INTERVIEW

“Genome-editing technologies now permit precise positioning of deletions, modifications, and transgenes within living cells. These ideas have led Zsolt Keresztessy, Ph.D., senior research fellow at Proxencell, to a method employing sequence-specific meganucleases and TAL effector nucleases to generate stable monoclonal cell lines expressing membrane-bound antigens, FCGR receptors, and monoclonal antibodies. TAL effector nucleases are novel sequence-specific nucleases, formed by fusing a transcription activator-like (TAL) effector DNA binding domain to the catalytic head of an endonuclease…

…Dr. Keresztessy explains that specific genome editing technologies are still in the initial evolutionary phase—true especially for TAL effector nucleases. “That means, in addition to requiring substantial optimization work, investigators must also innovate in the adaptation of commercially available systems from, for example, Cellectis Bioresearch or Life Technologies.”Uncovering effective ways to transfer and express sequence-specific nucleases (e.g., plasmid DNA, mRNA, or proteins) into your target cells or cell lines, together with accessory sequences including like templates for homologous recombination or genome editing reporter constructs, is critical. “As a result,  we were forced to develop new technologies for assessing genome modifications at early stages of TAL transfections, strategies and tools for detecting and enriching knockout cells, and new approaches for mapping TAL specificity in vivo in automated and high-throughput assays.”…

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 RECENT STAFF PUBLICATIONS

Jana Krenkova, Ákos Szekrényes, Zsolt Keresztessy, Frantisek Foret and András Guttman (2013) Oriented Immobilization of Peptide-N-glycosidase F on a Monolithic Support for Glycosylation Analysis. Journal of Chromatography A, 1322, 54-61(IF  4.6/2012) LINK

 CONFERENCE TALKS AND POSTERS

Keresztessy, Z. (2013) Genome Engineering Adventures in Cell Line Development: Reporters, effectors, producers. Informa Life Sciences: Cell Line Development and Engineering, 11 – 15 February 2013, NH Danube City, Vienna.

Recent specific genome editing technologies allow us to precisely position deletions, modifications, and transgenes in the genomes of living cells. Using various sequence-specific meganucleases and TAL effector nucleases engineered to recognise integrated target sequences or natural genomic loci of mammalian cells (Jurkat, HEK, YT, THP-1, CHO etc.), we have successfully generated a set of stable monoclonal cell lines expressing membrane-bound antigens, cell surface receptors, or monoclonal antibodies, to operate as target cells or effector cells in bioassays, or potential biopharmaceutical protein producers.

 Zsolt Keresztessy, Eva Nagy, Erzsebet Matyas, Jozsef Horvath, Zoltan Doro, Balint Laszlo Balint, Gabor Zahuczky, Laszlo Nagy (2013) Cell line development using genome engineering technologies: Reporters, effectors, producers.  Oral Presentation, O-060. Hungarian Molecular Life Sciences Conference, 5-7 April, Siófok, Hungary

Recent specific genome editing technologies allow us to precisely position deletions, modifications, and transgenes in the genomes of living cells. As a result, stable cell lines can be generated with controlled characteristics and predictable transgene functioning for the purpose of e.g. fundamental research, bioassays, and recombinant protein production. Using various sequence-specific meganucleases and TAL effector nucleases engineered to recognise integrated target sequences or natural genomic loci of mammalian cells (Jurkat, HEK, YT, THP-1, CHO etc.), we have successfully generated various stable monoclonal cell lines expressing membrane-bound antigens, cell surface receptors, or monoclonal antibodies, to operate as target cells or effector cells in bioassays, or potential biopharmaceutical protein producers. The concept of sequence-specific genome editing is based on the fact that genomes are ordered and controlled mostly by sequence-specific DNA binding proteins, transcription factors. Recently, thanks to the accumulating information on DNA sequence recognition by such proteins, the principles of those interactions  have been increasingly exploited to engineer tools for targeted genome modifications. Firstly, a nuclease is engineered to cleave the desired target sequence in the genome, creating a double-strand break (DSB). Secondly, the cellular mechanisms of DNA DSB repair, which are (i) non-homologous end joining (NHEJ) or (ii) homologous recombination (HR), operating in the absence and presense of a template DNA, respectively, are exploited to introduce the desired modifications at the chosen genomic location.  To date, engineered nucleases, which were successfully used in specific genome manipulations are restricted to Meganucleases at some extent, but mostly to Zinc-Finger Nucleases (ZFNs), and recently, to TAL-like effector nucleases (TALENs). This cutting-edge technology, which is fully operational in both cell lines and primary cells, including oocytes, has made us able to rationally disrupt target genes at single nucleotide resolution (sure knock-outs) via the introduction of small insertions or deletions at the endonuclease TALEN) cleavage site in a cell line. Also, we were able to insert the gene of a recombinant cell surface antigen into precisely-defined location for efficient expression without harmful effects on normal cell functioning in a cell line of choice. In addition, we specifically integrated therapeutic antibody genes into a highly expressive and well defined genomic locus of CHO cells to achieve efficient protein production. Specific genome editing technologies are still in the initial evolutionary phase (it is true especially for TAL effector nucleases and some upcoming new approaches), that means, besides the substantial optimisation work one need to carry out, one also has to innovate a lot in the adaptation of the commercially available systems (e.g. Cellectis Bioresearch or currently the System Biology Division of Life Technologies) to the specific applications. Therefore, and as a result, while going along our genome editing routes, we were  forced to implement and developed new technologies (i) to assess genome modifications at early stages of TALEN transfections, based on specific amlification and capture of modified sequences for more accurate quantification and sequencing, (ii) generally applicable strategies and tools for the detection of, and direct enrichment for assisted KO cells using e.g. flow cytometry, and (iii) new approaches to map TALEN specificity in vivo in automated and HTS assays. Most promisingly, we are keen (iv) to device and test some new/proprietary genome editing approaches, which are completely divergent from the current state of the art.

