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Definitions

• This invention relates generally to recombinant Chinese Hamster Ovary (CHO) cells for producing recombinant proteins.
• CLD CHO cell line development
• the inventors have surprisingly discovered a high-throughput screen that measures transgene transcription from precisely defined integration sites would provide a direct scoring metric, which could be used to prioritize sites for retargeting, in contrast to previous hotspot prediction methods relying on endogenous transcriptomic or epigenomic attributes.
• a recombinant Chinese Hamster Ovary (CHO) cell for producing a recombinant protein comprises a heterologous gene encoding the recombinant protein, wherein the heterologous gene is integrated into the genome of the recombinant CHO cell at one or more integration sites, wherein each of the one or more integration sites is within 200 kb of a target sequence in the genome, and wherein the target sequence is at least 80% identical to a gene sequence selected from the group consisting of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at the one or more integration sites in the one or more target sequences.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at the one or more integration sites within 200 kb of one of the one or more target sequences.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at the one or more integration sites in one of the one or more target sequences.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one of the one or more integration sites in one of the one or more target sequences.
• the recombinant CHO cell may be incubated in a culture medium and express the recombinant protein.
• the recombinant protein may be a monoclonal antibody.
• a method for producing a recombinant protein may comprise incubating the recombinant CHO cell in a culture medium, and expressing the recombinant protein by the recombinant CHO cell.
• the production method may further comprise producing the recombinant protein at 0.1-10 g/L based on the total volume of the culture medium or 1-10 pg per said recombinant CHO cell per day.
• the production method may further comprise purifying the recombinant protein from the culture medium.
• the recombinant protein may be a monoclonal antibody.
• a method for preparing a recombinant CHO cell comprises obtaining a host CHO cell comprising a genome having one or more target sequences, wherein each of the one or more target sequences is at least 80% identical to a gene sequence selected from the group consisting of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc; and integrating a heterologous gene encoding a recombinant protein into the genome at one or more integration sites within 200 kb of the one or more target sequences, whereby a recombinant CHO cell is prepared.
• the preparation method may further comprise integrating the heterologous gene into the genome at the one or more integration sites in the one or more target sequences.
• the preparation method may further comprise integrating the heterologous gene into the genome at the one or more integration sites within 200 kb of one of the one or more target sequences.
• the preparation method may further comprise integrating the heterologous gene into the genome at the one or more integration sites in one of the one or more target sequences.
• the preparation method may further comprise integrating the heterologous gene into the genome at one of the one or more integration sites in one of the one or more target sequences.
• the genome may comprise a landing pad.
• the preparation method may further comprise incubating the recombinant CHO cell in a culture medium, and expressing the recombinant protein.
• the preparation method may further comprise purifying the recombinant protein from the culture medium.
• the recombinant protein may be a monoclonal antibody.
• FIGS. 1A-F show a Thousands of Reporters Integrated in Parallel (TRIP) system summary.
• FIG. 2 shows mRNA expression of the CD4-FKBP transgene in the landing pad in sorted polyclonal pools and clonally-derived master host cell lines with verified on- target, single-copy integrations at eight Piggybac TRIP integration sites and the Ferll4 integration site.
• Absolute quantification of CD4-FKBP and RAB10 mRNA was performed by multiplexed ddPCR analysis.
• CD4-FKBP mRNA expression was driven by a CMV promoter in the landing pad and therefore considered to be representative of transgene expression at each site in the master host cell lines.
• RAB10 was chosen as a housekeeping gene based on its stable expression across several CHO host cell lines in an internal meta-analysis of public CHO RNAseq data (data not shown). Error bars represent Poisson 95% confidence intervals from three technical replicates.
• FIG. 3A-B show a summary of RMCE configuration optimization experiments with GFP-Puro r .
• Ins Insulator
• CpG Azinl CpG island
• ITR Piggybac inverted terminal repeat
• SV40L SV40 late polyadenylation signal.
• FIGS. 4A-D show a summary of mAb RMCE configuration optimization experiments with NISTmAb and Trastuzumab.
