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

Research output: Contribution to journalArticle

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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Isoantigens
Genetic Therapy
beta 2-Microglobulin
Vesicular Stomatitis
Product Packaging
Licensure
Major Histocompatibility Complex
Genes
Transfection
Glycoproteins
Genome
Viruses
T-Lymphocytes
Cell Line
Gene Editing

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Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy. / Milani, M; Annoni, A; Bartolaccini, S; Biffi, M; Russo, F; Di Tomaso, T; Raimondi, A; Lengler, J; Holmes, MC; Scheiflinger, F; Lombardo, A; Cantore, A; Naldini, L.

In: EMBO Molecular Medicine, Vol. 9, No. 11, 2017, p. 1558-1573.

Research output: Contribution to journalArticle

Milani, M ; Annoni, A ; Bartolaccini, S ; Biffi, M ; Russo, F ; Di Tomaso, T ; Raimondi, A ; Lengler, J ; Holmes, MC ; Scheiflinger, F ; Lombardo, A ; Cantore, A ; Naldini, L. / Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy. In: EMBO Molecular Medicine. 2017 ; Vol. 9, No. 11. pp. 1558-1573.
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AU - Milani, M

AU - Annoni, A

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AU - Russo, F

AU - Di Tomaso, T

AU - Raimondi, A

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N2 - 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

AB - 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

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