Monday, September 26, 2022

Chroma Medicine and Tune Therapeutics: Two companies take up epigenome editing

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Since the 1990s, attempts to manipulate the epigenome to fight diseases like cancer have relied on DNA-modifying drugs such as azacitidine. These drugs act more like therapeutic sledgehammers than precision medicines. Now, with decades of research on DNA-binding domains and a burgeoning number of enzymatic effectors that can write or erase epigenetic marks, precision epigenome editing is coming into view. These advances have not been lost on investors, who in 2021 poured $165 million into two epigenome-editing companies: Tune Therapeutics and Chroma Medicine. Earlier this year, a third firm, Epic Bio, spun out of Stanford University, with a $55 million A round.

It doesn’t hurt that the founders of Tune and Chroma are part of a veritable ‘who’s who’ of gene regulation, many of whom pioneered the design and refinement of molecular tools for targeting, binding and modifying DNA and the histone proteins that control access to it. Tune’s co-founders, Fyodor Urnov and Charles Gersbach, together have many decades of experience in the field, with the latter’s 2015 paper showing transcriptional activation of four endogenous genes after targeting histone acetylation of the promoters.

Fydor Urnov, (Innovative Genomics Institute) co-founder Tune Therapeutics.Credit: D.L. Anderson

Chroma Medicine also was founded by a group of scientific pioneers: Jonathan Weissman, David Liu, Luke Gilbert, Keith Joung, Luigi Naldini and Angelo Lombardo. In 2016, Naldini and Lombardo published groundbreaking work in Cell showing they could heritably and specifically silence genes by DNA methylation.

Both companies are working toward the same end. As Gersbach explains, “Our goal is fundamentally to have complete control of gene expression — to turn things on or off, for as much as we want, for as long as we want, in the cell types that we want.” Urnov also points to a large body of foundational work. An analysis of 150,000 human genomes in the UK Biobank by Kári Stefánsson, published earlier this year in Nature, shows conclusively what had been gleaned a decade earlier during the ENCODE (Encyclopedia of DNA Elements) project: that the vast majority (89%) of disease-causing variants occur in non-coding regulatory elements of the genome. Says Urnov, “Mother Nature wants to protect them because they matter. It means that when we get sick, these are diseases of gene control, not what the genes themselves say.”

Charles Gersbach (Duke University) co-founder Tune Therapeutics.Credit: D.L. Anderson

To create therapeutics that can act at specific genetic loci, both companies couple DNA-binding proteins — a ‘dead’ Cas9 (dCas9) protein with RuvC and HNH endonuclease domain mutations or a zinc finger — to different enzymatic effectors, including transcriptional activators (for example, VP16), transcriptional repressors (for example, KRAB), epigenetic ‘writer’ enzymes (for example, DNA methyltransferases or histone acetyltransferase) or epigenetic ‘eraser’ enzymes (for example, DNA demethylase, histone demethylase or histone deacetylase).

Tune co-founder Gersbach has been creating large numbers of CRISPR–dCas9-based editors to screen for editable sites in the genome. “Because it’s so easy to design thousands, hundreds of thousands, or millions of guide RNAs and to get them synthesized quickly, we can use them to target epigenome editors to over a million sites in the genome in a single sample and then measure the outcome on the cells for every epigenetic perturbation at every one of those thousands or hundreds of thousands of sites,” says Gersbach.

In recent years, the Gersbach group has been building epigenome editing screens at an ever-increasing scale. A 2016 Nature Biotechnology paper used dCas9KRAB repressor and dCas9p300 activator constructs to screen>10,000 gRNAs for activity against a several-megabase region around two genes (HBB, encoding β-globin, and HER2) in human cells to find genomic control regions. In a subsequent paper, the same team used dCas9VP64 and a library of>8,000 gRNAs targeting ~1,500 genes encoding human transcription factors to screen for loci that drive cell differentiation and maturation. In 2020, they showed that the same epigenetic effector targeted to the gene encoding transcription factor PAX7, which controls skeletal muscle cell differentiation, could drive human induced pluripotent stem cells (iPSCs) to mature into myogenic precursors. Gene activation lasted longer than when PAX7 was overexpressed directly as a transgene, corresponding with histone marks characteristic of expression including H3K4me3 and H3K27ac, which were absent in PAX7-overexpressing cells. In a recent preprint, they describe a screen using the dCas9KRAB transcriptional repressor and>1.1 million gRNAs to screen>100,000 regions of open chromatin in K562 human lymphoblast cells to identify regulatory elements involved in gene regulation and cell fitness.

Tune’s CEO Matt Kane has been in the gene-editing field for some time, having founded and run Precision BioSciences. He took the post at Tune because he believes epigenome editing is now sufficiently mature to make commercial therapeutic development feasible. “We understand diseases better, and how to deliver the actual editors better than we would have, even just a couple of years ago,” he says. “We know where all the obstacles are so we can see where the near-term opportunities might be, as well as laying some of the groundwork now for some of the longer term ambitions.” Urnov also believes decades of work in the gene-editing field will stand epigenome-editing in good stead. “The vision of human genetic engineering as disease therapy is 50 years old exactly this year. The only reason, for example, that the recent gene-editing approaches for sickle cell anemia have gone so well [is] because they are standing on the shoulders of 20 years of gene therapy; and gene therapy in turn is standing on 30 years of bone marrow transplantation,” he says.