• the present invention relates to a recombinant Chinese Hamster Ovary (CHO) cell for producing a recombinant protein, in which a heterologous gene encoding a recombinant protein is integrated into the genome of the recombinant CHO cell at specific integration sites, also referred to as "hot spots," for improving production of the recombinant protein by the CHO cell.
• CHO Chinese Hamster Ovary
• the invention is based on the discovery by the inventors of a compendium of thousands of candidate sites identified with a high- throughput screen method that would likely capture the entire range of positiondependent expression that is possible for a single-copy transgene driven by a particular promoter, and that one high-throughput screening experiment could therefore effectively reduce the hunt for the highest-expressing stable hotspots to the practice of retargeting a handful of highly ranked sites and evaluating their ability to express a product of interest in the CHO sublineage and culture conditions used in any particular lab.
• the inventors report the adaptation of Thousands of Reporters Integrated in Parallel (TRIP), a pooled high-throughput screening method capable of simultaneous measurement of mRNA expression, DNA copy number, and mapping information of barcoded transgenes integrated at thousands of genomic loci, for the application of identifying stable hotspots in the CHO genome.
• TRIP Reporters Integrated in Parallel
• Genome-wide position effects on CMV promoter-driven transgene expression strength and stability were evaluated using barcoded reporters compatible with Piggybac transposase-mediated and lentiviral integration systems, which both integrate their cargo with well-known preferences for transcriptionally permissive chromatin regions and have been used previously for either CHO CLD or hotspot screening.
• protein and “polypeptide” are used herein interchangeably, and refer to a polymer of amino acid residues with no limitation with respect to the minimum length of the polymer.
• the protein or polypeptide has at least 20 amino acids.
• the definition includes both full-length proteins and fragments thereof, as well as modifications thereof (e.g., glycosylation, phosphorylation, deletions, additions and substitutions).
• polynucleotide refers to a polymer of nucleotide residues with no limitation with respect to the minimum length of the polymer.
• the polynucleotide has at least 60 nucleotides.
• the polynucleotide may be a DNA, cDNA or RNA.
• a polynucleotide is native to a cell where the polynucleotide is naturally occurring in the cell.
• a polynucleotide is heterologous to a cell where the polynucleotide is not naturally occurring in the cell.
• variant of a protein or polynucleotide used herein refers to a polypeptide having an amino acid or a polynucleotide having a nucleic acid sequence that is the same as the amino acid or nucleic acid sequence of a target protein or polynucleotide except having one or more amino acids or nucleic acids modified, for example, deleted, inserted, or replaced, respectively.
• a variant of a protein or polynucleotide may have an amino acid or nucleic acid sequence at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to the amino acid sequence or nucleic acid of the protein or polynucleotide.
• host CHO cell refers to a CHO cell before a heterologous gene is integrated into its genome at one or more integration sites within a predetermined distance from a target sequence in the genome, which target sequence is a variant to a gene sequence selected from the group consisting of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc.
• the present invention provides a recombinant Chinese Hamster Ovary (CHO) cell for producing a recombinant protein.
• the CHO cell comprises a heterologous gene encoding the recombinant protein.
• the heterologous gene is integrated into the genome of the recombinant CHO cell at one or more integration sites. Each integration site is within a predetermined distance from a target sequence in the genome.
• the target sequence is a variant to a gene sequence selected from the group consisting of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc.
• Table 1 shows the mapping information of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc.
• the heterologous gene encoding the recombinant protein is a DNA not naturally occurring in a CHO cell.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one or more integration sites by any method.
• Each integration site may be within 200 kb, 100 kb, 50 kb, 25 kb, 10 kb, 5 kb, 1 kb, 500 bases, 100 bases, 50 bases, 10 bases, 5 bases or 1 base of the target sequence in the genome.
