A multi-landing pad DNA integration platform for mammalian cell engineering

Abstract

Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing.

Figure 1.

Twenty stable integration sites were discovered in CHO-K1 by lentiviral screen with an LP probe. (A) Schematic diagram of the lentiviral LP integration vector (pLV-LP) used for the lentiviral screen. Key components include 5′ and 3′LTRs necessary for lentiviral processing and integration, an attP attachment site for the BxB1 recombinase, a hEF1a constitutive promoter driving expression of an EYFP reporter and a hygromycin (Hygro) selection marker that are co-expressed using a 2A translation skip peptide, and a WPRE element (WHP Post-transcriptional Regulatory Element) for enhanced stability of the viral mRNA transcript. (B) Lentiviral infection and selection of candidate LP clones. Lentiviral particles were packaged with the LP vector and used to infect adherent CHO-K1 cells at a low MOI (0.05 and 0.005). EYFP expressing clones were picked with FACS (top ∼10 and ∼15% EYFP expressing cells), expanded and analyzed by flow cytometry. Clones with homogenous fluorescence profiles were isolated and subjected to further analysis. (C) ddPCR gene copy analysis of LP clones. Vertical axis denotes copy number for EYFP gene normalized to the housekeeping gene Cog1. Candidate clones isolated from the lentiviral screen were assessed by ddPCR analysis to select for single integration monoclonal LPs (named mLP2 to mLP19). Note that mLP1 is a double integration clone, and the F1 clone with a gene copy number of 1 was discarded due to genomic instability. (D) EYFP expression stability analysis of 18 lentiviral single integration monoclonal LPs. Cells were propagated for 2 months in the absence of antibiotic selection. Genetic stability was assessed weekly by monitoring EYFP expression with flow cytometry from which mean EYFP fluorescence intensity (top panel) and percentage of EYFP-positive cells (bottom panel) were derived. Fifteen clones exhibited >96% EYFP-positive cells after 2 months of stability assay, while three clones exhibited moderate stability (∼ 75–90% positive cells; see Supplementary Figure S3A-B and Table S1 for details). (E) Antibody expression stability analysis by pools of mLP clones integrated with the 1x-hEF1a-mAb circuit. The mAb circuit was integrated with a BxB1 recombinase, and pools of LP-mAb integrants were selected with puromycin. mAb titers were assayed weekly in conditioned media from polyclonal cell pools propagated without selection. Each data point represents one week of cell propagation and mean titer measurement with biological duplicates or triplicates (n = 2 or 3). A doubling time of 18 h was used to calculate the generation number of adherent CHO-K1 derived clones. The same symbols are used in panels D and E (see Supplementary Figure S4B and Table S1 for details).

Figure 2.

LP cell lines reconstructed with CRISPR/Cas9 maintain genetic stability and stable payload expression. (A) Schematic diagram of the LP donor vector used for CRISPR/Cas9 targeted insertion into the newly discovered stable integration sites of WT CHO-K1 cells. The LP cassette is similar to the lentiviral LP integration vector (pLV-LP, Figure 1A), but it includes locus-specific left and right homology arms (LHA and RHA) instead of lentiviral LTRs, and a polyA signal sequence instead of a WPRE. Single integration LP clones (sLPs) were constructed by targeted insertion of the LP probe into the genomic loci identified in the corresponding lentiviral mLP clones. (B) ddPCR gene copy analysis of selected LP clones constructed with CRISPR/Cas9. sLP1-1 and sLP1-2 are single integration lines bearing an LP cassette in the loci derived from the double integration clone mLP1. sLP2, sLP3, sLP6 and sLP8 are the reconstructed homologs of the lentiviral mLP clones. sLP20 and biLP20 are mono-allelic and bi-allelic integrants in the newly identified Rosa26 locus. Vertical axis denotes copy number for EYFP gene normalized to the housekeeping gene Cog1. (C) Southern blot analysis of sLP20 and biLP20 clones. gDNA was cleaved with BamHI (5′) or HindIII (3′) restriction enzymes and hybridized with an anti-hygromycin radiolabeled probe. Two sLP20 and two biLP20 clones were analyzed (B2, B12 and B7, B11, respectively). (D) EYFP expression stability analysis. Cells were propagated for 2 months in the absence of antibiotic selection. Genetic stability was assessed weekly by monitoring EYFP fluorescence intensity (top panel) and the percentage of EYFP-positive cells (bottom panel) by flow cytometry. All clones exhibited >99% EYFP-positive cells after 2 months of stability assay. (E) Antibody expression stability analysis. Cell pools of LP-mAb integrants stably expressing the 1x-hEF1a-mAb circuit were propagated in the absence of selection, and mAb levels were measured weekly in conditioned media. Each data point represents 1 week of cell propagation and mean mAb measurements with biological duplicates or triplicates (n = 2 or 3). A doubling time of 18 hr was used to calculate the generation number of adherent CHO-K1 derived clones. The same symbols are used in panels D and E.

