Engineering of the Caspase-3 Gene in Recombinant CHO Cells Caused More Apoptosis Resistance and enhanced Recombinant Protein Production Than the BAX Gene

Authors
Amirabbas Rahimi 1
Morteza Karimipoor 1
Reza Mahdian 1
Atefeh Alipour 2
Saadi Hosseini 3
Marzieh Mohammadi 1
Hooman Kaghazian 4
Hosein Shahsavarani 5
Mohammad Ali Shokrgozar 3
1 Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran
2 Department of Nano-Biotechnology, Pasteur Institute of Iran, Tehran, Iran
3 Laboratory of Regenerative Medicine and Biomedical Innovations, National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran
4 Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran
5 Department of Cell and Molecular Biology, Shahid Beheshti University, Tehran, Iran
10.61882/ibj.4934
Abstract
Background: BAX and caspase-3 are essential genes in the apoptotic pathway of cells, promoting the apoptotic cascade through different mechanisms. Inhibition of these genes can increase the longevity of cells in cell culture. This study aimed to compare the effects of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated knockdown of BAX and caspase-3 genes on apoptosis inhibition, cell lifespan, and erythropoietin (EPO) production in CHO cell lines.

Methods: The BAX and caspase-3 gene expression was evaluated in the recombinant Chinese hamster ovary (rCHO) cell lines producing EPO using the CRISPR-Cas9 method. Their anti-apoptotic effects and the level of EPO expression were also compared. In addition, oleuropein (OP) as an apoptosis inducer, was introduced to the manipulated cell line to assess the stability and viability of the manipulated cell lines.

Results: The rCHO cells with the manipulated BAX gene exhibited a higher cell density than those with the manipulated caspase-3 gene (152% vs. 142%). Despite the increased cell density in the cells with the BAX gene manipulation, EPO production was higher in the cells with the manipulated caspase-3 gene (1.58-fold increase in the BAX-manipulated cells compared to a 1.70-fold increase in the caspase-3-manipulated cells).

Conclusion: Our observations suggest that the downregulation of the BAX and caspase-3 genes using the CRISPR method, inhibits apoptosis and enhances the yield of recombinant EPO, even in the presence of an apoptosis inducer. Additionally, reduction of caspase-3 expression was proved to be more effective than BAX in extending the lifespan of cells and producing heterologous recombinant proteins.
Keywords
Apoptosis
Bax
Caspase-3
CRISPR-associated protein 9
Iranian Biomedical Journal
Volume 29, Issue 3 - Serial Number 118
May and June 2025
Pages 149-158