• Authors: Collaud, F., Bortolussi, G., Guianvarc'h, L., Aronson, S. J., Bordet, T., Veron, P., Charles, S., Vidal, P., Sola, M. S., Rundwasser, S., Dufour, D. G., Lacoste, F., Luc, C., Wittenberghe, L. V., Martin, S., Le Bec, C., Bosma, P. J., Muro, A. F., Ronzitti, G., Hebben, M., Mingozzi, F.
  • Year: 2019
  • Journal: Mol Ther Methods Clin Dev 12 157-174
  • Applications: in vitro / DNA / PEIpro
  • Cell type: HEK-293
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293, 293


Adherent HEK293 cells grown in roller bottles were transfected with the three plasmids containing the adenovirus helper proteins, the AAV Rep and Cap genes, and the ITR-flanked transgene expression cassette. GMP-like (ss)AAV8-hUGT1A1 vectors used in this study were produced in bioreactor and at different scales up to 200 liters by adenovirus-free transient transfection method. Suspension HEK293 cells were transfected with Polyethyleneimine (PEI) (PEIpro, Polyplus).


Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.