PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

Abstract

GEN BiotechnologyVol. 2, No. 2 News FeatureFree AccessPASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and PrimeAlex PhilippidisAlex PhilippidisE-mail Address: aphilippidis@genengnews.comSenior Business Editor, GEN.Contributing Editor, GEN Biotechnology.Search for more papers by this authorPublished Online:18 Apr 2023https://doi.org/10.1089/genbio.2023.29088.aphAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail MIT McGovern Institute researchers Omar Abudayyeh and Jonathan Gootenberg look to expand the universe of diseases treatable via genome editing. Will their patent touch off another legal wrangle for the field?A recently patented genome editing tool called programmable addition via site-specific targeting elements (PASTE) holds genuine promise for expanding the universe of treatable genetic diseases. The approach combines elements of CRISPR and prime editing with a pair of enzymes designed to enable the integration of large segments of DNA without incurring double-stranded DNA breaks.US Patent No. 11,572,556, assigned to MIT, covers systems, methods, and compositions for PASTE. The patent describes site-specific integration of a nucleic acid into a genome, using a CRISPR–Cas9 nickase fused to a reverse transcriptase (RT) and a serine integrase. These enzymes target specific genome sequences known as attachment sites, binding to them before integrating their DNA payload (Fig. 1).FIG. 1. PASTE editing uses a recombinase-prime editor and a pegRNA encoding the attachment site (termed atgRNA) to enable a two-step editing procedure in which attB is inserted first followed by donor DNA integration. (Adapted from Mahmood and Mansoor8). pegRNA, prime editing guide RNA.Invented by Omar Abudayyeh and Jonathan Gootenberg, a pair of McGovern fellows at MIT's McGovern Institute for Brain Research, PASTE can insert DNA fragments as large as 50 kilobases (kb). This places PASTE on a different plane compared with other genome editing tools such as prime editing.Abudayyeh and Gootenberg were both graduate students with CRISPR pioneer Feng Zhang, at the Broad Institute before moving across the road to the McGovern Institute in Kendall Square, Cambridge, MA, to set up their own joint laboratory in 2019. They first detailed PASTE in a preprint posted on bioRxiv in 2021. The peer-reviewed article was published in November 2022 in Nature Biotechnology.1Omar Abudayyeh (top) and Jonathan Gootenberg (bottom) are McGovern fellows at MIT's McGovern Institute for Brain Research.PASTE entails the engineering of Cas9, RT, and integrase linkers to create a fusion protein capable of efficient integration (5–50%) of diverse cargos at precisely defined target locations within the human genome with small stereotyped scars that can serve as protein linkers. The serine integrases used in PASTE can insert large DNA sequences by targeting specific “attachment” sites within the genome.“It has been very difficult for the [CRISPR] field to really put large edits in the genome without relying on things like homology-directed repair,” Gootenberg told me. “The concept of PASTE is actually quite simple: Instead of being able to try to do everything all at once in one conceptual aspect—and there are some technologies that approach that—we thought it would be easier to take something that can insert very efficiently into a constant sequence like an integrase and then put the constant sequence into the genome.”“It's a two-step process, where we first insert the constant sequence, which is a small insert—the site is between 38–46 nucleotides long—and it's easier to do. Then we use that constant sequence to put in a larger sequence. That's the concept in a nutshell,” Gootenberg added.The $64-Million QuestionJacob S. Sherkow, a professor at the University of Illinois College of Law and the Carle Illinois College of Medicine, and an authority on the long-standing CRISPR–Cas9 patent standoff,2 says that PASTE appears to address a key challenge in genome editing: How to achieve high fidelity site-specific insertion of an exogenous target sequence?Citing the three-prong standard for patentability—novelty, utility, and nonobviousness—Sherkow said MIT and the PASTE inventors can likely show their technology is novel and useful. But it is less clear if they can show nonobviousness should the PASTE patent be challenged someday. “That is the $64-million (sic) question,” said Sherkow. “To the extent that there's patent litigation, I assure you that on the PASTE patent, at least some of those arguments are going to turn on non-obviousness.”David Liu, The Broad Institute and Harvard University; Co-Founder of Prime MedicineJacob Sherkow, University of Illinois“You've got the older [David] Liu prime editing patent that claims prime editing. Then you've got [the] PASTE patent filed later, which claims 80% of what the prime editing patent claims, plus the addition of using an interface and an RT, to insert a large piece of DNA in a site-specific site.”