Study Of Site-specific Integration Of Therapeutic Protein Genes Into CHO Genome And Stable Expression

Chinese Hamster Ovary?CHO?cells are the first approved manufacturing cell line to produce biopharmaceutical products by FDA of United States,EMA of Europe Union,and cFDA of China.It was widely used as the host for many therapeutic proteins'production.CHO cells have very significant advantages in producing therapeutic proteins.The conventional cell line construction was based on random integration method or gene copy number amplification strategy,in which a high-throughput screening method was followed to select the monoclonal cell line with highest expressing level.Since this method was mainly achieved by selecting the monoclonal cells with highest expression,rather than selecting the monoclonal cells based on stable expression,the selected production cell lines may not keep expressing the target protein stably in a long term.The time to construct cells was also a great concern.Here,the apoptosis-resistant cell lines were first set up for the later research;one hot spot discovered were double-confirmed for its stability in expressing heterogenous genes;finally,different kinds of therapeutic recombinant protein expressing monoclonal cells were constructed and corresponding expressing stability were verified for different cells.?1?CHO-K1 cells were co-transfected with three expression plasmids:CD513B-Cas9,sgRNA-BAK and sgRNA-BAX.The target site of three selected BAK^-/BAX^-CHO-K1 cell lines all had insert/delete events confirmed by DNA sequencing.BAK^-/BAX^-CHO-K1 cell lines were not expressing BAK and BAX protein detected by western blot assay.The late-stage apoptosis rate of all three selected BAK^-/BAX^-CHO-K1 cells were stably around the range of 14%-19%after being treated with 5 mg/L of puromycin.However,the rate for wild type cells were increased from 13.3%into 41.3%.Both BAK^-/BAX^-CHO-K1?1d4?and CHO-K1 cells were adapted to suspension.Cell density of two cells were both around 6×10⁶cells/mL when doing batch culture.Compared to CHO-K1,BAK^-/BAX^-CHO-K1?1d4?can maintain the viability to be at least 83%,while the viability for CHO-K1 was less than 82%by day 4 and the viability was continuously decreasing later until around 72%by day 6.These results demonstrated BAK^-/BAX^-CHO-K1 cells were more resistant to apoptosis and could be stably cultured in a longer term.By optimizing the original CRISPR/Cas9 technology,we successfully avoid repetitive screening work and enhance the knock-out efficiency.The BAK^-/BAX^-CHO-K1 cell lines were obtained and could be used as the original cell line in the prospective research work.?2?By optimizing lentivirus random integration method together with genome walking method,a total of 6 hot spot within BAK/BAX double knock-out cell genome were disclosed.They were located at 691045^th base pairs of NW₀06882077.1,6874389^th base pairs of NW₀06883358.1,1235357^th base pairs of NW₀06880285.1,1969647^th base pairs of NW₀03613638.1,66645^th base pairs of NW₀06884764.1,1020651^st base pairs of NW₀06882456.1.One hot spot was chosen based on NGS sequencing results which predicted the CNV value of the spot.Corresponding cells which hold the spot located at1235357^th base pairs of NW₀06880285.1 were intensively studied.All cells?100%?could stably express the heterogenous gene over 50 passages;the mean value of fluorescence intensity was stable over 50 passages as well.When cells were adapted to suspension culture,more than 98%of cells can express Zsgreen1 fluorescence protein;moreover,around 93%-94%suspended cells can keep expressing green fluorescence protein over 50 passages and the mean value of fluorescence intensity was stable.By combining different technologies and technologies optimization,we successfully developed one novel way to discover new hot spot within CHO cell genome.We finally confirmed one spot can express heterogenous gene stably by applying functional experiments.?3?The specific integration site?NW₀0688025.1:1235284-1235429?was finally determined by using CCTOP CRISPR/Cas9 online predictor system.The donor plasmids of three different genes were designed and constructed successfully.All three donor plasmids contained complete target gene cassettes and positive screening gene cassettes?puromycin resistant gene and Dsred gene?.There were two 600 bp homology arms outside of the two cassettes mentioned above.Additionally,there was one cop-GFP gene cassette outside of the5'homology arm which was used for negative screening.The donor plasmids designed can ensure successful construction of knock-in cell lines.By transfecting a total of three expressing plasmids:DTU-Cas9,sgRNA and corresponding donor plasmids,one VEGF humanized antibody light and heavy chain gene?VEGF-hAb,150 kDa?knock-in monoclonal cell line,,three GLP-1-GLP-1-human serum albumin fusion protein gene?NGGH,75 kDa?knock-in cell line,and two human serum albumin gene?HSA,68 kDa?knock-in cell line were obtained.All hits were heterozygous.The expression level of all NGGH knock-in cell line was pretty close which was around 1.2 pg/cell/day at different cell passages in adherent cell mode.The expression level of all HSA knock-in cell line was pretty close which was around 0.8 pg/cell/day at different cell passages in adherent cell mode.The expression level of antibody knock-in cell line was around 0.007 pg/cell/day at different cell passages in adherent cell mode.Moreover,when NGGH,HSA and antibody gene knock-in cell lines were adapted to suspension culture,their expression level were around 17 mg/L,13 mg/L and 2.3?g/L over different passages at batch mode in 6 days.Finally,the transcription process was not the main reason to cause the antibody expression level being significantly less than other two recombinant protein expression level:the light chain gene transcription level of VEGF-hAB cell line was 48.9%of NGGH gene transcription level of NGGH cell line,while the heavy chain gene transcription level was 56.1%of NGGH gene.In summary,the donor plasmid designed can guarantee the target genes being precisely integrated into target site;the specific integration site located at 1235284-1235429 base pairs of NW₀0688025.1 can stably express different kinds of protein,including therapeutic antibody protein,fusion protein and human serum albumin protein.The results also reveal heterogenous genes will not be lost during cell passage and can be expressed stably.