• the target sequence may be at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to a gene sequence, which may be Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one or more integration sites in one or more target sequences in the genome, where each target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one or more integration sites, where each integration site is within 200 kb, 100 kb, 50 kb, 25 kb, 10 kb, 5 kb, 1 kb, 500 bases, 100 bases, 50 bases, 10 bases, 5 bases or 1 base of one target sequence in the genome, and where the target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one or more integration sites in one target sequence, where the target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one integration site in one target sequence, where the target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the recombinant protein may be any protein. Although the recombinant protein may be a protein naturally expressed in a natural CHO cell, the recombinant CHO cell may overexpress the protein as compared with the natural CHO cell.
• the recombinant protein may be heterologous to a natural CHO cell.
• the protein may be an antibody, for example, a monoclonal protein.
• the recombinant CHO cell may be incubated in a culture medium and expresses the recombinant protein.
• a method for producing the recombinant protein comprises incubating the recombinant CHO cell of the present invention in a culture medium, and expressing the recombinant protein by the recombinant CHO cell.
• the production method may further comprise producing the recombinant protein at 0.1-10, 0.5-10, 1-10, 5-10, 0.1-9, 0.5-9, 1-9, 5-9, 0.1-8, 0.5-8, 1-8, 5-8, 0.1-7, 0.5-7, 1-7, 5-8, 0.1-7, 0.5-7, 1-7, 5-7, 0.1-6, 0.5-6, 1-6, 5-6, 0.1-5, 0.5-5, 1-5, 0.1-1 or 0.1-0.5 g/L, based on the total volume of the culture medium, or 1-10, 1-9, 1-8, 1- 7, 1-6, 1-5, 1-4, 1-3 or 1-2 pg per said recombinant CHO cell per day.
• the production method may further comprise purifying the recombinant protein from the culture medium.
• the purification of the recombinant protein may be achieved by any method.
• the recombinant protein may be any protein.
• the protein may be an antibody, for example, a monoclonal protein.
• the recombinant CHO cell may be incubated in a culture medium and expresses the recombinant protein.
• a method for preparing a recombinant CHO cell comprises obtaining a host CHO cell.
• the host CHO cell comprises a genome having one or more target sequences.
• Each target sequence is a variant of a gene sequence selected from the group consisting of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc.
• the preparation further comprises integrating a heterologous gene encoding a recombinant protein into the genome at one or more integration sites. Each integration site is within a predetermined distance from a target sequence in the genome.
• the target sequence is a variant to a gene sequence selected from the group consisting of Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 and Ddc.
• Azinl Sartl
• Fosl2 Pde4a
• Doplb Doplb
• Vgll4 Vgll4, Khdc4
• Ddc Ddc
• the heterologous gene encoding the recombinant protein is a DNA not naturally occurring in a CHO cell.
• the heterologous gene may be integrated into the genome of the recombinant CHO cell at one or more integration sites by any method.
• each integration site may be within 200 kb, 100 kb, 50 kb, 25 kb, 10 kb, 5 kb, 1 kb, 500 bases, 100 bases, 50 bases, 10 bases, 5 bases or 1 base of the target sequence in the genome.
• the target sequence may be at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to a gene sequence, which may be Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the preparation method comprises integrating the heterologous gene into the genome of the recombinant CHO cell at one or more integration sites in one or more target sequences in the genome, where each target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to.
• the preparation method comprises integrating the heterologous gene into the genome of the recombinant CHO cell at one or more integration sites, where each integration site is within 200 kb, 100 kb, 50 kb, 25 kb, 10 kb, 5 kb, 1 kb, 500 bases, 100 bases, 50 bases, 10 bases, 5 bases or 1 base of one target sequence in the genome, and where the target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the preparation method comprises integrating the heterologous gene into the genome of the recombinant CHO cell at one or more integration sites in one target sequence, where the target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the preparation method comprises integrating the heterologous gene into the genome of the recombinant CHO cell at one integration site in one target sequence, where the target sequence is at least about 70%, 80%, 90%, 95%, or 99%, preferably at least about 80%, more preferably at least about 90%, identical to Azinl, Sartl, Fosl2, Pde4a, Doplb, Vgll4, Khdc4 or Ddc.
• the genome of the host CHO cell may comprise a landing pad.