“That is not an overlap issue at all, such that the patent office is going to be asked to, or have the authority to, cancel one patent in favor of the other,” Sherkow said. “Where the conflict is going to arise is not on the validity of these two patents. It's going to be on who is going to pay for them and who is going to use which technology over another technology.”Precision genome editing without the need for double-stranded breaks in DNA was first laid out by David Liu, and colleagues at the Broad Institute. In a pair of articles in Nature published between 2015 and 2016, Liu's laboratory developed base editing, a technique that can make certain classes of single-base substitutions without cleaving the double helix. Two former postdocs in Liu's laboratory, Alexis Komor (now on the faculty of University of California, San Diego) and Nicole Gaudelli, (Beam Therapeutics), led the development of base editing, which is being commercially developed by Beam Therapeutics.3Inspired by Liu's work on base editing, Andrew Anzalone conceived and led the development of an RNA-based “search-and-replace” genome editing technology dubbed prime editing, reported in a landmark 2019 article published in Nature.4 Prime editing can introduce targeted insertions, deletions, and all 12 possible nucleotide substitutions. Among roughly 75,000 catalogued pathogenic mutations in human genetic diseases, prime editing has the potential to correct up to 89% of them.Liu cofounded Prime Medicine (see Box 1) to commercialize prime editing based on Anzalone's groundbreaking work. (Anzalone is Prime Medicine's scientific cofounder and head of its prime editing platform; see the interview with Anzalone5 in this issue.)Box 1. Search and ReplaceLast year, Prime Medicine completed an initial public offering that raised ∼$180 million despite the bear market for newly public biotechs. In its prospectus, Prime Medicine revealed an 18-program pipeline and the in-licensing of U.S. Patent No. 11,447,770, which covers methods of using prime editors, was granted on September 20, 2022, and expires in 2040.“We are confident in the strength of our patent portfolio,” Prime Medicine said in a statement. “Broad Institute's in-licensed IP includes an issued U.S. patent broadly covering [prime editing] methods and an allowed U.S. application, which is expected to issue shortly, covering pegRNAs.” Prime Medicine says its patent portfolio includes “numerous in-licensed and Prime Medicine-owned patent applications in the U.S. and worldwide.” Both the Broad Institute and Prime Medicine have filed for patent protection covering technological advancements that will “greatly” expand the scope of prime editing.In addition to offering the full slate of nucleotide substitutions, prime editing can in principle engineer insertions and deletions as well as alter the regulatory regions of genes, insert or create premature stop codons, and modify splicing sequences.Prime Medicine has and will likely continue to in-license prime editing improvements from Liu's laboratory. “We believe these investments, along with our continuing relationship with Dr. Liu, establish Prime Medicine as a clear leader in prime editing,” the company added. “We plan to continue… reinforcing our leadership position and making fundamental progress towards better therapies for patients.”Prime Medicine recently announced the selection of its first development candidate—PM359, a treatment for chronic granulomatous disease, after observing long-term in vivo engraftment of prime-edited hematopoietic stem cells in a mouse model of the disease.“We look forward to advancing PM359 into investigational new drug (IND)-enabling studies later this year, while continuing to advance our broader portfolio toward additional preclinical proof-of-concept readouts,” Keith Gottesdiener, Prime Medicine's President and CEO, said in a statement.In January, Prime Medicine announced positive preclinical data for three of its development programs—candidates to treat Friedrich's ataxia, cystic fibrosis, and Wilson's disease. In addition, Prime Medicine said its PASSIGE platform achieved an ∼60% precise insertion of a 3.5-kb transgene at a single target site in primary human T cells, resulting in positive expression of the gene product.The 2019 article showed prime editing being used to install an integrase/recombinase landing site into a target DNA site. Earlier, in March 2019, Liu and colleagues filed for a U.S. patent that was granted in September 2022 (No. 11,447,770), which described several examples using prime editing to install integrase/recombinase landing sites, followed by targeted gene integration using an integrase/recombinase enzyme. In 2021, Liu's team published the use of “twin” prime editing to install an integrase/recombinase landing site into a target DNA site, followed by the catalytic integration of cargo DNA into that landing site.6 Prime Medicine is pursuing this technology, which it calls PASSIGE™ (prime-assisted site-specific integrase gene editing).PASTE Versus PASSIGELiu explains that one distinction between PASTE and PASSIGE is that PASTE fuses the prime editor and the integrase enzyme into a single molecule, whereas PASSIGE typically uses two separate proteins. Liu's verdict is that “the two proteins must act separately.”