By Hui-Chia Yu-Kemp and Drew Kelso, Technology Development Scientists, Life Edit Therapeutics, an ElevateBio company
Life Edit is leveraging a proprietary microbe collection and extensive metagenomic datasets to discover and develop unique gene editing systems for an innovative, next-generation gene editing platform. Our large and diverse collection of proprietary RNA-guided nucleases (RGNs) and base editors provide flexible editing options and further our mission of curing disease by making any edit, anywhere.
In a prior article, Life Edit presented a primer on gene editing technologies, including CRISPR-Cas9, and their potential for treating many human diseases. Here, we’ll dive deeper into the cool technologies we’re developing at Life Edit, what differentiates it from other higher-profile gene editing technologies, and the distinctive company culture that has grown up around our science.
Our technology includes an array of proprietary gene editing systems that have a number of desirable properties.
First, our nucleases are small, which allows for versatility when packaging our systems for therapeutic delivery. For example, our lead systems, along with one or two guide RNAs (gRNAs) and all necessary regulatory elements, fit into single adeno-associated viral (AAV) vectors. Our systems are also amenable to other modes of delivery, like lipid nanoparticles (LNPs), which are less affected by size restrictions.
Second, our nucleases have diverse and complementary protospacer adjacent motif (PAM) requirements that increase the genomic loci we can edit. This matters because the commonly used SpyCas9’s PAM is NGG (any nucleotide followed by two guanines). And, while this PAM requirement does enable editing with Cas9 at many genomic locations, not every region in the genome is located near an NGG and accessible.
One way to illustrate the importance of these features is to consider the genome as a book. In that context, this PAM requirement means you can edit any place in the book that is located near one particular word, e.g., “and,” but nowhere else. While many sites in the book meet this requirement, it’s possible the place you want to edit is not close to “and.” Critically, many loci in the genome that are known to be associated with debilitating genetic diseases and disorders are not near the NGG PAM required for Cas9 editing. Thus, there’s a need for new tools that can unlock editing at these important locations in the genome.
Life Edit is building a collection of nucleases with A-, C-, G- or T-rich PAMs that will allow us to edit across a wide range of genomic loci. Sticking with the above book analogy, rather than being locked to edits near “and,” we have tools that can edit near a number of small words throughout the book, including “and,” “in,” “of,” “the,” “to” and so on. This will open every locus in the genome to editing, because virtually every place in the “book” we want to edit will be located near a small word that one or more of our nucleases can work with.
Moreover, the PAM diversity of our RGNs is of particular importance when utilizing precise editing tools, such as our proprietary collection of base editors. With this type of editing, pioneered by David Liu’s lab, a specialized enzyme called a deaminase is fused to an RGN in which the nuclease (scissor) activity is limited to cutting only one strand of DNA while allowing the deaminase to modify the uncut strand. The deaminase removes an amine group on specific DNA bases for which there are two classes of base editors: cytosine (C) base editors and adenine (A) base editors. Once the amine group is removed from C or A by the respective deaminase, the natural repair machinery of the cell facilitates the change of the deaminated base to a new base: cytosine base editors change C to G, and adenine base editors change A to T. If we go back to the analogy of editing a book, then base editors allow us to change specific letters in words, such as changing “and” to “any.” Important to the base editing system, the fused deaminase can only function within a narrow window of bases away from the PAM, and because of that limited mobility, having RGNs with PAM-diversity is incredibly advantageous. Also advantageous is the ability to engineer our deaminases and PAM-diverse RGNs separately to reduce the off-targeting propensity of each, then “mix and match” them to produce base editors with the best possible selectivity for the target loci. These features contribute to the strength and differentiation of our platform at Life Edit and drew the attention of partners like Moderna and Novo Nordisk.
Third, our systems have high fidelity and have shown little to no off-target editing to date.
