Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy

M Milani, A Annoni, S Bartolaccini, M Biffi, F Russo, T Di Tomaso, A Raimondi, J Lengler, MC Holmes, F Scheiflinger, A Lombardo, A Cantore, L Naldini

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Abstract

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy. © 2017 The Authors. Published under the terms of the CC BY 4.0 license
Original languageEnglish
Pages (from-to)1558-1573
Number of pages16
JournalEMBO Molecular Medicine
Volume9
Issue number11
Publication statusPublished - 2017

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