“We have compared, side-by-side, fused and unfused prime editors plus integrases at several different target sites in human cells, and we have never observed any benefit from fusing the prime editor and integrase,” says Liu, who is also the director of the Merkin Institute of Transformative Technologies in Healthcare and a Howard Hughes Medical Institute investigator.“Indeed, for most of the target sites and integrase enzyme combinations we've tested in human cells, we observed that the fused prime editor–integrase proteins as reported in the PASTE paper substantially underperform the separated prime editor and integrase proteins as used in PASSIGE,” Liu said. The improved performance of separate prime editor and integrase proteins, rather than fusing the two proteins together, makes sense scientifically, Liu says, “because the prime editor must vacate the target landing site before the integrase enzyme can perform cargo DNA integration.”According to Liu, fusing the two proteins increases the chance that the target landing site is blocked by the prime editing protein and/or associated prime editing guide RNA (pegRNA), “because the integrase must compete with an always-nearby prime editor to access the target landing site when the two proteins are tethered.”In contrast, Liu says, fusing the DNA-nicking Cas protein and the engineered RT together into a prime editor makes sense “because the Cas protein must hold open—or melt—the two DNA strands in order for the nicked target DNA strand to prime reverse transcription, initiating the prime editing process,” Liu added. Fusing the Cas nickase to the engineered RT keeps the engineered RT nearby, “poised to act on the opened and nicked target DNA site.”Light Bulb MomentAsked about the potential for a challenge to the PASTE patent, Gootenberg acknowledged: “It's obviously going to be a complex situation with intersecting IP, as has been with Cas9 and other nucleases from the start. But that said, we're really excited about how we can develop these technologies and move these technologies forward to develop actual cures for patients.”Abudayyeh and Gootenberg are veterans of genome editing, having previously discovered Cas13 in the laboratory of Zhang, and subsequently in their own laboratory, the CRISPR effector protein Cas7–11. They shared a long-standing interest in targeted gene integration. “When we saw the [Anzalone] paper, a light bulb went off in our heads!” Abuddayeh recalled. “We saw these integrases that we were familiar with already. If we could somehow combine these systems together, [we could] basically use them to lay (at) the target site, too.”“When we first started down this path, Cas9 was the only thing people were focusing on for programmable double-stranded cleavage and editing. We asked the question of whether we can go beyond Cas9,” Abudayyeh added.Gootenberg and Abudayyeh have licensed the technology behind PASTE editing to a startup they cofounded, Tome Biosciences, which has reportedly raised more than $95 million from several big-name investors, including a16z, ARCH Venture Partners, Polaris Partners, and Google's GV. (The company is still in stealth mode.)“Were it not for the weak market, I suspect (the PASTE patent) would have propelled CRISPR-related stocks higher. But this also demonstrates that—while markets are weak—innovation in the space is still going strong,” Jeff Brown, founder and chief investment analyst for Brownstone Research wrote last September.Prime editing and PASTE are among numerous technologies that have developed in the rapidly evolving genome editing field as researchers and startups pursue curative therapies that engineer changes to the genome without introducing double-stranded DNA breaks. Last year, Intellia Therapeutics shelled out up to $200 million to buy Rewrite Therapeutics (a University of California, Berkeley startup) and its DNA writing technology.Patentability is among the key challenges for genome editing. For example, Tessera Therapeutics is pursuing a patent for its RNA gene writing technology, which uses a mechanism called target primed reverse transcription to write genes into the genome.7 The method involves four steps—binding RNA, binding DNA, nicking DNA, and priming reverse transcription. The company is among several whose intellectual property has been challenged, according to a recent article in STAT, as “bear[ing] strong similarity to prime editing—even if they use different terminology.”Blanket TherapyPASTE editing could potentially treat diseases caused by genes harboring a large number of mutations, such as Leber's congenital amaurosis or cystic fibrosis, where gene editing systems would have to be tailored for specific mutations and each subset of a disease population. In those diseases, programmable insertion of a wild-type gene could address most potential mutations and serve as a blanket therapy.