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

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.

Clinical use of lentiviral vectors

“…Alloimmune reactivity towards HLA class I proteins carried within the vector envelope have been described and can limit vector survival. Gene editing of packaging cells to generate lines that lack HLA class I can enhance the stability of lentiviral vectors in serum [ 81 ]. Surface engineering in addition to vector genome engineering will likely be critical for successful application of these vectors in vivo.…”

Genetic engineering of hematopoiesis: current stage of clinical translation and future perspectives

“…Finally, even within the setting of autologous HSC GT we should be aware of the potential emergence of immune response to the therapeutic gene product, which may act as foreign antigen in the recipient, and to the transduced cells, which may transiently express after transduction vector-derived components, such as allogeneic HLA molecules incorporated into the virions when budding from the plasma membrane of the human LV producer cell (Milani et al, 2017). Although the occurrence of such immune complications has not been reported yet in clinical trials, we should closely watch for them as the application of HSC GT broadens to new diseases and exploits milder conditioning regimens where an immune response may be more easily encountered.…”

Innovative Therapies for Hemoglobin Disorders

“…LVV preparations contain proteins captured from the packaging cell lines that can induce alloimmune reactivity and limit vector survival. Genome editing in packaging cell lines for the production of alloantigen-free LVVs has been shown to enhance LVV stability and may help to increase efficacy after in vivo injection [223].…”