Preview

PDF, English Download (9MB) | Terms of use

Abstract

In the last years, our laboratory has developed and refined a novel platform of episomal self-sustaining S/MAR DNA vectors for gene and cell therapy. Their non-viral and non-integrative nature avoids integrational genotoxicity problems, a known issue with currently used gene therapy vectors. Previous work demonstrated their ability to provide persistent expression in cell lines and primary T-Cells. Further work successfully applied them to genetically modify mouse stem cells. Without altering their pluripotency capabilities, sustained maintenance and expression during reprogramming, differentiation and chimaera formation were shown. In this project, we aimed to extend our knowledge and understanding of these episomal vectors and developed and applied the technology towards human induced pluripotent stem cells (hiPSCs).

As a first step, we demonstrate the potential of our generated S/MAR DNA vectors in cancer cell lines and implemented vector establishment protocols with antibiotic selection or by purification of expressing cells via FACS sorting. We then provided proof of principle evidence that we can genetically modify hiPSCs using our S/MAR DNA vector system and demonstrated their isogeneity and unaltered capabilities to act as genetically modified cell source for gene and cell therapy. Besides implementing xeno- free hiPSC culturing which can easily be transferred to a GMP conforming protocol, we used cells isolated from urine as a novel, non-invasive cellular source for the generation of hiPSCs.

For the first time, we then moved from using the stable expression of GFP as an easily trackable reporter gene towards the restoration of a functional transgene in these cells. The potential of our S/MAR DNA vectors for use in advanced cell models and as prophylactic gene therapy for Birt-Hogg-Dubé syndrome (BHD) was then investigated. BHD patients harbour germline mutations in the gene for Folliculin (FLCN). After second hit mutations, functional FLCN is lost which leads to the development of kidney cancer. Currently, the only treatment available is the surgical resection of these tumours. However, this treatment does not restrict the development of further second hit mutations and tumours. Previous work by our group and others indicates that the pathways in which FLCN is involved and the tumorigenesis can be avoided by introducing a functional copy of FLCN. Thus, one potential treatment for this disease would be the introduction of mutation-proof copies of Folliculin into cells before the tumorigenic event takes place.

Utilising CRISPR/Cas9 we generated FLCN-knock-out urinary derived hiPSC cell lines. By establishing GFP or FLCN encoding S/MAR DNA vectors in both WT and KO cells, we generated important cell models for the study of FLCN. Characterisation of the cell lines as well as single-cell RNA sequencing suggested little impact of the S/MAR DNA vector on the cells. Also, FLCN expression levels were shown to be not required for the exit of pluripotency of these hiPSCs, rendering them a developmentally earliest cell model to study FLCN expression in a variety of cell models in the future. We finally demonstrated their unaltered nature and persistent transgene expression by implementing a kidney organoid differentiation protocol as an advanced cell model to investigate BHD.

Together, our data demonstrate the versatile application possibilities of the combination of our S/MAR DNA vector platform in combination with urinary and fibroblast derived hiPSCs for functional pathway analysis, disease modelling, patient- specific drug screenings or future cell therapies with optimised, non-viral gene therapy vectors.