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CRACK IT Challenge

InMutaGene: A human IPS cell model for gene therapy vector safety evaluation

DNA replication

At a glance

Award date
July 2016 - December 2020
Contract amount


  • Replacement


As Gene Therapy (GT) becomes a clinical reality, there is an increasing demand for non-clinical tests to assess the safety of the therapeutic strategy for the patient. Animal testing is generally required by regulatory authorities for GT products prior to initiation of human trials and in vivo studies are generally performed on each individual GT product. However, the unique biological properties of individual integrating vectors means that careful consideration is required to define appropriate non-clinical studies relevant for the prediction of clinical outcome.  A robust in vitro/ in silico tool to evaluate the risk of insertional mutagenesis/oncogenesis will support risk assessment while limiting the use of animals. 

Genotoxicity by retrovirus (RV), lentivirus (LV) and adeno-associated virus vectors (AAV) has been demonstrated both in vitro and in vivo. In Phase 1 of this CRACK IT Challenge, the team at Brunel University London led by Dr Michael Themis along with GeneWerk, Germany, set up the basis of a standardised assay for an Individualised Genotoxicity testing (InGeTox) approach to test vector safety and efficacy. The team includes experts from leading researchers at University College London, King’s College London and The Natural and Medical Sciences Institute at the University of Tübingen, Germany. In this model, human induced pluripotent stem cells (hiPSC) are used for reprogramming liver cells in order to test vector associated genotoxicity. This approach, which may use cells derived from the patient, enables the individual’s genetic background to be accounted for in toxicological studies. Using established assays, vector-related factors that cause genotoxicity can be measured. These include the effects of the vector on cancer gene expression by vector promoters and enhancers, vector splicing with host gene to form aberrant transcripts and changes that alter host gene expression as a result of the host epigenetic response to gene transfer. In addition, these factors are scored alongside the therapeutic effect of the expressed transgene. During Phase 1, the team achieved:

  • The development of a gene therapy eligibility assay that measures the patient’s target cell’s capacity for repair of DNA damage caused by vector integration.
  • Optimised hiPSC differentiation to hepatocytes in 3D culture.
  • Efficient gene transfer by LV of iPSC.
  • Genome-wide vector integration site distribution SIN LV safety profile contrasts to unmodified LTR LV.
  • Robust epigenetic response to infection and integration.
  • Design of a protocol to identify human endogenous retroviruses in vector preparations intended for gene therapy.

During Phase 2 of the project, the team at GeneWerk GmbH led by Dr Manfred Schmidt will continue with the validation and standardisation of the Phase 1 results according to international quality control Standards (i.e. GCLP, DIN EN ISO/IEC 17025) to provide a modular service omics-platform that measures genotoxic factors by gene therapy vectors, a comprehensive package predicting patient eligibility and outcome before vectors are considered to move to the clinic.

Full details about this CRACK IT Challenge can be found on the CRACK IT website.


Scholz S et al. (2017). Lentiviral Vector Promoter is Decisive for Aberrant Transcript Formation. Human Gene Therapy, (10):875-885. doi:10.1089/hum.2017.162.