Tune’s headquarters in Durham, North Carolina, has recruited many employees from the Gersbach lab at Duke University. The company also has a second site in Seattle. Gersbach explains, “We got paired up with some pioneers in [the] cell and gene therapy field out there. There were introductions made to us from some of our investors. It’s one of our strengths that we are in an area that is less crowded with cell and gene therapy companies than other parts of the country.”

“We have an initial portfolio of programs, and the good news is that it is working really well. The bad news is that it starts getting more expensive,” Kane adds. Gersbach says, “We want to prioritize areas where some of the other technical risks are minimized.”

Chroma’s founders have created their own set of epigenomic editors, which they call ‘CRISPRoff’ and ‘CRISPRon’ editors. The technology was showcased in a 2021 Cell paper from the labs of co-founders Luke Gilbert at the University of California, San Francisco (UCSF) and Jonathan Weissman at the Whitehead Institute. According to Weissman, “We first saw the Lombardo paper as the starting point. Once it showed it was possible at least for a handful of genes, [we asked] could we make a robust system that we could program to any gene or least a majority of genes; then what would be the rules for silencing and how strict would it be?” The 2016 Cell paper by Lombardo and Naldini used a cocktail of CRISPR–dCas9 tethered to KRAB and/or the DNA methyltransferases DNMT3A or DNMT3L to silence several highly expressed genes in K562 cells.

Jonathan Weissman, the Whitehead Institute co-founder Chroma Medicine.Credit: Gretchen Ertl, Whitehead Institute

Building on this system, Gilbert and Weissman’s groups designed epigenome editors for>20,000 human genes, tested in several cell types (iPSCs, HeLa cells, U2OS human epithelial cells or K562 cells). These studies have shown that, in some cell lines, silencing is heritable over>450 cell generations and reversible with CRISPRon (which substitutes a demethylase for a methylase). “They took a lot of the foundational work done by Luigi [Naldini] and Angelo [Lombardo] and patched it into a single construct,” says Vic Myer, Chroma’s president and CSO.

Last year, Naldini and Lombardo joined the Chroma team when their company, Milan-based Epsilen Bio, merged with the US company. According to Chroma CEO Catherine Stehman-Breen, the buyout let Chroma leverage Epsilen’s experience — they had a year or so head start compared with the US startup. “Also, they had experiments already set up and running, so it gave us a running start. They also brought with them some early [intellectual property], which we also felt was important for the company.”

Catherine Stehman-Breen, CEO Chroma Medicine.Credit: WAITING

Co-founder Lombardo, continues to do groundbreaking work at the San Raffaele Telethon Institute for Gene Therapy in Milan, presenting results of in vivo epigenome editing in mice at this year’s American Society for Gene and Cell Therapy meeting held in May in Washington, DC. With a single administration of engineered transcriptional repressors programmed to target the PCSK9 locus, they were able to lower circulating levels of PCSK9 by 50% as compared with controls (mock edited) for over 200 days after administration, with a significant reduction in low-density lipoproteins as measured at day 30.

David Segal, a longtime gene-editing researcher at University of California, Davis, says that one constraint on epigenome editing (and indeed for gene therapy and gene editing) is the poor efficiency of in vivo delivery: it simply has been hard to fit epigenome effector proteins into company delivery vectors like adeno-associated virus (AAV). “This has been a major drawback and has led to a lot of innovation in the field,” he says. Lipid nanoparticles (LNPs), which don’t have packaging constraints, are being used already in clinical trials, but they are only efficient in targeting the liver. “So if you’re going after liver disease, it’s great. But if you want to go after anything else, it’s hard to do that with LNPs, ” says Segal.

Notably, the in vivo experiment by Lombardo used a commercially available LNP to target the liver, according to Myer.

Another issue, Segal says, “is any consensus on what kind of epigenetic editing would be required for a persistent, durable editing to increase gene expression. We know more about how to silence genes and keep them durably silent.” Whether a cell will tend to reverse the pattern being forced on it remains a nagging question. Weissman acknowledges that there have been fewer demonstrations of activating genes, but he says a priori, there’s no reason why it shouldn’t work. Besides, “at the moment we have plenty of targets that are interesting that we know we can get silencing, though there might be a subset that is recalcitrant to silencing,” he says.

Like Tune’s CEO Kane, Chroma’s Myer was previously involved with genome editing, being part of Editas Medicine’s team. “I thought I would never go back to genome editing,” he says. “Then this technology came along — and this is really game changing. What differentiates epigenome editing from gene editing is that epigenomic editors don’t break DNA, and don’t mutate irreversibly. It allows for turning things on to the right level or turning things down and potentially silencing completely,” he says. With gene activation, it is possible to modulate in a way that genes are still responsive to environmental signals — those are things that are uniquely possible with epigenome editing. “This is essentially the normal way cells regulate gene expression,” says Myer. Stehman-Breen agrees: “What I really loved was that … we are taking a highly evolved, highly conserved endogenous mechanism and leveraging it,” she says. Myer is confident that the company can surmount any remaining hurdles. “There have been no ‘Oh no’ moments yet,” he says.

Laura DeFrancesco, Pasadena, CA, USA

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