Figure 3.

The multi-LP platform enables parallel construction of various payload expressing clones by one-shot payload integration. (A) Schematic representation of mAb payload integration into the dLP1 cell line. The dLP1 clone harbors LP2 and LP15 sites, each bearing the BxB1 attP site. BxB1-mediated recombination occurs between the LP’s attP site and the payload’s attB site, yielding a mixture of single integrants (LP2-mAb and LP15-mAb) and the double integrant (dLP1-mAb). (B) Flow cytometry diagrams and selection schemes of different pools of LP-mAb integrants following mAb payload integration into the dLP1 clone. Site-specific integration of a mAb payload into an LP site triggers expression of the payload’s promoterless selection marker (puromycin), while expression of the LP cassette stops. Appropriate antibiotic selection schemes and sorting with FACS enable isolating three types of LP-mAb integrant pools where the mAb payload is integrated in only the LP2 site (LP2-mAb), only the LP15 site (LP15-mAb) or both (dLP1-mAb). Small residual populations of unintegrated LPs (e.g. an EYFP+ population in the LP2-mAb pool and an EBFP+ population in the LP15-mAb pool) can be removed by FACS sorting.

Figure 4.

Stable and increased mAb expression with the use of multi-copy mAb circuits integrated in the multi-LP platform. (A) Schematic diagrams of LP configurations in single (sLP2), double (dLP1) and triple (tLP1) LP cell lines used for the multi-mAb payload integration. (B) Weekly mAb titers of clonal sLP2-mAb and dLP1-mAb integrants carrying multi-copy mAb constructs in different LP sites. mAb constructs were integrated into dLP1, and different pools of LP-mAb integrants carrying mAb payloads in single (LP2 or LP15) or double (dLP1) loci were selected and sorted clonally. For example, dLP2-hEF1a-mAb with 1 and 2 mAb cassettes were constructed by selecting and sorting clonal integrants of 1x-hEF1a-mAb payload in single (LP2 or LP15) or double (LP2/LP15) loci, respectively. Clonal mAb integrants were propagated in the absence of selection, and mAb levels were measured weekly in conditioned media. Each bar represents 1 week of cell propagation and mAb measurements (mean ± SD, n = 2 or 3). (C) Antibody titers of clonal tLP1-mAb integrants stably expressing multi-copy mAb payloads integrated into single (LP2), double (biLP20) or triple (LP2/biLP20) loci in the tLP1 clone. mAb constructs were integrated into tLP1, and different pools of LP-mAb integrants carrying the mAb payload in single (LP2), double (biLP20) or triple (biLP20/LP2) loci were selected and sorted clonally. For example, tLP1-hEF1a-mAb with 1, 2 and 3 mAb cassettes were constructed by selecting and sorting clonal integrants of 1x-hEF1a-mAb payload in single (LP2), double (biLP20) or triple (biLP20/LP2) loci, respectively. Clonal integrants were propagated in the absence of selection, and mAb levels were measured weekly in conditioned media. Each bar represents 1 week of cell propagation and mAb measurements (mean ± SD, n = 2 or 3). The table under the graph indicates the LP loci integrated with the payload, the integrated mAb payload and the total number of the integrated mAb cassettes. mAb gene copy number of all clonal integrants was verified by ddPCR.

Figure 5.

Linear increase in mAb titers with the use of multi-copy mAb circuits integrated in the multi-LP platform. Antibody titers of sLP2-mAb and dLP1-mAb (A) and tLP1-mAb (B) integrants of hEF1a-mAb and CMV-mAb circuits are shown against the total number of the integrated mAb cassettes. Total number of mAb cassettes is the number of integrated LP sites multiplied by the number of mAb copies in the mAb payload vector. mAb titers were calculated from the mAb stability assay (Figure 4) as the average titer values over 5 weeks of the stability assay (mean ± SD).