Drug Discovery Genetically Modified Mouse Models in Drug Discovery Advances in the production of genetically modified mouse models – notably the ability to make multiple genetic changes in one mouse-line – has opened up new areas of application in drug discovery and development. By Dr Paul Rounding at Artemis Pharmaceuticals GmbH and Dr Tom Shepherd at CXR Biosciences Ltd Dr Paul Rounding is Managing Director, Business and Operations, at Artemis Pharmaceuticals GmbH; he joined the company in January 2000 to lead the business development operation. Dr Rounding has over 17 years’ experience working for Bayer AG where he held various positions in the pharmaceutical business group, including drug discovery for respiratory and cardiovascular diseases, cardiovascular technology, international co-operation and licensing, and strategic marketing for Bayer’s blockbuster antibiotic Ciprofloxacin. Dr Rounding has a BSc in Pharmacology from the University of Bath, and a PhD in Pharmacology from London University. Dr Tom Shepherd is Chief Executive of CXR Biosciences Ltd (Dundee, Scotland). Prior to moving back to his country of origin, he was Chief Executive Officer of Neurotech SA in France; earlier posts included Vice President positions at ICN Pharmaceuticals Inc and IntraBiotics Pharmaceuticals Inc, both in California. He has over 20 years’ experience in business development in the pharmaceutical industry and holds a PhD in Biochemistry from Strathclyde University (Glasgow, Scotland). The pharmaceutical industry is continually looking for time-consuming areas of vector construction, cell new approaches to increase the efficiency of R&D and biology and mouse breeding – have reduced these times improve the quality of the innovative new medicines such that today all types of genetically modified mouse that they produce. In recent years, innovations in the models can be generated within 12 months. Further ability to produce genetically modified mouse models improvements in technology – such as RNAi-induced have increased their use in drug discovery – both for the gene knock-down in vivo – have led to model generation evaluation of the function of genetic targets in vivo, and times of less than four months (Figure 1). These times the predictive testing of the efficacy, pharmacokinetic are now compatible with drug discovery programmes and side effect profile of potential therapeutic agents. Figure 1: Improvements in time to generate conditional knock-out mouse models from 1999 to 2004 THE GENERATION OF GENETICALLY MODIFIED MOUSE MODELS Time (Months) 0 3 6 9 12 15 18 21 24 98 Time and Cost Cell culture experiment Conditional knockout mouse 24 Historically, the production of a genetically modified 99 mouse model has been a very long and expensive process. 00 Industrial production line process 18 The large number of techniques and disciplines required 01 (high quality molecular genetics, ES (embryonic stem) ArteMiceTM Platform 15 cell culture, cell injection techniques and mouse Year 02 Designer mice 12 breeding) combined into a complex process that resulted 03 in mouse model generation times of up to 24 months; this was far too long a time to allow a significant 04 RNAi 4 Significant reductions in time and effort to contribution to decision-making in drug discovery 05 RNAi ind. 4 generate genetically modified mouse models programmes. However, recent improvements in project 06 management – as well as technical improvements in the Innovations in Pharmaceutical Technology 21 and mean that data from advanced in vivo models can be technique was used during the 1990s in particular for the made available in time to contribute to the decision- tissue-specific modification of gene expression. This making process, and thereby make a significant allowed a closer dissection of genetic function for a contribution to increasing the efficiency and output of particular tissue of interest. For drug discovery purposes, R&D drug discovery programmes. the more interesting and relevant application is an inducible gene modification. In order to achieve this, a Early Limitations suitable gene switch was required which, when During the 1990s, genetic modification in a mouse was introduced into a mouse, produced a high level of gene limited to the over-expression or knock-out of a gene knock-down and only upon a particular chemical from birth onwards. Experimenters introduced the stimulus. It has recently become apparent that the gene modification into mouse germ-line or embryonic stem switch of choice for this application is the CreER gene cells, which then developed into the genetically modified switch, which when introduced into a mouse can mouse model. This had the significant disadvantage that produce a suitable gene knock-down in all tissues of the the gene of interest was modified in all mice from body, only in the adult (2, 3). conception onwards. Associated problems – such as embryonic lethality (mice dying before birth due to the These advances – together with the advances in timing gene modification) or compensatory changes (changed described above – mean