Alexandre Regamey

Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.

The Tumor Suppressor CYLD Interacts with TRIP and Regulates Negatively Nuclear Factor κB Activation by Tumor Necrosis Factor

Cylindromas are benign adnexal skin tumors caused by germline mutations in the CYLD gene. In most cases the second wild-type allele is lost in tumor tissue, suggesting that CYLD functions as tumor suppressor. CYLD is a protein of 956 amino acids harboring a functional deubiquitinating domain at the COOH-terminal end. To shed more light on the function of CYLD, we have performed a yeast two hybrid screen using an HaCaT cDNA library that identified the RING finger protein TRIP (TRAF-interacting protein) as interactor with full-length CYLD. Mapping of the interacting domains revealed that the central domain of CYLD binds to the COOH-terminal end of TRIP. Far Western analysis and coimmunoprecipitations in mammalian cells confirmed that full-length CYLD binds to the COOH-terminal domain of TRIP. Because TRIP is an inhibitor of nuclear factor (NF)-κB activation by tumor necrosis factor (TNF), the effect of CYLD on NF-κB activation was investigated in HeLa cells. The results established that CYLD down-regulates NF-κB activation by TNF-α. The inhibition by CYLD depends on the presence of the central domain interacting with TRIP and its deubiquitinating activity. These findings indicate that cylindromas arise through constitutive NF-κB activation leading to hyperproliferation and tumor growth.

Using Matrix Attachment Regions to Improve Recombinant Protein Production

Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

The N-acetylmuramoyl-L-alanine amidase encoded by the Bacillus subtilis 168 prophage SPβ

Heat shock of Bacillus subtilis CU1147, a strain lysogenic for SP beta c2, a prophage with a thermosensitive repressor, results in phage induction and subsequent cell lysis. Cloning in Escherichia coli and sequencing of a DNA fragment of prophage SP beta led to the identification of blyA, the gene encoding a 367 amino acid polypeptide with a molecular mass of 39.6 kDa. Purified BlyA obtained from the E. coli clone exhibited an N-acetylmuramoyl-L-alanine amidase activity. Insertional mutagenesis confirmed that the latter enzyme was associated with SP beta-phage-mediated cell lysis. Analysis of the neighbouring sequence suggested that the two ORFs immediately downstream of blyA and belonging to the same operon encode polypeptides which may be involved in the release of the endolysin. The presence on the chromosomes of B. subtilis or related Bacillus spp. of other, similar genes, and their possible relationship, is discussed.

MAR Elements and Transposons for Improved Transgene Integration and Expression

Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1–68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1–68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2–4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168–166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPβ established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPβ, plays a key role in the formation of so‐called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of ‘transstamping’. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X‐ray‐induced, rearrangement.

The caspase-3/p120 RasGAP module generates a NF-κB repressor in response to cellular stress

ABSTRACT The nuclear factor κB (NF-κB) transcription factor is a master regulator of inflammation. Short-term NF-κB activation is generally beneficial. However, sustained NF-κB might be detrimental, directly causing apoptosis of cells or leading to a persistent damaging inflammatory response. NF-κB activity in stressed cells needs therefore to be controlled for homeostasis maintenance. In mildly stressed cells, caspase-3 cleaves p120 RasGAP, also known as RASA1, into an N-terminal fragment, which we call fragment N. We show here that this fragment is a potent NF-κB inhibitor. Fragment N decreases the transcriptional activity of NF-κB by promoting its export from the nucleus. Cells unable to generate fragment N displayed increased NF-κB activation upon stress. Knock-in mice expressing an uncleavable p120 RasGAP mutant showed exaggerated NF-κB activation when their epidermis was treated with anthralin, a drug used for the treatment of psoriasis. Our study provides biochemical and genetic evidence of the importance of the caspase-3–p120-RasGAP stress-sensing module in the control of stress-induced NF-κB activation. Summary: Mild caspase activation in cells subjected to non-lethal doses of stress generates an N-terminal p120 RasGAP fragment that inhibits NF-κB activity.

High performance CHO cell line development platform for enhanced production of recombinant proteins including difficult-to-express proteins

Background In an effort to improve product yield of mammalian cell lines, we have previously demonstrated that our proprietary DNA elements, Selexis Genetic Elements (SGEs), increase the transcription of a given transgene, thus boosting the overall expression of a therapeutic protein drug in mammalian cells [1]. However, there are additional cellular bottlenecks, notably in the molecular machineries of the secretory pathways. Most importantly, mammalian cells have some limitations in their intrinsic capacity to manage high level of protein synthesis as well as folding recombinant proteins. These bottlenecks often lead to increased cellular stress and, therefore, low production rates.