Can RENDER Move Epigenome Editing Closer to Therapeutic Applications?

Can RENDER Move Epigenome Editing Closer to Therapeutic Applications?

Epigenome editing via programmable transcriptional repressors/activators regulates gene expression in mammalian cells by altering the local chromatin environment (via DNA methylation or histone modifications) at target gene loci. To this end, epigenome editing tools typically comprise effector domains from transcriptional repressors/activators coupled to programmable DNA-binding proteins (McCutcheon et al.). Recent applications of this exciting technology include durable gene repression i) in the liver to combat hypercholesterolemia (a disorder known for excess low-density lipoprotein in the blood) (Cappelluti et al. and Tremblay et al.) and ii) in the brain to combat prion disease (Neumann and Bertozzi et al.). Unfortunately, the relatively large size of most epigenome editing tools poses a significant challenge to their ongoing use in research and their exploration as therapeutics for a wide range of human diseases.

To further improve epigenome editing technology and move this therapeutic approach closer to the clinic, researchers have begun exploring new means to safely and effectively deliver epigenome editing tools into cells. Delivery as ribonucleoprotein or RNP complexes (Raguram et al.) offers several benefits, including eliminating the need for transgene or viral vector-based expression (and the associated risk of viral DNA integration) and minimizing exposure time in target cells, which in turn decreases off-target editing. Delivering RNP complexes in virus-like particles or VLPs (Madigan et al.) also offers several unique advantages, including their increased cargo capacity and natural ability to enter cells.

Researchers led by James K. Nuez (University of California, Berkeley) recently developed Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins or RENDER to transiently deliver CRISPR-based programmable epigenetic repressors/activators as RNP complexes into human cells via engineered (e)VLPs (Banskota et al.) to modulate gene expression. They reported this advance in a recent Nature Communications paper, which may now significantly broaden the use of epigenome editing in basic research and therapeutic applications (Xu and Besselink et al.).

Paired-Tag technology from Epigenome Technologies enables the simultaneous profiling of transcriptomics and epigenetics in single cells. Could integrating this approach into this fascinating new study have yielded additional insight into the safety and effectiveness of this new epigenome editing platform? Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays while avoiding the need for cell sorting.

eVLPs and RNPs: RENDERing Epigenetic Editing in Human Cells a Straightforward Task?

In brief, a rational engineering approach led to the development of an initial version of RENDER that enabled the delivery of a diverse range of epigenome-editing tools as cargo. This included a CRISPR interference platform, enabling short-term and reversible gene repression via the Krppel-associated box (KRAB) transcriptional repressor domain, which recruits cofactors to deposit H3K9me3, and DNMT3A-3L-dCas9 to induce DNA methylation; and a CRISPRoff platform, enabling long-lasting gene silencing via combined deposition of H3K9me3 and DNA methylation. Furthermore, the team demonstrated the robust applicability and potency of RENDER in inducing efficient epigenetic silencing of endogenous genes, which persisted for weeks in various normal and cancer cell types, including donor-derived primary T cells.

Excitingly, the team also reported the additional optimization of RENDER for the CRISPRoff platform (RENDER-CRISPRoff v3); here, the increased protein and single-guide RNA cargo packaging supported a nearly five-fold increase in epigenetic silencing, which allowed a single dose of RENDER-CRISPRoff v3 to induce epigenetic silencing of multiple target genes for over four weeks in multiple cell types. Notably, the authors reported the use of this platform to suppress the expression of the V337M-mutated Tau protein in human stem cell-derived neurons harboring a pathogenic mutation, highlighting the potential of RENDER-CRISPRoff for the treatment of neurodegenerative diseases.

The Multiple Applications of the RENDER Platform for Advanced Epigenome Editing

Overall, the authors hope that their RENDER platform, which supports the safe, efficient, and transient delivery of CRISPR epigenome editors into human cells through the implementation of RNPs and eVLPs, will find broad applications in fundamental biomedical research and in vivo epigenetic therapies as disease treatments. Furthermore, the authors highlight the potential of RENDER to facilitate functional genomics applications through epigenome-editing perturbations.

The additional integration of simultaneous profiling of transcriptomics and epigenetics in single cells, afforded by applying Paired-Tag technology from Epigenome Technologies, could provide additional insights into the safety and effectiveness of the RENDER platform; furthermore, the implementation of RENDER in vivo will require detailed epigenetic and transcriptional analyses at the single-cell level to explore the full extent of induced epigenetic alterations.