P07

Generation and characterization of Xcell™ Eng-HEK293: a suspension cell line for rAAV production.

M Vona(1) I Bodenmann(1) L Nanni(1) R Daveau(1) P Kolcak Yasli(1) A Felix(1) M A Perrenoud(1) R Buchs(1) S C Hinz(1) T Schuepbach(1) I Fisch(1) E Guzman(1) N Mermod(1)

1:NewBiologix SA

The HEK293 cell line is widely used for the production of viral vectors, which serve as essential delivery vehicles in gene therapies. Several HEK293 derivatives are commercially available for R&D and GMP-manufacturing. However, we still have limited knowledge on the genetic composition of these cells and on how to improve their properties. Xcell™ Eng-HEK293 is a novel suspension HEK293 cell clone developed and characterized by NewBiologix SA. Deriving from a polyclonal parental HEK293 cell line, this clone was selected based on its high transfection efficiency, growth profile in suspension culture, suitability for efficient single-cell sorting, and ultimately for improved rAAV production efficiency following triple-plasmid transfection. Xcell™ Eng-HEK293 was fully characterized at the genomic level using PacBio long-read sequencing and Bionano's optical genome mapping, and at the transcriptomic level using high-throughput Illumina sequencing. Xcell™ Eng-HEK293 produced similar or higher rAAV titers as compared to either the parental polyclonal cell line or currently available leading HEK293 cell lines. This effect was demonstrated using various transgenes or culture volumes, and it was consistently observed over 55 generations of cultivation. In addition, Xcell™ Eng-HEK293 produced 2-fold higher ratios of DNA-containing vs empty capsids when compared to the materials produced by a leading HEK293 cell line, as well as comparable or higher degree of AAV genome integrity as assessed by high-throughput sequencing. In conclusion, we have generated and characterized Xcell™ Eng-HEK293, a novel cell line specifically optimized for the production of high quality rAAV gene therapy candidate vectors and which is available for licencing.