We apply our scientific know-how to engineer these proprietary gene editing systems in various ways to optimize their editing efficiency and application as gene editing therapeutics. One improvement involves shortening the gRNA while retaining editing efficiency in order to facilitate GMP manufacturing.
Additional improvements include modifying the gRNA’s nucleotide sequence to increase its stability and making chemical modifications to the gRNA that further increase its stability and prevent it from getting digested by ribonucleases (RNases).
All of the foregoing translates into a versatile gene editing platform. We have many options to offer partners, based on the region of the genome they want to edit, the edit (or even multiple edits) they want to make, and the delivery system they want to use.
The versatility Life Edit offers is also important because there are still some things that aren’t fully understood by the gene editing field. For example, two genomic loci with identical PAMs may be edited by the same nuclease with differing efficiencies; editing efficiencies can also be very different when one genomic locus has two PAMs very close to each other. It is unknown why this happens, but we are interrogating different theories we have.
We can help partners navigate this obstacle because we have optimized the efficiencies of other nucleases with a variety of PAMs. If a partner isn’t getting good editing efficiency at the site of interest with SpyCas9, which requires an NGG PAM, we could offer them a system that uses another nuclease with a different PAM that edits the same gene with better efficiency.
Furthermore, we have demonstrated the multiplexing capability of our base editors – that is, their ability to edit multiple genes in one cell. As an example, using cell-based analysis, we have demonstrated that our base editing systems are able to edit three therapeutically relevant genes at once, with efficiencies comparable to those achieved when editing the same three genes individually.
These options could influence how a client thinks about making a new medicine and even change its approach to solving a therapeutic problem. Life Edit’s versatility allows partners to step outside the technological limits of Cas9, escaping its “mental box” and imagining new possibilities.
Life Edit has a breadth of science and technologies that we can apply to each new system we investigate. We learn and combine different kinds of engineering approaches available in the field, including shortening gRNAs, swapping nucleotides in or out of them, and stabilizing them with chemical modifications. The engineering capabilities we have built set us apart from other companies that have had to carve out different, and more narrowly focused, gene editing approaches for IP purposes. It makes us unique among our peers in the gene therapy field. We have also built out – and will continue to expand – our capabilities in computational biology and high throughput robotics that enable us to engineer proteins, solve structures, and generate more experimental data faster.
Unlike most in the CRISPR field who are focused on optimizing the Cas9 system, we are developing platforms and techniques that can be used across systems. Each system we find is completely new, just like Cas9 was at one time; we get to figure out how it works, how to optimize it, and how to apply it to new technologies, such as base editing. We need a broad toolbelt to do all of this.
There are generally no universal rules; each system we find is unique and may have slightly or significantly differentiated properties as compared to previously known systems. We have to think of each system distinctly and become experts in it. That is a big part of why we think our science is so cool and intriguing, and that’s what brought us both to the company.
Whether it’s the science influencing the company culture or vice versa, Life Edit’s culture has a distinct “coolness” that differentiates the company and drives us. We work hard to protect and maintain that culture, even with our exponential growth, and we’re eager to share it with each new employee who joins the company.
We and other scientists at Life Edit have presented our research and technologies at national and international conferences. These presentations have highlighted the discovery of new nucleases and identification of their respective PAMs; the ability of these new systems to make knock-in and knock-out edits, including multiplex edits; and the high editing efficiencies achieved by chemically modifying the gRNAs. Furthermore, we’ve also presented exciting preclinical data demonstrating that our gene editing systems can enable allele-specific editing of the mutant huntingtin (mutHTT) gene in mouse disease models, leading to clinically meaningful reductions in striatal mutHTT protein.
Life Edit continues to develop the systems we’ve already found and to explore new gene editing systems from our proprietary collection of microbes. Future technological expansions could include new systems for epigenomic editing, other classes of nucleases, and base pair-specific editing. We’re pioneering discoveries in all of these areas and more, and we’re connecting our discoveries to the development of real and curative gene therapies for patients.
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