A POWERFUL GENE EDITOR, BUT DOES NOT CUT THE DOUBLE STRAND? A HIT-AND-RUN EPIGENOME EDITING.

 

Although editing our genes can solve many of our problems, be it genetic disorders at birth or cancers in our 50s, making a permanent change in our instruction manual seems daunting. What if we had a way around this? What if, the changes we make to our genome can be reversed? And Even better, what if we had a switch to turn it on or off? Well, look no further! Epigenetic editing, a gene editing system that plays around with DNA binding molecules, has been in play for a few decades now. Epigenetic editing refers to the modifications in histones and DNA that can regulate transcription. 

However, an obvious question arises, if the changes are not made in the DNA sequence itself, how would the information travel across replication? Research has also answered this question. DNA replication is a highly conservative process that involves disassembly and reassembly of chromatin. During this process, half of the histones carrying parental epigenetic information are deposited to the new strands, while the other half is newly synthesized. This re-establishment of parental epigenetic information in a DNA sequence-specific manner is of growing interest in the research world. A piece of recent evidence has also surfaced suggesting histone-modifying enzymes might be responsible for this transfer rather than histones themselves. 

Now that the transfer of epigenetic information through generations is sorted out, what about the efficiency? Will epigenetic editing have the same efficiency as editing the DNA sequence? If so, will it stand the test of time? These questions have been pondered in the research world for decades, to come up with an efficient, permanent, and durable epigenome editor. A recent study published in February 2024 from Italy has developed the same, A durable and efficient gene silencing in vivo by hit-and-run epigenome editing. Where the term Hit-and-run refers to a transient delivery that leaves to lasting impact.

Epigenetic Modifications

In the study, the researchers have designed an all-in-one configuration mRNA molecule delivered by Lipid nanoparticles and called it the EvoETRs, Evolved Engineered Transcription Repressors. Engineered Transcription Repressors (ETRs) have been familiar to scientists for quite some time. ETRs are molecular tools specifically designed for epigenome editing, they consist of programmable DNA-binding Domains (DBDs) such as catalytically deactivated Cas9, Zinc finger Proteins (ZFPs), or Transcription Activator like Effectors (TALEs) that can bind to specific sequences, and an Effector Domain (ED) derived from naturally occurring Transcription repressors such as Krüppel associated box (KRAB), catalytically dead DNA methyltransferase 3A (cdDNMT3A ), or DNA methyltransferase 3-like protein (DNMT3L). These components are co-delivered as separate mRNAs and they function together to achieve epigenetic silencing, whereas the design proposed in this study, is an all-in-one mRNA molecule, i.e. the DBDs, EDs, and other necessary compounds are fused into a single mRNA molecule. This simplifies the process, furthermore, Evo-ETRs are optimized for enhanced efficiency and specificity in epigenetic silencing, offering a streamlined approach.

In the design of Evo-ETRs, the components were optimized for the best repressive activity of the gene Pcsk9, a gene responsible for regulating cholesterol in the liver. The results were compared against repression induced by Pcsk9-targeted CRISPR-Cas editing. They discovered that ZFP-based ETRs showed the most downregulation of Pcsk9 and had the least off-target effects, this was attributed to the unintended docking of ZFP arrays at the off-target sites, causing less to no harm rather than the activity of the effector domains while freely floating. Therefore, ZFPs were chosen as the DBD and it was the N-terminal of the EvoETR. 

Structure of EvoETRs

At the C-terminal was the ED, and the ED selected for the EvoETR was KRAB, KRAB is a famous repressor among the epi-silencers known for its ability to induce robust gene repression in various cell types both in vivo and in vitro by recruitment of histone-modifying enzymes through conserved mechanisms making them attractive tools for clinical testing. Between these two domains is the component that further improves the efficacy of EvoETRs. The component is an obligate heterodimer between cdDNMT3A  and DNMT3L bound to the N terminal of the ZFP domain. Normally, DNMT3A is an enzyme that adds methyl groups to DNA. This typically represses gene expression, although, cdDNMT3A is catalytically dead and does not have any enzymatic action, but it can still bind to DNA and interact with other proteins. DNMT3L is a regulatory protein that does not have catalytic activity but can enhance the activity of  DNMT3A by forming a complex with them therefore forming an obligate heterodimer. The EvoETR mRNAs are further modified to enhance translation efficiency and minimize innate immune responses. The fused all-in-one mRNA is encapsulated in Lipid Nanoparticles (LNPs) which protect mRNA from degradation and is administered intravenously to the target organism. The LNPs reach the hepatocytes, where they deliver the EvoETRs and EvoETR proteins are transcribed, which in turn silences the Pcsk9 gene by epigenetic modifications.

The study also proved that partial hepatectomy, causing forced regeneration did not destabilize or eliminate the epi-silencing activity of ETRs in the liver of mice, proving that ETRs are durable and pass epigenetic information to subsequent generations (making them heritable). This newly designed approach only needs a single administration and is described as a “One-and-Done treatment” by the authors. A main advantage to this approach is that it showed equal efficacy to that of DNA sequence editing therapies (when compared to Pcsk9 silencing mediated by CRISPR-Cas), therefore no breaks need to be created in the DNA whose off targets could be fatal and side effects would be irreversible, whereas for the former it would be the opposite and a much safer therapeutic approach. 

The question we need to ask now is: Will this change the course of gene therapies, or is it too good to be true? 

REFERENCES 

1. Cappelluti MA, Mollica Poeta V, Valsoni S, et al. Durable and efficient gene silencing in vivo by hit-and-run epigenome editing. Nature. 2024;627:416-423. https://doi.org/10.1038/s41586-024-07087-8

2. Budhavarapu VN, Chavez M, Tyler JK. How is epigenetic information maintained through DNA replication? Epigenetics Chromatin. 2013;6(1):32. doi: 10.1186/1756-8935-6-32. PMID: 24225278; PMCID: PMC3852060.

IMAGE CREDITS:

1. Cover image: https://www.nature.com/articles/nm0817-900 
2. Epigenetic Modifications: https://www.whatisepigenetics.com/fundamentals/