title: Generation of a Host Cell line containing a MAR-rich landing pad for site specific integration and expression of transgenes
Creators: name: Oliviero, Claudia
Publisher: name: UNSPECIFIED
Year: 2023

description: In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. 
The aim of this work was to generate a host cell line containing several copies of a Matrix Attachment Region (MAR) rich landing pad for site-specific integration of Gene Of Interests (GOIs). The developed system is based on the integration of dual landing pads containing the same MAR, two different orthogonal recombination sites for the serine integrase BxB1 (AttB wild type, and AttB with GA mutation) and two different reporter genes (EGFP and DsRed). 
The first part of the work focused on the generation of landing pad host cell lines. Three different typologies of dual landing pads were developed and integrated into the CHO-S genome: ones containing the chicken lysozyme 5' MAR, ones containing the human 1-68 MAR and a control ones without the MAR sequence. Clones integrating both landing pads can be selected by antibiotic selection and by following the dual reporter gene expression. Landing pad clones were selected and characterized to evaluate the effect of MAR sequence on the number of copies of LPs integrated into the genome, the expression of reporter genes, and the stability of the clones. 
The second part of the work focused on the site-specific integration of GOIs into the selected host cell lines, using BxB1 intregase. The sequence of a monospecific human antibody (msAb-Fer) was used for this proof of concept. To further validate the system and demonstrate its versatility, a bispecific antibody (bsAb-Fer) was also expressed using the landing pad system. Clones were selected and analyzed in order to verify the successful integration of donor vectors and their correct expression. These experiments highlighted the possibility of using the developed landing pads system as a "chassis" for the expression of different genes. In addition, various insights were obtained regarding points to be optimized to achieve a more robust expression platform.
The last part of this work focused on improving culture conditions in fed batch and a culture scale up. Different commercial basal media were tested in combination with different feeding strategies to evaluate the impact in cell growth curves and antibody titer. Improvements in culture conditions allowed to increase culture duration and antibody titer significantly compared to the initial tested conditions. The best conditions found were used during the scale-up of the system, which was proven up to 5L in a shaking bioreactor.
In summary, this work provides methods for the implementation and development of a platform for the expression of several genes of interest at the same time. The dual landing pad system, associated with different reporter genes and a MAR sequence allows the efficient selection of stable host cell lines. The landing pad design, coupled with the promoter trap strategy, allows efficient and rapid selection of producig clones once recombination and integration of GOIs has occurred. This system has been proven for expression of both monospecific and bispecific antibodies but has the potential for rapid and efficient expression of other molecules as well. Therefore upon optimization, this system could be used in the future for generating stable cell lines for production but also for rapid expression of different biologics without having to go through transient transfection steps or long selection processes.