Comparative proteome analyses of HEK-293-derived cells in adherent or suspension culture and during recombinant adeno-associated virus production with plasmids or Minicircles

For gene replacement therapies adeno-associated viruses are preferred carriers. Luxturna, Zolgensma, Upstaza, and Beqvez are produced in mammalian human embryonic kidney cells (HEK). The HEK-293 cell line is the origin of several derivatives like Freestlyle 293-F. To my knowledge, there are no proteomic studies that compare the parental HEK-293 cell line with the Freestlyle 293-F cell line. Therefore, in this work a comparative proteomic approach of the adherent HEK-293 and the suspension Freestyle 293-F cells (HEK-F) cell line was investigated to understand the differences of these cell lines on the protein level. Additionally, suspension HEK-F cells were re-adapted to adherent culture for a more extended interpretation of relevant cellular events. This comprehensive study revealed basic differences between those two cell lines. The analysis of differential expressed proteins and identification of regulated biological processes revealed three pathways: Clathrin-mediated transport, glycolysis and gluconeogenesis and protein processing in the endoplasmic reticulum. Besides the differences between producer cell lines, the investigation of the impact of rAAV production on the producer cells due to transfection and rAAV production is of great interest to detect both bottlenecks and players crucial for the rAAV production process. In this work the nuclear proteome of not transfected HEK-293 cells was compared with rAAV producing and mock transfected HEK-293 cells with a focus on the nuclear protein fraction. In this experiment proteins that were regulated due to transfection were identified and proteins known to play a role in the response to viruses, and proteins known to be influenced by adenoviral proteins were investigated. The analysis of relative transcript levels revealed decreased transcript levels for some of the targets investigated in treated cells. In combination with the proteomic data, this provides the hypothesis of nuclear accumulation of some of these proteins in the nucleus. Regulated proteins identified in the proteomic experiment may influence rAAV production. Four of these proteins became targets for a knock-out experiment. These genes were targeted in a CRISPR/Cas9-mediated knock-out experiment in HEK-293 cells and rAAV production in these generated cell pools was investigated. In these experiments an increased rAAV production yield was detected by qPCR in whole cell lysates for some pools. The plasmids that are used for rAAV production contain bacterial sequences beside the specific DNA sequences that are necessary for rAAV production. This work investigated if the reduction of the transfected DNA amount by the Minicircle (MC) system by PlasmidFactory reduces the cellular immune response and leads to increased rAAV production yields. For this purpose three self-constructed MCs for rAAV production were designed in cooperation with PlasmidFactory. Different combinations of MC constructs and original plasmids were tested in rAAV production. The influence of MC constructs on the host cell proteome was analyzed and revealed no significant impact. The production of rAAV with the constructed MCs was successful and by increasing the transfected DNA amount the rAAV production yield was increased. Capsid assembly is one important step in the production of rAAVs and this process is still not completely resolved. In general, assembled capsids in rAAV producing cells are visualized with the A20-antibodies, with no option to analyze rAAV production in one cell over time. In this work, a new method based on the incorporation of fluorescent proteins into the capsid proteins was developed enabling it to produce a FRET signal. This signal can be detected when these proteins are incorporated in the capsid next to each other at a suitable distance. This work presents a suitable FRET approach for the detection of rAAV assembly in fixed cells, with high potential for the detection of rAAV capsid assembly in living cells. The technique developed may contribute to understanding the process of capsid assembly in future experiments. Overall, this work provides information that contributes to a deeper understanding of the rAAV production process. The proteins identified in this work may also have an impact on rAAV production and their manipulation may lead to increased rAAV production yields.