title: Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells.
abstract: Mesenchymal stromal cells (MSC) constitutively express low levels of human leukocyte antigen-G (HLA-G), which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immuno-modulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1) and a viral system (Lv-HLA-G1) using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK) cell-mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR)9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA ; Institute for Biotechnology and Bioengineering and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Joana S
authors.initials: JS
authors.lastname: Boura
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Melisa
authors.initials: M
authors.lastname: Vance
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Weihong
authors.initials: W
authors.lastname: Yin
authors.affiliation: Institute for Biotechnology and Bioengineering and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Madeira
authors.affiliation: Institute for Biotechnology and Bioengineering and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Lobato da Silva
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Porada
authors.affiliation: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Graça
authors.initials: G
authors.lastname: Almeida-Porada