Studies On The Site-specific Integration Loci And Application For Stable Expression Of Exogenous Protein In CHO Cell

Chinese hamster ovary（CHO）cells are widely recognized by regulatory bodies such as the FDA and NMPA as the best cell lines for the production of biotech drugs.Currently,therapeutic biological products are being utilized in the treatment of chronic and geriatric diseases.However,due to changes in medical insurance and personal payment methods,the price of these products has become a limiting factor in market expansion.To address this challenge,it is crucial to focus on reducing production costs through technological advancements while ensuring high quality and efficiency in the production process.Consequently,optimizing CHO engineering cells using specific technologies has emerged as a key area of innovation.Considering the regulatory requirements for monitoring the production of stable-expressing cell lines and the proven cost benefits of continuous production,it is imperative to identify stable expression sites within CHO cells that can sustain the long-term expression of therapeutic proteins.This entails developing targeted integration technologies that can be practically implemented and significantly extending the production cycle of CHO engineering cells.These optimizations hold substantial value for the advancement of CHO engineering cells.The objective of this research article is to enhance the proficiency and reliability of recombinant CHO cells in expressing therapeutic proteins with stability.In the preliminary phase,we utilized lentiviral transfection combined with chromosome walking to identify potential expression sites for foreign proteins.We comprehensively evaluated the stability of expression,efficiency of integration,expression levels of foreign proteins,and adaptability of expression.Furthermore,we developed a practical technology capable of targeted integration of exogenous genes into stable integration sites.Additionally,we improved the growth and expression characteristics of CHO cells by enhancing the expression of telomerase reverse transcriptase（TERT）.The main findings of our research are as follows:（1）In accordance with the quality standards set by drug regulatory agencies for master cell banks,production cell banks,and production,as well as the previously surveyed potential sites by our research team,we developed a method to evaluate the stability of targeted integrating sites in CHO cells.To achieve this,we utilized CRISPR-Cas9 to integrate the reporter gene,EGFP,into potential sites on the CHO chromosome.Subsequently,single clone cell lines were selected through puromycin screening and sorted using flow cytometry.The suspension-cultured cells were then cultured continuously for 60 generations,with fluorescence intensity measured throughout.We successfully identified two stable expression sites in CHO cells,namely 2b2（located at position 1204505 base of NW＿003614095.1,in the first exon of Sclt I）and 3 g10（located at position 1969647 base of NW＿003613638.1,in the first intron of Cdk6）.By integrating the human serum albumin（HSA,68KD）gene into 2b2 and 3 g10 respectively,we achieved stable expression of HSA,confirming the efficacy of identifying stable expression sites in CHO cells.These findings indicate that 2b2 and 3g10 can serve as targeted integration sites for stable expression of foreign proteins in CHO cells.（2）Building upon the aforementioned 2b2 and 3g10 sites,as well as the previously discovered stable expression sites 2c6（located in the nc RNA of LOC103162981）and 1 g11（a poly structure of Cob II1）by our research team,we conducted an assessment of these four sites in terms of integration efficiency,adaptability for expressing therapeutic proteins,and expression levels.Through targeted integration using CRISPR-Cas9,we observed significant differences in integration efficiency among these sites.Notably,2b2 exhibited the highest efficacy（>40%）,followed by 2c6（>30%）,while 3g10 displayed the lowest efficacy（6%）.These results underscore the need to develop a practical targeted integration technology.Moreover,all four sites,2b2,2c6,1 g11,and 3 g10,demonstrated stable expression of HSA,interferonα2b（INFα2b,12 k Da）,coagulation factor VII（r FⅦ,55 k Da）,and vascular hemophilia factor lyase（ADAMTS13,190 k Da）,indicating their broad adaptability for expressing therapeutic proteins.Analysis of HSA expression in a laboratory setting revealed that 2c6 exhibited the highest expression level（100 mg/L）,followed by 2b2 and 1g11（15 mg/L）,while 3g10 showed the lowest expression level（5 mg/L）.These findings suggest that 2c6 could serve as the primary site for expressing therapeutic proteins,with 2b2,1g11,and 3g10 serving as auxiliary sites for stable expression in recombinant technology.（3）Due to the observed variations in integration efficiency among the 4 stable expression sites discussed above,we utilized the Bxb I attp sequence as a universal recognition sequence for site-specific integration.Through CRISPR-Cas9,technology we integrated this sequence into the stable expression sites,creating a cassette exchange landing pad CHO technology platform.Notably,the landing pad technology platform exhibited significantly higher integration efficiency compared to CRISPR-Cas9 targeted integration.For instance,the efficiency of integrating TERT（90 k Da）into the 3 g10 site surpassed 40%,far exceeding the6%achieved with CRISPR-Cas9 targeted integration.Similarly,the efficiency of integrating IFNβ-HSA（85 k Da）into the 2c6 site was 78.3%.Moreover,the integration efficiency of exogenous genes into the 2b2 and 1 g11 sites also significantly increased using the landing pad technology platform.These findings indicate that the landing pad technology platform effectively enhances the integration efficacy of genes that express therapeutic proteins.Additionally,this platform is user-friendly,promoting its practical application.（4）This investigation revealed that during the cultivation of high-density CHO cells,a significant factor contributing to differences in productivity was the accumulation of cell death caused by apoptosis and cell senescence.By utilizing the landing pad technology platform as the central technology,we achieved enhanced expression of TERT at the auxiliary 3 g10 site in CHO cells,thereby improving cell growth and the stable expression of therapeutic proteins.The CDS sequence of TERT was specifically integrated into the 3g10 site of CHO-K1 and CHO-Ie3(an anti-apoptotic CHO ^{Bax–/Bak–}cell line),and recombinant CHO cells,K1-TERT and Ie3-TERT,with enhanced TERT expression were identified through bleomycin selection combined with flow cytometry.q PCR analysis demonstrated that the transcription level of TERT in K1-TERT and Ie3-TERT cells increased by more than 7 times.The cell-doubling time shortened from 20.6 h in CHO-K1 to 17.5 h in K1-TERT,and cell cycle analysis indicated a reduction in the G0/G1 and G2/M phases in K1-TERT,while the S phase showed no significant change.During fed-batch culture in shaking flasks,we observed that the VCD of K1-TERT cells exceeded 25×10⁶ cells/m L,which was twice that of CHO-K1.Additionally,the effective culture duration extended from 13 days to 19 days,with similar trends observed for Ie3-TERT.Analysis of apoptosis and cell senescence revealed that increasing the expression of TERT effectively delayed the senescence and apoptosis of CHO cells,while enhancing their resilience to environmental changes when cultivated at high densities.Furthermore,we integrated HSA into the 2c6 site of CHO-K1 and K1-TERT,generating cell lines K1-HSA and KT-HSA expressing HSA.Comparative analysis of fed-batch culture demonstrated that the maximum cell density of KT-HSA was twice that of K1-HSA,reaching 18×10⁶ cells/m L.The culture duration increased from 11 days to 14 days,and the expression of HSA was about 783 mg/L for KT-HSA,which was 2.12 times that of K1-HSA.