prof. dr. H.J. Haisma
CRISPR/Cas9 knockout of EGFR in cancer
Recent advances in genome editing based on CRISPR/Cas9 system have triggered a revolution in gene targeting or gene editing research and application, since it is much easier to construct and more efficient than the previous nucleases (Meganucleases, ZFN, TALENs). However, efficient and safety delivery to target cells or tissues remains a bottleneck for CRISPR/Cas9 as well as other nucleases, when they are actually applied in clinical gene therapy.
This research will mainly focus on using AdV mediated CRISPR/Cas9 to structurally knockout the mutant/overexpressing EGFR gene in order to obtain a therapeutic effect. Furthermore, the selected high efficiency AdV-EGFR-CRISPR/Cas9 vectors will be redirected to EGFR positive tumor cells by a bispecific fusion protein (bispecific scFv 425-S11) in vitro and in vivo.
Targeting TNF-related apoptosis-inducing ligand to cancer cells
Genetic fusion of sTRAIL to a tumor-selective single-chain Fv (scFv) antibody fragment can optimize the efficacy of TRAIL-based therapy. Specifically binding of an scFv:sTRAIL fusion protein to the predefined target antigen leads to selective tumor cell accretion. In addition, the soluble and conditionally inactive scFv:sTRAIL is hereby converted into a membrane-bound and active form of TRAIL that can activate both DR4 and DR5. Importantly, target antigen-bound scFv:sTRAIL can induce bi-/multicellular activation of TRAIL receptors resulting in apoptotic activity toward proximal tumor cells.
We will investigate the specificity and efficacy of adenovirally expressed fusion proteins containing EGFR-targeted TRAIL mutants. To this end, we will use different peptides with selectivity for EGFR and determine their specificity in the context of a TRAIL fusion protein. A specific avenue for research will be the use of TRAIL mutants, designed to reduce binding to decoy receptors and improve DR5 tumor-specific TRAIL receptor binding.
CRISPR/CAS9 system optimization
Targeted genome editing using engineered nucleases such as Zinc finger and Transcription activator-like effector (TALE) nucleases has rapidly evolved from being a niche technology to a mainstream method. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities.
Here we propose to expand our ongoing research line on the development of HAT and HDAC inhibitors in relation to their effects on gene expression. We hypothesize that HDAC inhibitors cause unwinding of the target DNA by the addition of acetyl groups to the histones, and thus improving the accessibility of the DNA for gene editing using the CRISPR/CAS9 system. This would enable binding of the nuclease to the desired target sequence and cut the DNA. In the current project we propose to evaluate HAT and HDAC inhibitors with various isoenzyme selectivity's for their effects on CRISPR/CAS9 mediated gene editing.
Gene doping detection by next generation sequencing
Gene doping represents a threat to the integrity of sport and the health of athletes. The anti-doping community has been focusing efforts on developing a test for its detection. As the sequences of DNA of Epo and other doping genes are known, it is relatively easy to aggravate this test, which will then result in a false-negative result.
We propose to develop a new gene doping detection assay that will overcome this problem. The test is based on targeted sequencing of doping genes with potential to detect any doping gene in any context with a very high sensitivity. Using an in-house designed next generation sequencing assay, we developed a gene doping detection assay for DNA of EPO. We proposes to evaluate and further develop a multiplex ‘gene doping detection panel’ which targets genes for among others insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. The panel allows simultaneous detection of several ‘sport-specific’ genes in one sample, reducing the test’s cost and turn-around-time. This research is crucial in the development of a reliable routine method for detection of gene doping with several genes that could be potentially used in all sports.
|Last modified:||15 September 2021 4.25 p.m.|