a report by

D r s Y v e s d e C h a s t o n a y , S t e f a n S e l b e r t and Do r o n S hme r l i n g

PolyGene, Inc.

I n t r o d u c t i o n

Fuelled by the genomics revolution, pharmaceuticaland biotechnology companies are blessed with anunprecedented wealth of potential drug targets.

Current therapies predominantly act at the receptor,hormone, ion channel, nuclear receptor, various factorand DNA levels, i.e. on approximately 500 drug targetsknown and dealt with today. With additional insightgained from different fields of research in the post-genome era, the number of tractable targets is likely toincrease significantly, and a prioritised subset of orphanreceptors having the greatest therapeutic potentialalone, could factor down to as many as 300 candidates.

With skyrocketing costs of drug development, theexistence of interesting scientific hypotheses isinsufficient to support the levels of commitmentrequired to bring new drugs to market. Potentialtargets need to be validated beyond findings such asthose based on differential messenger ribonucleicacid (mRNA) expression patterns. Modulation oftarget level and/or activity in cell culture and animalsmust be shown to significantly ameliorate the diseasephenotype. Hence, one of the most significantchallenges (particularly with respect to targetvalidation) is the provision of models truly predictiveof the disease under investigation.

With the on-going identification of genes, geneproducts and biochemical pathways potentiallyinvolved in the susceptibility to (and/or progressionof) human diseases, a key question is how to selectthe appropriate intervention points amenable topharmaceutical development.

Irrespective of whether a systems or a molecularapproach is chosen (starting from patients or animalmodels and from clinical or cell samples,respectively), the drug discovery process entailsvalidating targets in both cell and animal models.Ultimately, transgenic and knock-out mice remainthe option of choice in target selection and validationbecause they represent the best available model forinvestigating the complex interactions that underliepathophysiological responses.

This article presents two solutions to significantlyenhance the current molecular validation toolbox –Transgenic Targeting in mice and embryonic stem(ES) cell differentiation, designed to minimise failureat later stages by allowing more reliable selection andvalidation of just the most promising candidates earlyin the drug development process.

T r a n s g e n i c T a r g e t i n g i n M i c e

Transgenic Targeting is a procedure in whichtransgenes are inserted into specific and predeterminedgenomic loci (e.g. via Cre/lox recombination).

A model mouse, in which a lox-flanked marker isplaced under control of a given promoter, isgenerated and the transgene expression patternthoroughly assessed. Generating further mouse linesby inserting transgenes (replacing the marker) viarecombinase mediate cassette exchange reproduciblyyields identical expression patterns.

The UBI-mouse, which is now available, is the firstin a series of Transgenic Targeting models. In theUBI-mouse, transgenes are specifically placed undercontrol of the β-actin promoter. The bornheterozygous animals are phenotypically normal andthe cassette exchange procedure reliably yieldsstrong, constitutive and ubiquitous transgeneexpression (see Figure 1).

The following benefits apply to this mouse model:

• Predictability – transgenes are specificallyintroduced at predetermined genomic loci,warranting defined and precharacterisedexpression patterns (full documentation on theexpected level and pattern of expression isavailable before project initiation).

• Cost-efficiency – a single mouse line is needed forany one transgene. Since experiments areperformed with fewer mouse lines, mouse housingand manpower costs are reduced significantly.

• Speed – all required vectors and genetic screeningprocedures (polymerase chain reaction, Southern,

Target Se lec t ion and Va l idat ion wi th Transgen ic Target ing and In V i t roEmbr yon ic S tem Ce l l D i f f e rent ia t ion – The Po lyGene Advantage

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Technology & Services

Dr Yves de Chastonay is ExecutiveDirector of PolyGene, Inc., wherehe is responsible for sales,marketing and businessdevelopment. Before joiningPolyGene, he gained broad insightin the pharmaceutical and medicaldiagnostics industries, acceptingaccountability in positions rangingfrom local Product & MarketingManagement to InternationalMarketing, Affiliate Management andBusiness Development. Dr deChastonay holds a PhD in MolecularBiology from the University ofFribourg, Switzerland, and an MBAfrom the European University inGeneva.

Dr Stefan Selbert is Senior ProjectManager at PolyGene, Inc., wherehe is accountable for theimplementation of customer andresearch and development (R&D)projects. From his prior paths inacademia and industry, he acquiredsolid knowledge in mousetransgenesis as well as inembryonic stem cell manipulationand in vitro differentiation and inthe generation and analysis ofrecombinant mice (in particular,animal models of human diseases).Following his studies at theUniversity of Freiburg, Dr Selbertearned his PhD at the Max-Planck-Institute of Biochemistry inMartinsried and held post-doctoralpositions in Edinburgh and Lübeck.

Dr Doron Shmerling is ManagingDirector and Delegate of the Boardof PolyGene, Inc., heading overalloperations and R&D. Before co-founding PolyGene, he gained in-depth knowledge in neurologicaldisease at Charles Weissmann’slaboratory and, more recently, hedeveloped innovative mousetransgenesis technologies forfunctional genomics applications atNovartis. Dr Shmerling has an MScfrom Tel Aviv University and earnedhis PhD at the University of Zurich.

primers, probes, controls) are pre-established, thusreducing mouse generation time.

