Syncytins for genome engineering in B cells – SYN-B

Project lasting 3 years and organized with the following 4 scientific worckpackages:
WP1: Optimization of CRISPR/Cas gene editing tools in B-cells.
WP2: Development of iASR strategies in primary B cells.
WP3: Functional validation of iASR in mice.
WP4: iCSR and IgH constant region engineering in hybridomas.

The interim report at M18 shows that we have reached the first milestones without difficulty, i.e. implementing the tools and methods and obtaining the first results of iASR and iCSR in B cells. Only a milestone originally planned in WP3 for experiments in mice has been adapted to take into account the rapid advances of the field.

The work underway in WP2 and WP3 aims to optimize cell culture conditions to increase the genomic editing level of primary B lymphocytes. In addition, work is underway to develop the conditions for the use of pseudotyped IDLDs with human syncytins (Syn1 or Syn2) for the contribution of HDRT in B lymphocytes.
The future prospects for WP4 are the realization of retro-switch in hybridomes with a comparison between the RNP/HDRT technique and RNP/IDLV in order to establish a robust protocol.
The work is progressing well and initial publications and communications have been carried out.

At M18
1 article submission ongoing
1 ongress abstract

B cells produce antibodies (Ab), play an essential role in the immune system and are important therapeutic targets. As hybridomas, B cells are also key for biotechnological production of recombinant monoclonal immunoglobulin (Ig) reagents. The possibility of engineering the B-cell genome for Ig production has potentially far-reaching interests in human health through vaccines, cancer, auto-immunity, infectious or genetic diseases and biomedical diagnosis applications. However, primary B-cells remain difficult to modify genetically. Gene editing technology is not commonly used in B cells to induce the production of a desired Ab instead of the cells’s own Ig. We have recently discovered a new system for efficient gene delivery to human and murine B-cells, which we propose to exploit for CRISPR/cas9 genome editing. Three laboratories will collaborate to develop tools and therapeutically-relevant applications in human and murine models. The efforts will initially be focused on editing the Ig heavy chain gene locus. One goal is to precisely redirect antibody specificity by induced antigenic-specificity replacement (iASR). Well-described Ab will be used to test iASR strategies in vitro and in vivo. Another goal is to modify IgH constant region to force a specific class switch recombination (iCSR). This strategy will be developed to generate IgM Abs for diagnostics and blood cell typing. Overall this project’s ambition is to overcome technological limitations to obtain an efficient platform for B-cell enginering and vectored antibody therapy.