Site-Directed Engineering of Defined Chromosomal Sites for Recombinant Protein and Virus Expression

ABSTRACT

In cell line development, the integration of transgenes into the chromosomal DNA of the host cell is a crucial step. Targeted introduction has many advantages over classical random integration procedures because of strong influences of the chromosomal surroundings on the expression of transgenes. Site-directed integration leads to predictable expression properties, avoids screening, is fast, provides high safety, and is thus the method of choice for the targeted integration of transgenes. Recombinase mediated cassette exchange (RMCE) using heterologous recombinases has been described as an efficient and reliable method to target (integrate and replace) transgenes by site-directed engineering of defined chromosomal sites for recombinant protein and virus expression. The article reports on the exploitation of defined chromosomal sites for the consistent production of highly biologically relevant proteins, namely the expression of viral vectors and antibodies.

Genetically modified cells are used for a wide variety of purposes, ranging from basic research to elucidate gene functions (e.g., in transgenic mouse models) to biotechnological applications like manufacturing antibodies. Efficient production of proteins and virus particles using mammalian cell lines usually relies on stable, albeit random integration of an expression construct into the cell's chromosomal DNA. After a construct is incorporated, its expression levels will be determined by neighboring genetic elements.^1,2 To exploit a favorable genomic locus for expression of a given construct, intensive screening usually is required to identify integration sites that support high-level protein production. Frequently, screened clones are even engineered to maximize their expression potential. Gene amplification is a standard technology that aims to increase the gene copy number and thereby improve the production properties of a given cell clone. The genomic instability associated with gene amplification is a major challenge for the establishment of a stable and fully characterized producer cell line. Further, these current state-of-the art protocols are usually time consuming and importantly, must be repeated for each and any transgene to be expressed.

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In comparison to the integration of numerous copies, a single copy of the gene of interest represents an advantageous scenario because of the possibility of full characterization of the integration site and the increased genomic stability of the producer cell line. The production of biopharmaceuticals from a single integration site is indeed a possibility, as already shown by the authors of this article and others. Single copy integration of transgenes in selected chromosomal sites provides the required expression strength and stability.^3–6

The biotechnological exploitation of producer cell lines containing a single copy of the transgene of interest gained considerable importance following the introduction of RMCE. As specified below, this technology permits the rapid exchange of expression cassettes of choice in defined genomic surroundings. In this way, RMCE allows the manipulation of a single chromosomal locus that supports the desired expression level of the relevant protein and renders its repeated re-use feasible. In this way, this technology represents an excellent way to exploit defined genomic sites and is a breakthrough in cell engineering.

To enable exploitation of a favorable chromosomal integration site, an initial primary (preliminary) genomic modification to first mark or tag the site must be performed (Figure 1). This, in turn, creates a genomic platform that supports subsequent modifications of that particular site. Although the first generation of tag-and-targeting approaches (so-called flip-in approaches) has limitations with respect to efficiency, RMCE is considered the method of choice for targeted integration mainly because of the lack of reversibility, the lack of insertion of bacterial sequences, and the elimination of the tagging cassette (as summarized in Gama-Norton, et al.).⁶

Figure 1. The recombinase mediated cassette exchange (RMCE) principle and strategy for efficient selection of correctly targeted subclones. A single copy of a tagging cassette is initially integrated into the genome of the cell of choice through random integration or viral mediated delivery of the DNA sequence (the tagging step). A reporter gene can be used to identify tagged cell clones with desired expression levels. The cassette exchange-targeting step through RMCE is performed by co-transfecting an Flp-encoding vector and a targeting vector containing the gene of interest (GOI). The latter is flanked by two heterospecific Flp recognition target sites (FRT) that are identical to those flanking the tagging cassette. Additionally, the vector contains an ATG start codon to restore the nonfunctional Δmarker-gene of the tagging cassette. Once the Flp enzyme has exchanged the tagging for the targeting cassette, the ATG is placed in frame with the marker gene and cells can easily be selected by the addition of the respective marker drug.10 Importantly, because of the incompatibility of the two heterospecific FRT sites, excision reaction of the tagging cassette is excluded.

