How Can Drug Discovery Programs Benefit From Genetically Engineered Models?

Target validation through tissue-specific expression of the target gene:

• Models for ADC: cell-specific expression of the antigen targeted by the antibody, expression level, and distribution are crucial to the design of a relevant model, enabling one to study the effect of ADC on the tumor microenvironment (ADC targeting TAM)

Target validation through gene ablation, the most commonly used strategy for target validation:

• Enables one to predict the consequence of a target-complete inactivation, but should be limited to target, with very little interaction with other pathways (ion channels, secreted proteins, etc.)

Target validation through point mutation:

• Becoming the standard for dissecting between functions of proteins displaying dual activity, such as scaffolding and kinase activities (RIP1, RIP3, JAK, STAT, etc.)

Tissue/time-specific inactivation of a target gene:

• Ideal for MOA studies and investigation of targeted therapies (immune check-point, complexes involved in effector functions, and more)

Tissue/time-specific induction of catalytically inactive enzymes/kinases:

• To inactivate a given function of an enzyme while preserving its expression and interaction with partners; mimics what a small molecule (DNA sensor, PKC, PK…) would do

The design of the humanized model must take into consideration the family of the target gene and type of therapeutics developed.

Beside its primary function, does the target gene interact with partner molecules? If yes, would a humanization of the target abolish these interactions?

Examples:

• IL-4Rα interacts with the γc chain to create functional IL-4 receptors, but also interacts with IL-13Rα1 to create the IL-13 receptor
• Recognition of TLR2 by its ligands requires an heterodimerization with TLR1 and TLR6

Is it required to humanize the whole target gene or shall we humanize only the epitope recognized by the antibody and preserve the interaction with other subunits of a complex?

Example:

• CD3ε partners with CD3γ, ζ and δ to create a functional CD3 complex
• Lack of interaction with partners would results in immune-compromised models

Shall a humanized model express only the canonical form of the target (appropriate for efficacy studies) or the canonical and secreted forms that could act as decoys? (More relevant for PK and PD studies)

The type of therapeutics developed influences the model design:

• Use a chimeric molecule to enhance the signaling of the human protein into mouse cells if often suggested but not adapted to GPCR targeted by small molecules since it could modify the binding pocket

What type of models should be used for ADMET studies?

• Selection of the species to be used for the study and its genetic background are essential parameters to be taken into account
• Easy readouts, early response detection, and enhanced throughput could be obtained using in vivo monitoring of selected biomarkers

How to reproduce the human pharmacokinetics?

• Humanization of pathways known to be crucial for PK and PD assessment of biologics and small molecules increases the predictability toward human data: humanization of HSA and FcRN (compound recycling binds HSA complex) closely mimic human pharmacokinetics (hFcRn/HSA off-the-shelf model)

How to detect off-target activities in genetically engineered models?

• Physiological relevancy of the model is indispensable to avoid false positives; i.e., most standard KO models exhibit genetic compensation and pathway deregulation
• Most studies are done without the appropriate control models, yielding false results and misleading conclusions

Understanding of the etiology of the disease in humans is paramount to designing a relevant translational model.

Are the SNPs identified through GWAS causal agent of the diseases?

What are the consequences of those mutations at the gene and protein levels:

• Defect in gene-splicing process resulting in no expression of the protein
• Defect in gene-splicing process resulting in expression of a mutated protein
• Defect in the gene expression level and pattern
• Defect both gene expression level and expression of a functional protein

Answers to these questions are often unknown, but a thorough bio-informatics analysis coupled to in vitro assessment of some of the hypothesis could clarify the context. This dictates the design of the in vitro or in vivo model recapitulating the human situation.

How can the time to first P.O.C be shortened?

• Use of new embryology technologies combined with in vitro phenotyping
• State of the art time requested to get first POC data is less than one year (which includes model design and creation, animal production, and POC experiment)

Could the cost of in vivo studies be reduced?

• Over the several years of use, well-designed genetically engineered models showed reduced expenses in both human resources and expenses, i.e., less animals per experiments, while meeting ethical considerations
• Delivery of cohorts of ready to use, fully validated animals combined with optimized screening methodologies
• In vivo monitoring of biomarkers reduces budget in kits and consumables and strongly decreases per experiment cost