Towards therapeutic modulation of the epigenome in females with X-linked liver disease using Adeno-Associated Viral vectors

Abstract

Allele-specific reactivation of healthy gene copies on the inactive X chromosome (Xi) has the potential to restore expression of absent proteins for therapeutic benefit as a method of treating X-linked disorders. Elimination of the master regulator XIST RNA may be required for success, however subsequent chromosome-wide Xi reactivation may lead to dosage imbalance of X-linked genes. Other approaches such as the recruitment of transcriptional activators using CRISPRa at the target loci offers the advantage of targeted effect. Here, we utilised our recently published novel model system, chimeric mouse-human FRG livers produced from hepatocytes derived from a heavily skewed female patient liver explant with the X-linked metabolic liver disorder, OTC deficiency, to investigate the effects of XIST RNA elimination and recruitment of a transcriptional activator in vivo. We observed that deletion of XIST in female patient hepatocytes in vivo, led to reactivation of the healthy OTC gene from the Xi. However, we also observe that elimination of XIST RNA causes potentially abnormal consequences on the epigenome environment of the human X-chromosome. To overcome the challenges and limitations associated with targeting the entire X chromosome, a locus-specific strategy was also explored to target the OTC promoter through the recruitment of a transcriptional activator. This strategy of modulating the epigenome induced expression of OTC in vitro and proved that the locus is amenable to editing, however no transcriptional activation was detected in the patient derived primary human hepatocytes. Additional analysis elucidated challenges inherent in delivering epigenome editing reagents efficiently to primary human cells in an in vivo setting. This thesis lays early foundations for overcoming hurdles associated with translating liver-targeted epigenome editing strategies from bench side to bedside, through bridging the gap between the gene therapy and the epigenome editing fields.