In silico Design of Plasmid to Generate Stable Cell Line Expressing Monoclonal Antibodies with CRISPR Cas9 for Precision Gene Integration

Abstract

Therapeutic mAbs are the class of innovative therapeutic molecules with the highest growth (mAbs). They have great potential for the treatment of many diseases, including cancer and chronic inflammatory conditions. In 1975 in order to develop monoclonal antibodies 2 scientists, Kohler and Milstein developed a technique in the fusion of myeloma cells with immunized mouse immune cells resulting in hybridoma cells capable of producing mAbs. However, this conventional method of producing mAb is not only costly and time demanding, but it also results in a decrease in the amount of viable antibodies because more than 99% of the cells do not survive the fusion process. The antibodies produced through hybridoma are also not a 100% human antibody gene but a mix of mouse or rodents which may possess immune reaction and reduced bioavailability. Over the course there have been several ways to improve the production of monoclonal antibodies namely using genetic vectors such as plasmid to integrate the mAb gene into the target expression system. This could reduce production cost and also reduce toxicity and prolong bioavailability. Genome editing technology has been to insert genetic material at specific spots into the human genome for practically a while, this insertion ensured that the desired gene would be stably expressed in the expression system. Here, I have designed plasmid vectors for monoclonal antibody gene insertion into HEK293 cells using CRISPR technology. First, the AAVS1 locus was recognized as the precise location where the mAb genes would be cloned. For this, a donor vector bearing certain HAs and an all-in-one Cas9-expressing vector are required. As a result, a donor vector was created by fusion cloning the mAb genes into the HA-containing, empty donor vector. Finally, gRNA spacer sequences were cloned into the Cas9 expressing plasmid to direct the Cas9 system to cut in the required particular location.