Next Phase Newsletter

Juan Carlos Lopez

Andy Marshall

Commercial interest in targeted epigenetic therapies — agents that target specific genes without altering bases in their sequence or causing double-strand breaks or even single nicks in the DNA — continues to grow, as underscored by the latest financing announced by Epigenic Therapies. The unique selectivity and specificity of targeted epigenetic therapeutics offers compelling advantages over small-molecule epigenetic drugs, which target a specific epigenetic reader, writer or eraser, but affect genes across the genome and affect many diverse tissues, leading to narrow therapeutic windows that make them difficult to develop for conditions outside of cancer.

Today, Haystack is aware of at least eight private companies (nChroma Bio (resulting from a merger of Chroma and Nvelop), Encoded Therapeutics, Epigenic Therapeutics, Epitor Therapeutics, Moonwalk Bio, Navega Therapeutics, Regel Therapeutics, and Tune Therapeutics), and two public companies (Modalis Therapeutics and Sangamo Therapeutics) that are pursuing the targeted epigenetic approach against disease (let us know if you know of any others). Another company, Flagship Pioneering’s Omega Therapeutics, went out of business in August after filing for bankruptcy in February. A smaller set of companies are also pursuing targeted epigenetic therapies against RNA modifications.

All of these therapies are designed around an alluring set of simple principles: take a gene-specific DNA-binding domain — zinc-finger proteins (ZFPs), ‘dead’ Cas9 (dCas9) with mutations in its RuvC and HNH endonuclease domains, or transcription activator-like effectors (TALEs) — and tether it via an amino acid linker to an enzymatic effector module. This effector is either an enzyme that directly places or removes a specific epigenetic modification (e.g., TET, histone demethylases or the histone acetyltransferase p300) or a transcriptional activator (e.g., VP16) or repressor (e.g., KRAB).

A particularly compelling application for such treatments is genetic disorders of haploinsufficiency (like Dravet’s) or imprinting disorders (like Angelman’s or Prader Willi). There are also many of these diseases where the therapeutic genes would be too large (>4.0 kb) for a traditional AAV gene-therapy approach; in contrast, epigenetic editing machinery can be packaged into an AAV vector.

Currently, the diseases being pursued by companies include hepatitis B, hypercholesterolemia, epilepsies (SCN1A (Dravet syndrome) and SCN2A), chronic pain, and muscular dystrophies. Those with the most advanced programs are Encoded’s AAV-9 intrathecally delivered SCN1A-targeting zinc finger protein linked to a VP16 activation domain in phase 1 testing for Dravet and Sangamo’s AAV- STAC-BBB-delivered SCN9A-targeting zinc finger protein linked to a KRAB repressor domainin a phase 1/2 trial for patients with chronic pain. In this context, two papers published in the past couple of weeks represent important proofs of the efficacy of targeted epigenetic therapies.

In a first paper published in Nature, the groups of Kevin Bender and Nadav Ahituv at UCSF (scientific co-founders of Regel Therapeutics) sought to test a targeted epigenetic therapy in patients with SCN2A mutations that exhibit decreased NaV1.2 function. These individuals have impaired action potentials, synaptic transmission and manifest diverse neurological symptoms and seizures, with few therapeutic options, beyond symptomatic anti-seizure medications that have a dizzying range of debilitating side effects.

The UCSF teams leveraged conditional genetic knock-in technolgoy or CRISPRa technology — an AAV-delivered SCN2A-promoter-targeting dCas9 fused to a VP16 activator domain — to upregulate transcription of the SCN2A gene. Using either approach, they were able to boost transcript levels from the healthy SCN2A allele, ameliorating electrophsiological deficits and chemical-induced seizure activity in Scn2a^+/− mouse models. Importantly, these effects were seen in adolescent mice, which conventionally have been thought to be too old to respond to treatment. This suggests that rescue of normal dendritic excitability with epigenetic agents at later stages of life might be capable of restoring neuronal function, with implications for patients.

