An In Ovo expression system for therapeutic proteins
With the global therapeutic protein market estimated to reach 316 billion USD by 2025, it is to no surprise that there is a peaked interest in research into alternative protein bioreactors. Currently, Chinese Hamster Ovarian cells are the most widely used vector for therapeutic expression, however, chickens present a potentially higher yielding and more cost-effective vehicle compared to this mammalian system. To fully utilize this avian bioreactor, gene drive technology in combination with CRISPR Cas9 needs to be employed. This will allow for the targeted editing of the Ovalbumin and Ovomucoid loci allowing for the maximum expression of a therapeutic protein in the albumin. To accomplish this, we first constructed a gene drive, with restriction digest, that contained CRISPR Cas9, the therapeutic protein human clotting Factor 9 (FIX), homology arms, and a meiosis-specific promoter. Primordial Germ Cells (PGCs) were then isolated from the blood of HH 14-17 embryos and cultured for 4 months to be the vehicle for gene transfer into the chicken. After this, kill curves were performed and the best concentration for selection with puromycin and neomycin of PGCs, was determined. The best concentration of busulfan for depleting native PGCs in gonads while conserving viability as much as possible, was also determined through IHC staining. The gene drive constructs were then electroporated and lipofected into PGCs and integration was assessed with Homology Directed Repair and Non-Homologous end joining specific primers. Separately, PGCs edited with an mRFP-Tol2 tracer were injected into HH 13-15 embryos and repopulation was assessed on these busulfan ablated embryos. The use of 75μg of busulfan in 50μl of sesame seed oil and DMSO was determined to be most effective at ablating native PGCs and conserving viability while 1.5μg/ml and 400μg/ml was determined to be the ideal selection concentration for puromycin and neomycin, respectively. While integration into the PGC genome was unsuccessful, transfection conditions were established that successfully transferred the constructs into PGCs. Future work will need to include treatment with HDR promoting factors in cell culture or gRNA redesign to ensure successful integration of the gene drive into PGCs. To address unsuccessful repopulation, different methods of injecting, such as using a microinjector, could be attempted or the technique could be repeated with more cells. Nonetheless, this study provides important preliminary data to create a transgenic chicken bioreactor for the expression of FIX at a lower cost than current methods.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023