By tagging the genomic locus of interest, the heterotypic and incompatible recognition targets such as the Flp recognition target (FRT) and FRTmut are introduced into a genomic locus (Figure 1).^6,7 This creates a cassette acceptor allele, i.e., this tagged locus can now be used to integrate and exchange different DNA cassettes of choice.

To target the genomic locus, a targeting vector containing the desired transgene flanked by the same set of heterotypic recognition target sites as the tagged cassette is transfected together with an expression plasmid that encodes for the recombinase. A double reciprocal crossover recombination event will lead to the exchange of the reporter cassette with the cassette of interest, the targeting cassette. This strategy can hence be termed the tag and target strategy.⁸

Screening at the tagging stage (step 1) makes it possible to identify the integration sites that provide the desired expression properties, i.e., high, stable, or regulated expression of the transgene. A successful selection process might require screening large numbers of cells. Once this cell line is established, it will be further characterized for stability and safety. Further, the optimal culture conditions that support maximal productivity will be defined. In this state, these cells can constitute a platform that can be exploited to predictably express other proteins in a short time. This is illustrated in Figure 2.

Figure 2. Timelines to generate tagged high expression clones and constitute a producer cell line after targeting with the gene of interest. Standard protocols to generate conventional producer cell lines rely on the establishment of a different cell line for the expression of a different product. Commonly, more than 24 months are needed for the development and full characterization of a biotechnologically relevant cell line. In contrast, the establishment of different cell lines that express different products with biotechnological value by the application of RMCE technology can be achieved one-month after targeting of an already tagged cell clone. This represents a tremendous advantage and opens up the possibility of creating a panel of cell lines that express different genes of interest in a predictive manner.

The examples given below illustrate the performance of this technology for the establishment and exploitation of biopharmaceutical producer cell lines.

USING RMCE TO PRODUCE VIRUSES AND PROTEINS

We screened various cell lines including human 293 cells, mouse NIH3T3, Chinese hamster ovary (CHO), and baby hamster kidney (BHK) cells for integration sites that meet the requirements of stable and high expression. For this purpose, we implemented an advanced site-specific cassette replacement strategy that combines retroviral tagging and a positive selection trap with the Flp/FRT recombination.^9,10 To select exclusively cells that underwent the site-specific recombination, the tagging cassette contains a transcriptionally inactive selection marker that is only activated by correct site-specific integration. This strategy makes it possible to correctly identify recombined clones with an efficiency level of 90% or better.¹⁰ We established a set of cells with singly tagged chromosomal sites and used green fluorescent protein (GFP) to identify potent integration sites. Various tagging protocols were successfully applied.¹¹

After the sites are tagged and characterized, they can be used to target expression cassettes of choice. Figure 3A gives examples for a GFP-tagged cell line into which the lacZ expression cassette was integrated. Targeting eliminates the GFP and includes lacZ. Importantly, the clones generated upon targeting have homogeneous expression profiles as expected from the integration into a given chromosomal site.

Figure 3A. Predictable and homogeneous product expression after targeting a tagged locus A. The GFP-containing tagging cassette of a master cell line has been replaced by a targeting vector encoding lacZ. The GFP expression analysis (measured by FACS) and a lacZ staining protocol were performed before and after RMCE. Whereas the tagged cell population exhibits a high level GFP expression but is negative in lacZ staining, resulting subclones after RMCE homogeneously lost the GFP signal but gained the expression of lacZ.

We exploited this homogenous expression to generate retroviral producer cells. The production of viruses, or viral vectors, can be seen as a special case of protein expression. Retroviral vectors constitute a powerful tool for stable gene transfer into mammalian cells. They can be used to efficiently infect cells of different origin of various species. Vectors derived from murine leukemia virus (MLV) are used as gene delivery systems in clinical gene therapy trials.¹² Considerable success in the treatment of several inherited diseases by integrating an expression unit for the therapeutic transgene(s) in the cellular genome delivered by retroviral vectors has been achieved, demonstrating the enormous potential of viral vectors in clinical therapy.^13–15

Figure 3B. Predictable and homogeneous product expression after targeting a tagged locus B. Seven individual clones of the RMCE experiment described in A were analyzed by Southern blotting to confirm a correct cassette exchange. Therefore, chromosomal DNA of the clones was digested with DNA endonucleases, blotted, and probed against the marker gene. Only successfully targeted clones would show a 3 kb fragment and be clearly distinguished from the tagged parental clone The lacZ expression of these clones was quantified and compared to the parental cell line. As shown in the diagram, the expression behavior is highly homogenous among all analyzed isogenic subclones.

