technology. This bachelor’s final year project aims to study: safety issues in Wi-Fi technology, wireless mesh network to detect existing deficiencies and problems that may arise in terms of performance, efficiency, safety and reliability. Also proposing, justifying and implementing actions in order to improve it and assess these enhancements to achieve the integration of new services. Thus, a correct operation of the infrastructure and resources of the network can be performed

Millora de la xarxa sense fils municipal Wi-Fi MESH de Viladecans

El creixement en els darrers anys de les xarxes d’accés sense fils ha generat la necessitat d’algunes ciutats d’implantar xarxes ciutadanes amb la finalitat d’apropar a l’usuari a l’ús d’Internet, moltes d’elles de tecnologia Wi-Fi Mesh. L’Ajuntament de Viladecans és el propietari i ens gestor d’una xarxa Wi-Fi Mesh desplegada sobre una infraestructura d’accés troncal amb fibra òptica. Els dispositius instal·lats al carrer que donen servei d’accés sense fils són equips Tropos 6320 del fabricant Tropos Networks. Aquesta xarxa es va implantar per a millorar l’accés de la ciutadania a la Societat de la Informació i a l’Administració Electrònica i, d’aquest mode, situar a la ciutat en l’avantguarda tecnològica. Aquest treball fi de carrera té com a objectius: estudiar aspectes de seguretat en tecnologies Wi-Fi, estudiar la xarxa d’accés sense fils mallada existent per tal de detectar les mancances i els problemes que pugui presentar a nivell de rendiment, eficiència, seguretat i fiabilitat. Així com proposar, justificar i dur a terme actuacions de millora de la mateixa, avaluar aquestes millores i aconseguir que el propietari integri nous serveis. D’aquesta manera, es realitzaria una correcta explotació de la infraestructura i dels recursos de la xarxa.English: In recent years, the growth of wireless networks has created the needed for cities to implement citizen networks in order to bring the Internet access to the users, many of these networks are Wi-Fi Mesh technologies. The Viladecans Cityhall is the owner and manager of a Wi-Fi Mesh access infrastructure deployed on a fiber optic backbone. The devices installed on the street providing wireless access are Tropos 6320 equipment of the manufacturer Tropos Networks. This network was set up to improve public access to Information Society and e-Government, thus, the city is located at the forefront technology. This bachelor’s final year project aims to study: safety issues in Wi-Fi technology, wireless mesh network to detect existing deficiencies and problems that may arise in terms of performance, efficiency, safety and reliability. Also proposing, justifying and implementing actions in order to improve it and assess these enhancements to achieve the integration of new services. Thus, a correct operation of the infrastructure and resources of the network can be performed

Millora de la xarxa sense fils municipal Wi-Fi MESH de Viladecans

El creixement en els darrers anys de les xarxes d’accés sense fils ha generat la necessitat d’algunes ciutats d’implantar xarxes ciutadanes amb la finalitat d’apropar a l’usuari a l’ús d’Internet, moltes d’elles de tecnologia Wi-Fi Mesh. L’Ajuntament de Viladecans és el propietari i ens gestor d’una xarxa Wi-Fi Mesh desplegada sobre una infraestructura d’accés troncal amb fibra òptica. Els dispositius instal·lats al carrer que donen servei d’accés sense fils són equips Tropos 6320 del fabricant Tropos Networks. Aquesta xarxa es va implantar per a millorar l’accés de la ciutadania a la Societat de la Informació i a l’Administració Electrònica i, d’aquest mode, situar a la ciutat en l’avantguarda tecnològica. Aquest treball fi de carrera té com a objectius: estudiar aspectes de seguretat en tecnologies Wi-Fi, estudiar la xarxa d’accés sense fils mallada existent per tal de detectar les mancances i els problemes que pugui presentar a nivell de rendiment, eficiència, seguretat i fiabilitat. Així com proposar, justificar i dur a terme actuacions de millora de la mateixa, avaluar aquestes millores i aconseguir que el propietari integri nous serveis. D’aquesta manera, es realitzaria una correcta explotació de la infraestructura i dels recursos de la xarxa.English: In recent years, the growth of wireless networks has created the needed for cities to implement citizen networks in order to bring the Internet access to the users, many of these networks are Wi-Fi Mesh technologies. The Viladecans Cityhall is the owner and manager of a Wi-Fi Mesh access infrastructure deployed on a fiber optic backbone. The devices installed on the street providing wireless access are Tropos 6320 equipment of the manufacturer Tropos Networks. This network was set up to improve public access to Information Society and e-Government, thus, the city is located at the forefront technology. This bachelor’s final year project aims to study: safety issues in Wi-Fi technology, wireless mesh network to detect existing deficiencies and problems that may arise in terms of performance, efficiency, safety and reliability. Also proposing, justifying and implementing actions in order to improve it and assess these enhancements to achieve the integration of new services. Thus, a correct operation of the infrastructure and resources of the network can be performed

CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch

With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive Alu retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an Alu-specific single-guide RNA, were integrated into the genome of target cells via Sleeping-Beauty-mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future