Drug Discovery

toolbox also opens new possibilities for perform- ing experiments aimed at target and hit validation. Phenotypic rescue experiments can be improved

by exploiting the CRISPR-Cas9 technology in var- ious ways (Figure 1). Complete abolishment of target gene expression using CRISPR-Cas9 pro- vides the maximal window of a phenotypic effect for the particular target without the confounding residual expression from incomplete knockdown by RNA interference. Thereby interpretation of knockout and rescue experiments is more straight- forward. Furthermore, the impact of a certain mutation on a disease phenotype can be assessed in a physiologically relevant system by precise cor- rection of the mutation at the endogenous locus, thus not affecting expression levels. Restoration of a disease-associated mutation is also a valuable approach for drugs that supposedly impact only the mutated and not the wild type form of a tar- get: a resulting insensitivity of the cell line to the drug confirms specificity for the mutated form and rejects off-target effects. Similarly, the involvement of a target in a drug-sensitive phenotype can be studied in more detail by precise mutation of its putative active site. Mutating the hypothesised drug interaction site can help to elucidate a drug mechanism, but also to determine specificity of a drug. Although in general these are effective approaches for sound target and hit validation, caution should be exerted for cancer lines specifi- cally since correction of a driver mutation may be technically challenging as it could result in loss of cellular fitness in vitro. All such target and hit validation strategies using

CRISPR-Cas9 can be reproducibly performed in several independent cell lines containing their own unique background mutations to confirm robust- ness in various patient populations. In principle, the same toolbox can be repeatedly used for all cell lines, albeit some loci seem to be more resistant to editing in one cell line compared to the other. Also, delivery of the system is challenging in some cells and tissues. So even though the addition of CRISPR-Cas9 technology to the phenotype rescue toolbox is a great add-on, there are some limita- tions to take into account.

The way forward to success In order to reduce the enormous costs of drug development there is an urgent need formore strin- gent filtering of putative drug targets and candi- date compounds in early drug discovery. This can be achieved by investing more time and effort in characterisation and validation of the drug-target interaction on a disease phenotype. As outlined in

Drug DiscoveryWorld Summer 2019

this article, rescue of a disease-relevant phenotype by genetic restoration using CRISPR-Cas9 is a valuable and recommended tool to use next to the more established methods for target identification, hit finding and validation. As all interventions and model systems have drawbacks and pitfalls of their own, it is strongly preferred to use a combination of approaches for detailed assessment of a putative clinical candidate prior to moving forward, with a strong focus on target and hit validation after the initial discovery. A robust validation procedure could entail: 1. Validation in different model systems, eg cell lines from various tissue types, several individual patients, 2D versus 3D cultures. 2. Confirmation of results generated using RNA interference methods by CRISPR-Cas9 gene edit- ing and vice versa. 3. Reproduction of the genetic ablation-induced phenotype by intervention with small molecules. 4. Rescue experiments by genetic restoration of the disease-associated mutation, abolishment of a tar- gets’ active site and/or putative drug-target interac- tion. Increased awareness of such gold standard

approach in drug discovery will help to boost prof- itability of a drug development programme by early failure of poor candidates.


Dr Anne-Marie Zuurmond is a Director at Charles River. She has been active as project leader in a CROenvironment for more than 14 years, manag- ing projects in various therapeutic areas, including neurology, fibrosis, arthritis and metabolic syn- drome. She was responsible for the implementation of the CRISPR/Cas9 technology at Charles River and is now leading genome engineering projects using this technology to support in vitro drug dis- covery. She earned her doctoral degree in molecu- lar biology and MSc in biology from University of Leiden.

Dr Geraldine Servant is Senior Scientist at Charles River. She received her PhD from Pierre andMarie Curie University in the field of Molecular Biology. As a molecular biologist with 12 years of experi- ence in France and the United States, she has been a key player in innovative research. Currently, her dream would be to work on research and develop- ment of bio-medicines and targeted therapy for the treatment of cancer or rare diseases. She is also striving to work as a projectmanager to pilot inno- vative scientific projects, develop systems that analyse living organisms and original and efficient


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