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EMBO Mol Med. 2017 Nov;9(11):1558-1573. doi: 10.15252/emmm.201708148.

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

Author information

1
San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.
2
Vita Salute San Raffaele University, Milan, Italy.
3
IRCCS San Raffaele Scientific Institute, Milan, Italy.
4
Baxalta (former Baxter) Innovation GmbH, Vienna, Austria.
5
Sangamo Therapeutics, Inc., Richmond, CA, USA.
6
San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy luigi.naldini@hsr.it.

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.

KEYWORDS:

MHC‐I; gene therapy; hemophilia; lentiviral vectors; stable producer cell line

PMID:
28835507
PMCID:
PMC5666310
DOI:
10.15252/emmm.201708148
[Indexed for MEDLINE]
Free PMC Article

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