A human IPS cell model for gene therapy vector safety evaluation

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.

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CRACK IT Challenge

Award date:

Jul 2016

Contract amount

£700,000

Primary 'R'

Replacement

Scientific Discipline

Safety
Toxicology

Technologies/approach

In vitro techniques

Keywords

Genotoxicity
Vector