BALTIMORE—It is a sign of the impressive growth of the gene therapy field in general—and the American Society of Gene and Cell Therapy (ASGCT) annual conference in particular—that the first sight upon entering the Baltimore Convention Center was Brad Loncar, the founder of Biotech TV. Loncar was setting up his camera and tripod for a series of televised interviews this week with various gene therapy luminaries and executives. It is Loncar’s first visit to the ASGCT conference, and unlikely to be his last.
This year’s ASGCT conference began with a series of workshops that set the stage for a busy week of scientific presentations and networking. One of the morning workshops, co-organized by Julian Grunewald, MD, assistant professor at Technical University of Munich, and Holly Rees, PhD, head of lead discovery at Beam Therapeutics, focused on the “Delivery and Development of Precision Genome Editing Technologies.”
Topics ranged from novel delivery strategies such as GMP-grade lipid nanoparticles (LNPs), engineered virus-like particles, pipeline updates, and computational approaches for designing novel DNA-binding proteins.
Persistence pays
Y. Esther Tak, PhD, Arena Bioworks, discussed work conducted until recently at Mass General Hospital in Boston as a member of the Keith Joung lab, on next-generation epigenetic editing. CRISPR-based methods for activating or inhibiting gene expression have been developed for the past decade, but one of the drawbacks of these approaches is that gene activation efficiency wanes over time due to dilution effects. “There is still a need for strong and robust activation methods,” Tak said. “No general mechanism has been identified in mammalian cells to induce de novo stable gene activation.”
PERSIST-On provides a strategy for inducing stable gene activation using a hit-and-run approach, with the degree of activation variable using different TF motifs. Tak said the method can be adapted to induce cell-type-specific target gene activation. And it “provides a feasible therapeutic modality for diseases where long-term activation is desirable” such as haploinsufficiency diseases or genomic imprinting disorders.
Samagya Banskota, PhD, Boston University, delivered an excellent talk on the rapidly emerging field of engineered virus-like particles (eVLPs) for in vivo delivery—work that began in the lab of David R. Liu (Broad Institute).
Traditional viral vectors have proven very successful for gene therapy with sustained gene expression and the ability to target specific tissues. But sustained expression is not so desirable in the context of gene editing because of potential off-target or oncogenic risks. LNPs are short-lived, but have shown limited target scope beyond the liver in vivo. An ideal delivery vehicle for in vivo gene editing would combine the merits of both systems. eVLPs resemble viruses although they lack viral genetic material. Banskota said advantages include high transduction efficiency, tissue tropism (by modifying surface glycoproteins), and short-lived exposure.
Rearranging the genomic architecture of the nuclear localization sequence helped the system to assemble particles in the cytoplasm of producer cells but still access the nucleus in recipient cells. “Version 4” of the eVLP significantly improved delivery of base editors and Cas9 and showed almost undetectable off-targeting at three tested gene sites. “Despite being a new modality, we’re seeing comparable results to AAV delivery,” she said. While on-target efficiency is comparable, it is the improved off-target editing numbers that are most striking.
Speaking of the Liu Lab, where prime editing was developed five years ago, Jonathan Levy, PhD, Prime Medicine, offered an update on the company’s progress, which includes the recent news of the FDA’s approval of an IND application for its lead program in chronic granulomatous disease (CGD). Since the foundational prime editing paper in 2019, the toolbox has expanded to include dual-flap prime editors (PEs) that can engineer larger insertions and deletions, and PASSIGE, a system for the integration of large genetic payloads by utilizing a recombinase target sequence.
The company’s clinical targets include diseases of the eye, liver, lung, blood, and muscle, using all established delivery vehicles—electroporation, LNPs, and adeno-associated viruses (AAVs). Levy said the company is intent on developing novel LNPs, searching a proprietary “prime lipid” library of more than 800 lipids. “We’re very excited about the potential of novel LNPs for hepatic and non-hepatic delivery,” Levy said. The goal is to develop a “universal” LNP that reduces biodistribution to secondary organs and can be used across multiple indications.
Moving on
The session closed with two more compelling presentations from postdocs who are both about to establish their own independent laboratories.
Makoto Saito, PhD, Broad Institute, discussed groundbreaking work in the lab of Feng Zhang, PhD, on fanzors. CRISPR is only found in prokaryotes, but Saito and colleagues elected to search for related “sister systems” in eukaryotes. As Saito and colleagues published last year, fanzor is a eukaryotic homolog of TnpB with a similar 3D structure that can function as a programmable RNA-guided gene editor.
The Zhang lab has focused on extracts from Spizellomyces punctatus (Spu), a fungus that is easy to grow and extract RNA. The genome contains 19 copies of SpuFz1 (Fanzor 1). This fanzor is attractive for therapeutics because it is roughly half the size of Cas9. However, it also has a shorter (15-nucleotide) target sequence, which makes off-target effects more likely. Saito and colleagues have engineered a triple mutant that has greater activity and are pursuing other tactics to increase the utility of fanzor.
As a result of this work, Saito said we now know that “all three domains of life are equipped with programmable RNA-guide gene editing systems.” Saito will continue this work next year in his lab at the RIKEN Institute in Japan.
Building on the engineering theme, Cameron Glasscock, PhD, University of Washington, in the lab of David Baker, PhD, discussed the application of novel computational design tools to develop novel DNA-binding proteins. Scientists have designed novel protein-binding proteins, small-molecule binding proteins, fold-shifting proteins, assemblies, and novel enzymes, but protein-nucleic acid interfaces have proven problematic.
Using a suite of tools, Glasscock presented workflows and tools including Rosetta Design software coupled with deep learning approaches, producing a tool called LIgandMPNN for sequence design. The result is the creation of several novel DNA-binding proteins that are quite different from those found in nature.
Glasscock is also launching his own lab in 2025, although he declined to say where.