that for the first time drug expression of non-modified genes in the embryo discovery researchers have the ability to produce animal compensating for the absence or over-expression of the models, in a reasonable period of time, in which their gene of interest) – complicate the phenotypic analysis of gene of interest is only modified in the adult. This such mice. For drug discovery teams who were interested provides significant advantages in terms of the type and in looking at the effects of gene modification in the utility of the data obtained. In particular, it allows adult, these techniques were – at best – only surrogates. discovery teams to obtain direct information regarding drug target efficacy only in the adult and, if the target Figure 2: Efficient RNAi knock-down in vivo in the mouse deletion is made once a disease state has developed, then direct information can be obtained about the therapeutic relevance of the target. In addition, discovery teams can 100 obtain information about any non efficacy-related effects 80 of the target, such as what degree of on-target toxicity 60 could be expected from a future inhibitor. All of this data is obtained after gene modification only in the adult, and 40 is therefore unencumbered by any compensatory or other Relative expression level 20 effects which may complicate interpretation of the results if the gene expression is modified also in the embryo. 0 Liver Lung Brain Heart Testis Kidney Spleen Muscle RNAi Gene Knock-Down Pancreas In recent years, RNAi knock-down in vitro has become a Salivary Gland Control Suprarenal Gland popular tool to study the effects of genetic modification. shRNA knock-down However, despite some early successes, it has not yet shRNA knock-down become widely usable in vivo. There are still many questions relating to repeatability and breadth of gene knock-down, as well as to the potential for off-target Conditional Gene Modification effects in vivo. Nevertheless, advances are being made in In the mid 1990s, Professor Rajewsky and his group in this field. The use of a targeted transgenesis approach in Cologne, Germany, identified a method to allow the ES cells, together with generation of the mouse model ‘conditional’ modification of gene expression in a mouse via injection into tetraploid blastocysts, may have (1). The intention was to provide the investigator with provided a breakthrough (4). Seibler’s group has shown the possibility of modifying gene expression in a mouse that the incorporation of a single siRNA sequence into a either in a particular cell of interest (leaving gene specific genetic locus in the mouse leads to reproducible expression in all other cells unaffected), or at the time of and broad deletion of the gene of interest (Figure 2). interest (for instance only in the adult mouse). This When combined with advanced methods of tetraploid 22 Innovations in Pharmaceutical Technology injection, the group can produce adult mouse models LEAD SELECTION AND OPTIMISATION within a four-month period. Potentially this can provide a revolution in the use of mouse models for discovery Traditionally, the major application of genetically purposes. For the first time, investigators can obtain modified mouse models in drug discovery has been relevant models relatively cheaply and within a very in the areas of modelling human disease, target short period of time. By reducing these twin constraints, identification, target validation and the testing of more genetic targets can be validated in vivo earlier candidate compounds to evaluate in vivo efficacy. in the drug discovery process, thereby increasing the Although standard rodent models are extensively used to quality of decision-making and having a positive impact study the safety and pharmacokinetic properties of on R&D efficiency. compounds, the use of genetically modified mouse models has been relatively limited in this area. Humanisation of Mouse Proteins In Vivo A further use of genetically modified mouse models, in This is due to a range of factors including: the generally addition to gene validation, is for drug testing. Recent more conservative nature of the drug development advances in the technique of ‘humanisation’ have process as opposed to drug discovery; the extensive contributed to the utility of mouse models for drug existing experience of using in-bred rodent species to testing applications. Using advanced cloning techniques evaluate risk to man; the preference in toxicological such as ET cloning (5), it is now possible to easily modify studies for the rat as a species, while the mouse is the large pieces of human DNA and build them into gene preferred species for genetic modification; and the vectors. By homologous recombination, these can be perceived difficulty in establishing with regulatory introduced into the mouse genome at the appropriate authorities the value of genetically modified animals in site, and the resulting human protein can be expressed in predicting the human situation. the mouse. This protein will be active, expressed at the correct level and in the correct tissues.