To translate the application of viral vectors into the clinic, it is mandatory to produce high titers of viral vector from a fully characterized producer cell line. For each therapeutic vector, a specific producer cell line must be generated. The titer critically depends on the site of chromosomal integration of the therapeutic vector. Thus, tremendous screening efforts must be undertaken to isolate clones that express the vectors in high concentrations and stably over time—a process that can take several months when conventional retrovirus packaging cell lines are used.¹⁶ We established a strategy based on RMCE to establish a new generation of producer cell lines. Upon selection for a chromosomal locus that supports retroviral vector insertion, a flexible retrovirus producer cell line was established that simplifies the isolation of highly productive producer clones.^3,4 This approach reduces laboratory time, increases safety, and in particular makes it possible to adjust viral vectors to the requirements of individual loci.¹⁷

With these modular packaging cells, infectious particles can be efficiently produced with high reproducibility, achieving titers up to 2 ×10⁷ IU/10⁶ cells in 24 hours (Figure 3C). Importantly, this technology can be used to produce clinically relevant viral vectors. Indeed, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within three weeks without screening.³ Because the master cell line is fully characterized with respect to retroviral vector production conditions, the establishment of a producer cell line is reduced to the replacement step to introduce the vector of interest.

Figure 3C. Predictable and homogeneous product expression after targeting a tagged locus C. Based on a chromosomal site tagged with a retroviral vector, a packaging cell line was established.3 The tagging vector was replaced by various viral vectors. The productivity of subclones obtained by the targeting of one of those vectors reveals that the expression levels are strictly homogenous among all analyzed isogenic subclones.

Finally, the high capacity of this strategy is demonstrated by its use in the predictable expression of antibodies. For this purpose, we used single-step vectors that encode both the heavy- and the light-chain genes. Upon targeting into the prescreened integration sites, antibody expression clones are generated. Importantly, they show homogeneous antibody expression levels which are expected from the defined genetic modification (Figure 3D).

Figure 3D. Predictable and homogeneous product expression after targeting a tagged locus D. To prove the flexibility of the RMCE system, an antibody expression cassette was targeted into the tagged chromosomal loci of clones of different origin, including Chinese hamster ovary cells (CHO), and human embryonic kidney cells (HEK293). The expression of the targeted subclonal producer was measured by ELISA and, although different between the species, is homogenous amongst each other.

CONCLUSION

The illustrated data presented here give evidence that this technology is a powerful method for achieving defined and reproducible genetic modification of cells, thereby making it possible to establish producer cells in a short period of time. After a potent integration site is tagged and identified on screening, characterized with respect to safety, and optimized for production, it constitutes a platform for integrating other expression cassettes of interest. Thus, it makes it possible to establish expression clones with predictable properties in a minimal time. Apart from this, it makes it possible to adjust expression cassettes to the requirements of the individual chromosomal sites.^11,17 In this way, it contributes to a predictable modification of the mammalian genome and its exploitation for the production of biopharmaceutical molecules.

Dagmar Wirth, PhD, is head of the research group Model Systems for Infection and Immunity (MSYS), +49 531 6181 5040, dagmar.wirth@helmholtz-hzi.de This group is associated with the division of Molecular Biotechnology at the Helmholtz Centre for Infection Research, Braunschweig, Germany. Leonor Gama-Norton is a research fellow in MSYS, and Kristina Nehlsen, PhD, and Roland Schucht, PhD, are scientists in MSYS and this division, respectively. Leonor Gama-Norton is also a member of the Instituto de Biologia Experimental e Tecnológica, Universidade Nova de Lisboa (IBET/ITQB/UNL), Oeiras, Portugal.

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