• the preparation method may further comprise swapping a partial or full sequence of the landing pad with the heterologous gene in the integration step such that the genome of the prepared recombinant CHO cell comprises the integrated heterologous gene and excludes the partial or full sequence of the landing pad.
• the preparation method may further comprise incubating the recombinant CHO cell in a culture medium, and expressing the recombinant protein.
• the recombinant protein may be produced by the recombinant CHO cell at 0.1-10, 0.5-10, 1-10, 5-10, 0.1-9, 0.5-9, 1-9, 5-9, 0.1-8, 0.5-8, 1-8, 5-8, 0.1-7, 0.5-7, 1-7, 5-8, 0.1-7, 0.5-7, 1-7, 5-7, 0.1-6, 0.5-6, 1-6, 5-6, 0.1-5, 0.5-5, 1-5, 0.1-1 or 0.1-0.5 g/L, based on the total volume of the culture medium, or 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 pg per said recombinant CHO cell per day.
• the preparation method may further comprise purifying the recombinant protein from the culture medium.
• the purification of the recombinant protein may be achieved by any method.
• the recombinant protein may be any protein.
• the protein may be an antibody, for example, a monoclonal protein.
• Example 1 A compendium of stable hotspots in the CHO genome
• Piggybac (pPB-GFP) and lentiviral (pLV-GFP) base, non-barcoded cargo plasmids were assembled using standard molecular cloning techniques. Additionally, the tamoxifen-inducible Piggybac transposase described by Akhtar et al. (Nat. Protoc. 9, 1255-1281 (2014)) with minor modifications to replace rarely used codons in CHO was inserted into an in-house plasmid backbone to generate pPBase-IRES-mCherry.
• Barcoded plasmid pools (pPB-GFP-bc and pLV-GFP-bc) were generated by restriction digestion/ligation cloning followed by high-efficiency transformations with Endura Electrocompetent E. coli (Lucigen) using a GenePulser Xcell (BioRad).
• Bxbl-compatible RMCE cargo plasmids were derived from a custom Golden Gate destination vector. Entry vectors were cloned by insertion of interchangeable plasmid parts (i.e.
• CMV promoters CMV promoters, transgene coding sequences, polyA signals, other regulatory elements flanked by PaqCI recognition sequences into pCRTM-Blunt II-TOPOTM using a Zero BluntTM TOPOTM PCR Cloning Kit (Thermo Fisher). Assembly reactions with the destination and entry vectors were performed following the standard thermocycling protocol described by New England Biolabs for Golden Gate assembly.
• the pLV-GFP-bc plasmid library was packaged into lentiviruses by transient transfection of adherent HEK293LTV cells (Cell BioLabs) with LipofectamineTM 3000 (Thermo Fisher) in a vented T75 flask (Corning) following a protocol from Thermo Fisher for lentiviral production modified to replace ViraPower packaging plasmid mix and lentiviral vector with an equal total mass of pMDLg/pRRE (Addgene plasmid #12251), pRSV-Rev (Addgene plasmid #12253), pMD2.G (Addgene plasmid #12259). Harvested, filtered supernatant was aliquoted into cryovials without concentrating and stored at -80°C. Infectious titer was quantified as described in Supplementary Methods.
• RNA and genomic DNA were extracted from cells growing in mid-exponential phase on Day 2 of the first passage exceeding each PDL milestone.
• NGS libraries were prepared from RNA and genomic DNA extracted at each timepoint as described in Supplementary Methods to quantify mRNA expression and DNA copy number and map the integration site of each barcode.
• Raw TRIP sequencing data was processed using a custom analysis pipeline. Forward reads from the expression and normalization libraries and reverse reads from the mapping libraries were trimmed with Cutadapt and counted for each barcode using custom R scripts. Forward reads in the mapping library were trimmed to exclude the end of the 3' Piggybac or lentiviral terminal repeat used for primer binding and to eliminate chimeric sequences arising from ligation of multiple short DpnII/N lain fragments within the sequenced region. Trimmed reverse mapping reads shorter than 8bp were discarded.