• Flexibility – additional new mutations are easilyintroduced (truly comparable mouse lines withfurther transgenes can be established, as required).

• Control – insertional effects (e.g. attribution ofphenotypes to gene disruption as a consequence offortuitous transgene integration) can be excluded.

Offsetting the major drawbacks of conventionaltransgenics, Transgenic Targeting is ideal for suchapplications as the following:

• Gain of function experiments – since theprocedure is swifter, fully reproducible andreliable with respect to expression pattern(particularly noteworthy for ubiquitous orinducible expression).

• Parallelisation – evaluating a series of targets (onemouse line being sufficient for each transgene).

• Line comparison – be it for judging the effects ofvarious single nucleotide polymorphisms (SNPs),different oncogenes or a series of small interferingRNA (siRNA) molecules.

Exchanging the lox-flanked marker sequence isfeasible as well via co-injection (in oocytes) as via co-electroporation (in ES cells) of new floxed-transgene-harbouring constructs with Cre-proteinand thus, different procedural strategies (as outlinedin Figure 2) have evolved.

E S C e l l I n V i t r oD i f f e r e n t i a t i o n

Establishing ES cell lines before transgenic mice aregenerated allows early validation steps to be performedin cell culture (including in vitro differentiation), inturn providing important information for decision-making in the target selection process.

ES cells, under defined culture conditions, have thecapacity of differentiating into so-called embryoidbodies (in many aspects similar to 11.5-day embryos),and further specifically into a great variety of celltypes, including any kind of neuronal cell,haematopoietic or epithelial cells, beating cardiacmyocytes, smooth or skeletal myocytes, adipocytes,chondrocytes, etc.

With the broad range of differentiation markers nowavailable, ES cell investigations can be monitored atboth the mRNA and the protein level. Applicationsinclude the following:

• Testing drugs with unknown cell specificity –embryoid bodies are the ideal model system toscreen for anti-angiogenic factors, as well as to testproliferative, cytotoxic or embryo-toxic potentialsof compounds such as cytokines, growth ordifferentiation factors.

• Isolating cell types for pharmacological studies – invitro differentiation is a reliable procedure toestablish and purify given cell types such ascardiomyocytes in large quantities for subsequentin vitro testing.

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Technology & Services

Figure 1: UBI-mouse Expression Pattern

Green fluorescence in mouse organs of the UBI mouse with a green fluorescent protein gene inserted at the β-actin locus, in comparison to wild-type organs.

Target Se lec t ion and Va l idat ion

• Dealing with a newly identified gene orregulatory (e.g. promoter) element – thistechnique (for example complemented byimmunofluorescence microscopy) allows you toassess spatial and temporal regulation of expression(in which cell types and at which stages ofembryonic development).

• Having established a new knock-out or knock-inES cell line – in possible anticipation ofdevelopmental abnormalities or embryoniclethality, in vitro differentiation is a rapid andreliable method for comparing differentiability ofwild-type and genetically modified ES cells.

S umma r y a n d P r o s p e c t s

Reproducibly yielding predictable transgeneexpression patterns, Transgenic Targeting is the mostreliable procedure for mouse transgenesis. Thetechnique is perfectly suited for large-scale targetevaluations by knock-in. Moreover, as an effectivecellular delivery technique, Transgenic Targeting isthe solution of choice for overcoming the limitations

of target knock-down (e.g. siRNA) strategies.Combined with in vitro ES cell differentiation forprecocious target selection, Transgenic Targetingsaves considerable time and money by astute targetvalidation in the drug development process.

A first Transgenic Targeting model, for strong,constitutive and ubiquitous expression, is now availableat PolyGene, Inc. Launch of models for tissue-specific(neuron-, pancreas- and adipocyte-) expression isanticipated for the second or third quarter of 2004, andanother model, for inducible transgene expression, iscurrently under development. ■

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Figure 2: Transgenic Targeting Strategies – Cloning Scheme for the Production of Mice via Transgenic

Targeting

A lox-flanked gene (tag) is targeted into a given locus, yielding targeted ES cells and tagged mice via subsequent blastocyst injection (blue arrows). Cassette replacement

(substituting the tag with a marker gene as specified below) yields model mice in which the expression pattern is thoroughly assessed. Custom mice (and the model mouse),

harbouring a new transgene (or a reporter gene, respectively), are produced either by micro-co-injection (green arrows) or via the ES cell route (violet arrows). The targeted

ES cells are perfectly suitable for a variety of tissue culture validations or high-throughput screening assays (red arrow).

Contact Information

PolyGene, Inc.

Riedmattstraße 9

CH-8153 Rümlang

Switzerland

Tel: (41) 1 828 63 80

Fax: (41) 1 828 63 81

e-Mail: info@polygene.ch

http://www.polygene.ch