In a separate set of experiments, the authors showed that their epigenetic approach was able to rescue neurophysiological activity in haploinsufficient neuron-like cells from SCN2A-knockout human embryonic stem cells. This cross-species reproducibility provides further confidence that CRISPRa-mediated upregulation could be translated into human treatments.

​In a second paper in Nature Biotechnology, a team from Epigenic Therapeutics (Shanghai, China) describes the design and validation of optimized epigenetic regulators (EpiRegs) to silence genes in a precise, durable way without altering genomic DNA. Epigen’s Shaoshai Mao and his collaborators at the Chinese Academy of Sciences and the First Affiliated Hospital of Anhui Medical University tested combinations of TALE- and dCas9-based systems, systematically optimizing effector domains and fusion architectures, looking for effective regulators of gene expression. The best-performing variant, EpiReg-T (a TALE-based system, which eliminates the need for a guide RNA), achieved 98% silencing of target genes in mice, substantially outperforming dCas9-based versions.

Using lipid nanoparticles (LNPs) for delivery, a single administration of EpiReg-T in macaques induced long-term repression of the PCSK9 gene, which encodes a validated target for the treatment of hypercholesterolemia. EpiReg-T reduced PCSK9 expression by >90% and LDL-cholesterol by about 60%, with effects persisting for nearly a year (343 days).

Mechanistically, the team used whole-genome bisulfite sequencing and cleavage under targets and tagmentation (CUT&Tag) to show that EpiReg-T induced stable DNA methylation and repressive histone marks at the PCSK9 promoter. The silencing persisted even after liver regeneration and could be reversed by targeted epigenetic activation. Multiomic analysis in mice, macaques and human hepatocytes confirmed high specificity of the manipulation and minimal off-target effects. Overall, these finding, as well as similar results reported in April by Chroma Medicine, establish epigenetic editing as a promising therapeutic platform for durable and reversible gene silencing.

Overall, targeted epigenetic therapies offer clear safety advantages over small molecules that indiscriminately target all genes under the control of an epigenetic eraser or writer enzymes. They avoid the potential risks associated with creating single- or double-strand DNA breaks associated with CRISPR/Cas9 gene, base or prime editing therapies. And they avoid the insertional mutagenesis risks associated with traditional viral gene therapies. What’s more, in applications requiring gene upregulation in haploinsufficient disease, these approaches maintain the endogenous regulatory context of the functional allele. This is in stark contrast to traditional gene-therapy replacement approaches, where overexpression of an introduced therapeutic gene can often lead to toxicities and immunogenecity.

Of course, questions still linger around the persistence of the changes elicited by these epigenetic agents. Will they persist in patients for long periods — for years or even decades? If they can, then epigenetic therapy may offer compliance advantages over small molecules, antibodies, ASOs or even siRNAs, which have treatment durations of six months or less.

Like all genetic medicines, though, delivery remains the key headache. Thus far, AAV vectors, lipid nanoparticles or ribonucleoproteins (RNP) have all been explored to deliver epigenetic therapies (with some evidence that RNPs might have advantages because they can result in higher dCas9 dosages within target cells). For AAV vectors, the fact that targeted epigenetic therapy might only need to be given once might be an advantage in terms of immunogenicity/neutralization concerns against the vector.

A broader point is that the safety profile of targeted epigenetic editors may offer advantages if AAV vectors are used as delivery vehicles: if the epigenetic agents themselves can be delivered at high dosage (given their intrinsic favorable safety profile and presumed maximal tolerated dose), perhaps AAV vector dosages could be lower than current practice. With many current gene therapies requiring dosages of 10¹³ or more viral particles/kg in patients, it is increasingly becoming clear that unacceptable liver toxicities arise from the virus at these levels in clinical studies. It will be interesting to follow this space as more agents enter human testing.

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