• mapping reads were mapped to the Chinese hamster PICRH genome assembly (CriGri-PICRH-1.0, GCF_003668045.3) with the Burrows-Wheeler Aligner and low-quality alignments (MAPQ ⁇ 10) were filtered out of the dataset.
• Forward mapping reads with each TRIP barcode were re-paired with their reverse reads with associated mapping information (chromosome coordinate and strand) using a custom R script. Mapping tables for each timepoint were then merged into one summary table, keeping only the mapping with the highest read depth for each barcode.
• Master host cell lines with single-copy integrations at each target site were generated by co-transfection of CHO-K1 host cells with a plasmid expressing an HDR donor-targeting sgRNA and hCas9oioA and a landing pad plasmid modified with site-specific homology arms and a genomic locus-targeting sgRNA expression cassette in its backbone.
• Transfection-grade landing pad plasmids were prepared using a Plasmid Midiprep kit (Zymo Research) following the centrifugation protocol.
• CD4+ cells in the initial transfected pool were enriched on Day 8 post-transfection by one round of MACS with a DynabeadsTM CD4 Positive Isolation kit (Thermo Fisher).
• Sorted CD4+ cells were recovered in a static 6-well plate and then scaled up to a lOmL working volume in spin tubes over 6 days. Single-cell cloning was then performed by plating cells from the enriched pools in ClonaCell ACF semi-solid media (Stem Cell Technologies) in 96-well plates with a target of one cell per well. After twelve to fourteen days, clonally-derived colonies were picked manually, deposited into a 96-deep well plate, and shaken for four days.
• Genomic DNA samples were prepared for ddPCR analysis by a preliminary digestion step with Hindlll-HF (New England Biolabs) and analyzed on a QXOne (BioRad) with ddPCR Supermix for Probes (no dUTP) following manufacturer instructions.
• Cell lines with the desired genotype were progressively scaled up and allowed to stabilize in spin tubes over two to three 3-day routine passages before banking and extracting RIMA.
• mRNA expression analysis with ddPCR was performed using One-Step RT-ddPCR Master Mix following manufacturer instructions.
• RMCE stable pools were generated by co-transfection of master host CHO-K1 cell lines with pCAG-NLS-HA-Bxbl, a gift from Pawel Pelczar (Addgene plasmid #51271), and a cargo plasmid. Transfected cells were passaged every three to four days under selection with 500pg/mL Geneticin (Gibco) for sixteen days. RNA extractions from selected RMCE pools were performed using an RNA 96 Extraction kit (Zymo Research) with on-column DNase treatment. Top-performing stable pools for mAb expression were subjected to negative selection with IpM 5-fluorocytosine with the continued presence of Geneticin and passaged for another seven days at which point the pools had fully recovered. The selected pools were then passaged for an additional six days in fresh media without selection agents and scaled up to 125mL shake flasks for fed-batch analysis.
• the 4-day batch productivity screen was performed using Geneticin-selected mAb-expressing stable pools in 24-deep well plates. Batches were initiated at a seeding concentration of 0.5 x 10 6 /mL using cells split off from selecting pools after recovery from Geneticin selection and before addition of 5-fluorocytosine. Batches were performed in the absence of any selection agent. On Day 4, viable cell concentration was measured and cell culture supernatant was harvested by centrifugation for secreted mAb concentration quantification. Fed-batches were performed in 125mL shake flasks with daily measurements of viable cell concentration and viability. Glucose and lactate concentrations in the cell culture supernatant were also measured daily using a YSI metabolite analyzer.
• the lower initial genome coverage in the lentivirus dataset is likely attributable to bottlenecking during lentivirus packaging, an inaccurate lentivirus infectious titer measurement, or variability in the transduction procedure.
• the absolute genome coverage of each pool would be challenging to estimate, as the final barcode count is the result of several filtering steps including integration site mappability and minimum expression cutoffs.
• Piggybac and lentiviral integration sites are expected to be subjected to similar biases during this filtering process.
• the final barcode count should therefore scale similarly with original genome coverage across methods.
