"I received the Young Investigator Award, given for the best oral presentation. The title of the presentation was ‘Targeted epigenetic modifications as a therapeutic approach to treat HIV infection’. The research aims to investigate the efficacy and safety of epigenome editing as a novel therapeutic strategy based on gene activation and may contribute towards establishing a safer therapy against HIV infection".
Targeted epigenetic modifications as a therapeutic approach to treat HIV infection
Tafadzwa Mlambo1,2, Jana Block1,2, Marianna Romito1,2, Tatjana Cornu1,2, Toni Cathomen1,2, Claudio Mussolino1,2
1Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany
2Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany
The human immunodeficiency virus (HIV) is a major global health burden which has claimed over 25 million lives in the past 30 years. While drug-based treatments have been effective in the management of infection, they do not represent a cure. Thus, focus has been directed towards therapies based on genetic engineering and cell manipulation. The CCR5 co-receptor is necessary for HIV entry into host cells and individuals that are homozygous for inactivating mutations in the CCR5 gene are largely protected from infection. Therefore, this receptor has gained interest as a possible target for gene therapy against HIV infection since its inactivation in T cells may render them resistant to viral infection. Genome editing strategies are currently being explored with the aim of creating patient-derived T cells resistant to HIV infection, but the genotoxic potential of designer nucleases has thus far restricted this approach to short-lived CD4+ cells. We hypothesize that transcriptional repression via epigenetic modification of the CCR5 promoter may provide a safer alternative to inactivate CCR5 expression compared to genome editing as the genomic sequence remains unchanged and as a consequence the severity of off-target effects may be reduced.
We have generated a panel of transcription activator-like effector-based repressors (rTALEs) and TALE-based designer epigenome modifiers (DEMs) and tested their activity in a reporter cell line in which a 320 bp proximal promoter fragment of the CCR5 gene drives the expression of a green fluorescent protein (GFP). Both platforms were able to efficiently modulate reporter gene expression with the most efficient rTALEs resulting in GFP silencing in 20% of transfected cells. Interestingly, delivery of CCR5-specific DEMs resulted in more efficient and stable GFP silencing in up to 80% of the target cells for a period of 23 days highlighting the potential of DEMs in inducing long-term epigenetic modifications which are maintained during cell division. GFP-negative cells arising upon DEM delivery were FACS-sorted and CCR5-specific TALE-based transcriptional activators or the demethylation agent 5’-Aza-2’-deoxycytidine (5’-AZA) used to restore GFP expression demonstrating that the targeted epigenetic marks are reversible. To highlight the translation potential of DEMs, their functionality was tested in CD4+ primary T cells. We observed a 50% reduction in CCR5 gene expression compared to controls following mRNA delivery. Investigations into the extent of DNA methylation via bisulfite sequencing and transcriptome-wide changes including off-target effects via RNA-seq are currently ongoing. This study aims to dissect the efficacy and safety of epigenome editing as a novel therapeutic strategy based on gene inactivation and provides a leap towards establishing a safer therapy for HIV infection.