title: Down-regulation of CD81 tetraspanin in human cells producing retroviral-based particles: tailoring vector composition.
abstract: 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.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, P-2781-901 Oeiras, Portugal.
authors.forename: A F
authors.initials: AF
authors.lastname: Rodrigues
authors.forename: M R
authors.initials: MR
authors.lastname: Guerreiro
authors.forename: V M
authors.initials: VM
authors.lastname: Santiago
authors.forename: C
authors.initials: C
authors.lastname: Dalba
authors.forename: D
authors.initials: D
authors.lastname: Klatzmann
authors.forename: P M
authors.initials: PM
authors.lastname: Alves
authors.forename: M J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: A S
authors.initials: AS
authors.lastname: Coroadinha
mesh_terms: D000818:Animals; D001688:Biological Products / Q000008:administration & dosage / Q000737:chemistry* / Q000302:isolation & purification; D002460:Cell Line; D015536:Down-Regulation; D055785:Gene Knockdown Techniques / Q000379:methods; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D012190:Retroviridae / Q000737:chemistry* / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D060225:Tetraspanin 28 / Q000032:analysis*
chemical_list: D001688:Biological Products; C554798:CD81 protein, human; D060225:Tetraspanin 28