• Age-dependent library quality deterioration was the result of competitive outgrowth, with fast-growing subpopulations in the cell pool outgrowing other subpopulations leading to low expression and normalization read counts for a significant fraction of barcodes in the PDL72 samples. This shift in barcode representation over time within each pool led to sequencing bias across timepoints, which was filtered out during integration site stability analysis by cross-sample normalization.
• TRIP barcodes exhibited stable expression over time. Over 90% of Piggybac and lentivirus barcodes had an absolute log-fold change less than one between PDLO and PDL36s. Additionally, only 627 out of 6,322 Piggybac barcodes and 5 out of 1,353 lentivirus barcodes were significantly differentially expressed (FDR ⁇ 0.1) between any of the three culture ages. Differential expression analysis of PDLO and PDL36 samples processed independently from the lower quality PDL72 data identified 1,031 out of 9,663 Piggybac barcodes and 27 out of 1,645 lentivirus barcodes with differential expression (FDR ⁇ 0.25).
• Hotspot features defined based on DNA sequence motifs alone could provide a rough map of desirable integration sites for any cell type if it has an associated high- quality genome assembly.
• the highest-expressing Piggybac barcodes exclusively integrated within the boundaries of genomic CpG islands and a strong distancedependent relationship was also observed in which the average expression of Piggybac barcodes decreased with increasing distance between the barcode integration site and the nearest CpG island (FIG. ID). This relationship was not observed for the lentiviral integration sites, which exhibited consistent average expression across all distances, aside from the two barcodes that integrated within CpG islands.
• Piggybac- and lentivirus-mediated integration methods had distinct preferences for different histone modification combinations, with Piggybac barcodes primarily integrating in regions marked by H3K4mel, H3K4me3, and H3K27ac and lentivirus barcodes overlapping with H3K36me3 and H3K4mel (Table 4). Elevated Piggybac barcode expression was also associated with above-average H3K27ac and H3K4me3 ChIP enrichment signals (FIG. IF).
• Piggybac and lentivirus barcodes also differed in their bias toward hypomethylated genomic regions (Ikb tiles with less than 10% CpG dinucleotide methylation), with integration frequencies of 17.6% (1,701/9,663) and 1.1% (18/1,645), respectively.
• TRIP integration sites retargeted for landing pad integrations exhibited clonal variability and locus-specific interactions with exogenous regulatory elements
• TRIP integration sites with median to maximum TRIP barcode mRNA expression and the Ferll4 integration site used by Pfizer were retargeted for integration of a landing pad to evaluate the reproducibility of transgene expression from TRIP integration sites in a context directly translatable to a commercial CHO CLD platform.
• the eight TRIP sites were classified into "High” (Azinl, Sartl, Fosl2), "Mid” (Pde4a, Doplb), and "Low” (Vgll4, Khdc4, Ddc) expression groups based solely on their TRIP barcode expression levels, with arbitrarily defined boundaries between groups.
• the genetic and epigenetic characteristics of this limited set of loci vary widely and are shared by other integration sites in all three expression groups.
• Cas9 target sites in a range of lOOObp surrounding the Piggybac insertion site were chosen based on computationally predicted high off-target stringency and on-target efficiency. All of the retargeted TRIP integration sites had equal or higher CMV-driven transgene mRNA expression than Ferll4 in equivalent culture conditions (FIG. 2). Additionally, transgene mRNA expression from integration sites other than the hypervariable Doplb and Ddc loci followed a similar trend to the TRIP predictions.
• Clone-specific chromosomal rearrangements surrounding the Doplb locus could potentially also play a role in determining transcriptional activity in individual clones, given the inherent plasticity of the CHO genome.
• the consistency of the 50/50 split in expression bins across two cloning campaigns indicates that these rearrangements were not random or non- reproducible, if they occurred.
• Experimental factors such as potential sources of variability in culture conditions and cell health early in the single-cell cloning and outgrowth process could also have a long-term impact on the epigenetic characteristics of the locus. Additional experiments will be needed to determine if clone-specific regulation of transgene expression at the Doplb locus can be associated with local genetic or epigenetic properties.
