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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD55 Antigens / metabolism
  • Cell Line
  • Factor IX / genetics
  • Factor IX / metabolism
  • Gene Editing / methods*
  • Genetic Therapy
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism*
  • HEK293 Cells
  • Hemophilia B / therapy
  • Humans
  • Isoantigens / immunology
  • Lentivirus / genetics*
  • Membrane Cofactor Protein / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Receptors, Complement 3b / metabolism
  • Transfection


  • CD55 Antigens
  • Isoantigens
  • Membrane Cofactor Protein
  • Receptors, Complement 3b
  • Factor IX