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Drug Discovery


simply deliver guide RNAs by either nanoparticles, lentiviral viral vectors or adeno-associated viral vectors to the organ of interest and induce mutage- nesis to promote disease. This then allows us to make in vivo somatic mutations rapidly without further engineering. Furthermore, the financial and temporal cost of generating new mouse alleles and incorporating them into increasing complex mouse models will be dramatically decreased. This was recently demonstrated in a model of pancreatic ductal adenocarcinoma (PDAC), which unfortunately is a leading cancer killer in the US39. Whereas existing in vivo models failed to model the stepwise progression and adult onset of pancre- atic cancer, the Winslow lab at Stanford University School of Medicine was able to induce targeted genomic deletion of Lkb1 in adult mice specifically in the pancreas by retrograde pancreatic ductal injection of virus containing sgRNAs to a Cas9 expressing mouse. In combination with oncogenic Kras expression, Lkb1 deletion led to the rapid for- mation of pancreatic tumours, confirming its role in pancreatic tumour development.


This approach is game-changing to the cancer biologist. By utilising this CRISPR/Cas toolkit for transgenic in vivo mutagenesis, this approach should make loss-of-function experiments in vivo no more difficult than altering those genes in vitro, meaning this system should enable the rapid func- tional investigation of any gene of interest in a live animal. Conceptually, sgRNA-directed Cas9 cut- ting combined with tet-inducible RNAi will enable multiplexed gene inactivation to rapidly give rise to disease and evaluation of therapeutic targets via RNAi-mediated gene silencing in the same animal. Beyond animal models, the synergy of RNAi and CRISPR/Cas9 enables genetic in vitro manipula- tion of human primary cell models, often patient- derived or with disease-associated genotypes edited in, enabling side-by-side comparison of data to improve the accuracy of these predictive models. Recognising the potential efficiencies this presents for drug discovery researchers, CROs such as Mirimus and Charles River have invested in sophisticated genome editing platforms to enable their partners to combine RNAi and CRISPR tech- nologies to streamline the discovery process for tar- get identification, validation and disease modelling both in vitro and in vivo.


Conclusion


When we think about all the new and exciting ways we have of probing the molecular make-up of cancer and other diseases, it is important to keep all of these tools in context. The CRISPR frenzy


14


Dr Prem Premsrirut is Founder and Chief Executive Officer of Mirimus, which uses RNAi and CRISPR/Cas9 technologies to engineer mouse models.


Drug Discovery World Fall 2017


notwithstanding, none of these by themselves are the magic bullet, but used together not only can we broaden our understanding of disease mechanisms, we might be able to determine whether a designat- ed target that looked promising in the earliest stages of discovery indeed has the kind of efficacy and limited toxicity that succeeds in patients. It might also help us to wage a winning war against those high drug prices.


Grateful acknowledgement to Qiantong Hu for contributing to the research of this article. DDW


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