• Monoclonal antibody productivity from the novel Doplb hotspot was benchmarked using a master host cell line chosen based on high transgene mRNA expression and RMCE donor cassettes containing various combinations of 5' regulatory elements found to enhance transgene expression in the initial GFP-Puro r screen (FIG. 4A).
• GFP-Puro r pre-screen and mAb batch screen were not possible at configuration level due to the use of two transcription units for mAb expression.
• the demonstrated ability of many of the tested regulatory element combinations to increase cell-specific productivity relative to the "baseline" constructs (NA/NB for NISTmAb and TA/TB for trastuzumab) implied that GFP-Puro r was an effective screening tool to identify regulatory elements with the potential to enhance mAb expression.
• NISTmAb configuration F had the highest bulk productivity of any of the tested stable pools with a final mAb concentration of 1.996 ⁇ 0.012 g/L, a 2.8-fold improvement over NISTmAb construct A.
• Cell-specific NISTmAb productivity between fed-batch days 5 and 10 also increased from 2.93 ⁇ 0.06 pg/cell/day with construct A to 8.23 ⁇ 0.22 pg/cell/day with construct F.
• Trastuzumab constructs K and A exhibited a greater than 2-fold difference in mAb productivity in the first four days but reached similar final mAb concentrations of 0.987 ⁇ 0.012 g/L (Day 5 - 10 qP: 4.37 ⁇ 0.22 pg/cell/day) and 0.966 ⁇ 0.012 g/L (Day 5 - 10 qP: 4.63 ⁇ 0.11 pg/cell/day), respectively (FIGS. 4C-D).
• the high-throughput screen described here evaluated thousands of integration sites in the CHO genome for expression strength and stability over time. Over 90% of these integration sites maintained stable transgene expression levels over an extended culture aging timeline of 36 to 72 PDLs, in agreement with previous reports that transposase- and lentivirus-mediated integration methods in general lead to stable expression.
• the Piggybac system was a more effective screening tool for identifying stable hotspots than lentiviruses, which were insulated from both positive and negative position effects. Additionally, the integration bias of the Piggybac transposase toward active regulatory elements led to the identification of a set of loci in which the median mRNA expression level exceeded that of an industrial reference hotspot known to be capable of supporting multi-gram per liter volumetric mAb productivity.
• lentiviruses were not biased toward canonical epigenetic markers of active regulatory elements, including CpG hypomethylation, and could be avoiding the position effect through some other method, possibly by viral regulatory elements within the lentivirus package acting as chromatin domain insulators.
• the absence of position-dependent effects on transgene expression from lentivirus integrations could be explained by a lack of variety in the epigenetic environments surrounding the surveyed lentiviral integration sites. Regardless of how lentiviruses achieve position-independent expression, these results imply that the Piggybac transposase integration system is a more appropriate method than lentiviruses to use when searching for the highest-expressing hotspots with reproducible expression upon retargeting.
• the distance-dependent relationship between Piggybac barcodes and CpG islands in such a small range indicates that the size of a hotspot region capable of maximizing transgene expression is on the order of 1 to lOkb. Defining hotspots using genomic CpG islands alone limits the total search space to specific regions accounting for less than 3% of the Chinese hamster genome, which could be filtered further using H3K27ac/H3K4me3 histone modifications and CpG hypomethylation profiles, the best epigenetic predictors of high transgene expression out of any of the predictors evaluated here.
• Table 3 NISTmAb and trastuzumab fed-batch feeding schedule. Table 4. Integration counts and frequencies of Piggybac (PB) and lentivirus (LV) barcodes overlapping specific histone modifications.
• PB Piggybac
• LV lentivirus
• Histone modification ChlP-seq profiles used for overlap analysis were originally collected by Feichtinger et al. (Biotechnol. Bioeng. 113, 2241-2253 (2016)) and reprocessed as described previously by Hilliard and Lee (Biotechnol. Bioeng. 118, 659- 675 (2021)).

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