Lilla Ozgyin, Ádám Pallér, Jozsef Horvath, Zsolt Keresztessy, Gabor Zahuczky, Balint Laszlo Balint, Laszlo Nagy (2013) ChIP experiments with spike controls. Poster, P-110. Hungarian Molecular Life Sciences Conference, 5-7 April, Siófok, Hungary.

Chromatin immunoprecipitation is a widely used, valuable method for the detection of protein-DNA interactions in vivo, primarily for transcription factors and histone modifications. Coupled with real- time quantitative PCR and deep sequencing methods, ChIP would be a promising tool to reveal disease-associated changes in transcription factor recruitment thus providing us a deeper insight into the chromatin-level variances in the case of many multifactorial diseases and cancer. Our aim is to develop a method that allows the introduction of chromatin immunoprecipitation into clinical research. Several protein-DNA complexes were tested, and shown to be able to serve as spike-in controls. Such controls will allow us to monitor the immunoprecipitation efficiency, to titrate the epitope-binding capacity of an antibody and also to monitor batch-to-batch variation of policlonal antibodies. By now, we have controls for HDAC1, MECP2, CTCF, RXR and ER. We tested a polyclonal RXR-specific spike control on chromatin samples from 293T HEK cell line, and also carried out epitope-selection to further improve specificity.

Zsolt Keresztessy, Eva Nagy, Adrienne Gyongyosi, Balint L. Balint, Gabor Zahuczky, Laszlo Nagy (2013) CUSTOM CYTOKINE PRODUCTION SERVICES DEVELOPMENT OF A RESEARCH CYTOKINE PRODUCTION PIPELINE. Poster, P-123. Hungarian Molecular Life Sciences Conference, 5-7 April, Siófok, Hungary.

Cytokines are widely used in the field of immunology and stem cell research but the cost of such biological reagents can mount to extremities in case of complex systems and can consume a large proportion of grant budgets. Purified recombinant cytokines tend to be hugely overpriced partly due to marketing and shipping costs. Locally provided cytokine products produced by university core facilities may be a way of reducing research expenses or re-allocating financial resources. In our laboratory, we aim to provide the research community with high quality cytokines at affordably low prices. We established an infrastructure and a development/production workflow suitable for the generation of cytokine products with thorough biochemical, toxicological, and standard biological activity testing and quality control. In collaboration with a number of departments and labs within the university, we established a network of independent/external research laboratories testing our cytokine products in their own experiments. Recombinant human cytokines are produced from optimised synthetic genes in HEK293T cells (DMEM, 10% FBS) via transient transfection or cytokine expressing stable HEK293 AD cell lines secreting the proteins into the medium. The proteins are glycosylated, contain affinity and epitope tags, specific cleavage sites for tag removal. Cytokines are available as conditioned medium or as purified by Ni-affinity chromatographic technique. We set up a series of standard biological assays in the labs to test and ensure quality of our recombinant cytokines. These assays are widely adapted by worldwide manufacturers, and therefore, we are able to make direct comparison with commertially available cytokine products e.g. with respect to specific activity. In summary, we are offering high quality, highly active, mammalian cell derived  cytokines (IL-4, GM-CSF, IL-2, LIF) for research use, at an affordably low price on a non-profit bases. We provide thorough biochemical and biological characterisation data for the products: cytokine conditioned media or purified proteins. Also, we are open up our cytokine development pipeline for your specific needs – you name a cytokine – we produce it for you within a couple of months.

Keresztessy, Z., Attila Horváth, Ádám Pallér, László Steiner, József Horváth, Gábor Zahuczky, Endre Barta, Laszló Nagy, Bálint L. Bálint (2012) Developing immunoprecipitation/next generation sequencing-based technologies and tool for antibody characterisation: Specificity, batch-to-batch production QC, and full process controls for research, therapeutic, and diagnostic applications. PEGS Europe 2012, 6‐7 November 2012, Vienna. Austria.

Immunoprecipitation (IP) is a widespread method of purification of specific proteins (or co-purification of those with associated molecules  in Co-IP) from complex samples including cell lysates or whole tissue extracts. Chromatin immunoprecipitation (ChIP) is a method to study protein–DNA interactions, typically in-vivo interactions. The method combined with next generation sequencing (ChIP-NGS) is one of the most important functional genomic methods that had a significant impact on both gene regulation research and the field of epigenetics. One of the biggest limitations of the method is the lack of standards and controls providing clear results for the procedure. This generates significant variability and makes it difficult to introduce the method into clinical research. Here we aimed to develop novel approaches to standardise immunoprecipitation methods to monitor full processes up to the final analysis steps like NGS in ChIP-sec experiments. With the set of solutions in our hands, we not only offer (i) ways of robust standardisation and validation of clinical diagnostic protocols, but also (ii) highly sensitive analysis tools of antibody specificity down to molecular level, and (iii) quantitative approaches to antibody production QC, potentially with great impact on the development of  bispecific and next generation therapeutic antibodies.

Ákos Szekrényes, Jana Krenkova, Zsolt Keresztessy, Frantisek Foret, András Guttman (2012) ORIENTED IMMOBILIZATION OF PNGASE F ON A POROUS POLYMER MONOLITH FOR RAPID N-GLYCAN RELEASE. CECE Junior 2012 Interdisciplinary Meeting on Bioanalysis, 31st October, Brno, Czech Republic.

 We demonstrate a simple and rapid method to fabricate a porous polymer-based monolith reactor for rapid N-glycan release using a simple approach for the oriented immobilization of glutathione S-transferase (GST) tagged peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase F (PNGase F) endoglycosidase enzyme. The monolith is contained in a capillary format, while the applied glutathione based affinity immobilization allows fast oriented and replaceable enzyme coupling to the monolith surface. Glycoproteins in native form (Ribonuclease B, Fetuin from calf serum and human serum Immunoglobulin G) perfused through the PNGase F reactor were shown to be effectively deglycosylated on a time-scale of low minutes using mg/mL concentrations. Analysis of the released glycan pool was performed off-line by matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF) and by capillary electrophoresis with laser induced fluorescent detection (CE-LIF).

 Keresztessy, Z., Bálint, B.L., Zahuczky, G. and Nagy, L. (2011) Development of better antibody solutions for functional genomics and epigenetics. PEGS 2011 The Essential Protein Engineering Summit, Boston MA, USA. 9-13 May 2011.

In the field of functional genomics, there is a lack of reliable sources of antibodies and solutions for gene expression analyses based on chromatin immunoprecipitation techniques (ChIP, ChIP-Seq, and ChIP-on-Chip). The challanges antibody developers have to face include how to obtain antibodies capable of recognizing functional epitopes on proteins sequestered in large protein complexes regulating chromatin functions. Our major aim is to develop and optimize innovative antibody design algorythms, which are based on structural as well as functional information available on the target proteins, and involve integrated ensemble approaches to rational epitope prediction, to make possible the generation of highly target and application specific antibodies. Via the establishment of state of the art facilities in house, we produce polyclonal and monoclonal antibodies based on our rational epitopes, and the applicability and reliability of the resulting “better antibody solutions” are rigorously tested and characterised from a wide range of aspects including their effectivity in Western boltting, ELISA, immunoprecipitation, immunohystochemistry, mobility shift assays, ChIP, ChIP-Seq, ChIP-on-Chip applications, and quality controlled. Successful results demonstrated here are for cases of nuclear hormon receptors such as RXR and PPAR, and nuclear receptor co-activator/co-repressor complex members such as SMRT as our antibody targets, in comparison with commertially available counterparts.

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