“Complete replacement of genes at their natural sites can be contemplated instead of creating variant-specific treatments,” Muhammad Arslan Mahmood and Shahid Mansoor, both of Pakistan's National Institute for Biotechnology and Genetic Engineering, observed in a recent commentary published in The CRISPR Journal.8“These exciting results enhance the versatility of the CRISPR-based gene editing along with the LSRs [large serine recombinases] that provide the opportunity for genome engineering, combinatory screening of bulky DNA libraries aiding to spread these applications for treating the diseases in both animals and plants, which are caused by defective genes,” the authors concluded.The Cystic Fibrosis Foundation is among the entities that have funded PASTE editing research, through a pioneer award designed to fund “ambitious basic research projects aiming to utilize cutting-edge techniques and strategies that have the potential to discover new genetic-based therapies for cystic fibrosis.”“We've been working on actually being able to insert the genes at the locus, and things are looking promising… That's one of the main funded applications we're looking at right now,” Gootenberg said.On a RollFeng Zhang's entrepreneurial spirit appears to have rubbed off on his two proteges. In addition to Tome, Gootenberg and Abudayyeh have also cofounded Sherlock Biosciences, which recently gained rights to a U.S. patent for diagnostic use of a CRISPR system based on Cas12; Proof Diagnostics, a CRISPR-based COVID-19 molecular test developer; and Moment Biosciences, a precision microbiome therapy developer.In the 2022 PASTE study, Abudayyeh, Gootenberg, and colleagues described delivery of genes ranging from 779 to 36,000 base pairs—a range that would enable insertion of >99.7% of human cDNAs as transgenes—to a variety of human cell types. Tests with more than a dozen payload genes revealed insertion success rates ranging from 5% to 60%, with “minimal” formation of indels at nine different integration sites.The researchers also reported inserting genes in “humanized” livers in mice, consisting of ∼70% human hepatocytes, with PASTE showing a much lower percentage (as high as 2.5%) of successfully integrating new genes—with average insertion ranging from 0.3% to 1.4%.Can PASTE transport genes larger than 36 kb? “We've not done larger, but there's no reason I think why you couldn't do larger,” Abudayyeh said. “It becomes a delivery problem: How do we get [50-kb] templates into a human cell? The reason we're able to max out at approximately 36 kb is because we're using an adenoviral construct. Adenoviruses are approximately a 36-kb genome, so it's easy to get that into the cell.”Abudayyeh said they have been approached by researchers interested in testing inserts of 50–100 kb. “We just haven't gone down that path,” he said. On its minimalist website, however, Tome Biosciences suggests it can handle DNA segments of that size range: “Using CRISPR, our technologies allow us to insert any genetic sequence of any size at any location into any genome.”Beyond Patent ValidityAlthough users of prime editing do not likely need a license covering PASTE, the converse might not be true. Sherkow states: “By contrast, if you are using the PASTE technology, there is, I think, an outstanding question as to whether you will also need to obtain a prime patent license.”How companies answer that question may well depend on how they plan to use both PASTE and prime editing technology. “This is more just, frankly, garden-variety technology, improvement licensing stuff that happens in a variety of other fields,” Sherkow said. “It is not this cataclysmic dispute with respect to who got their first and validity, the way that we had in the CRISPR 1.0 context.”Sherkow has written extensively on the twists and turns in the long-running CRISPR–Cas9 patent saga. In that case, he says, “in some conceptual sense either the [University of California] patents are valid and the Broad Institute's are not, or vice versa. That's just not necessarily true here between Harvard with prime editing and MIT with PASTE,” Sherkow said. “One could be valid, one could be not. One could be infringed, one could be not.”“It's going to come down to whether a company that wants to develop a product in this area is going to be counseled to take a license to one or the other, or both. I think that's going to be a function of what they think the best fit for their technology is,” Sherkow added.As a result, he believes researchers will be inclined to use prime editing or PASTE—and thus pursue licenses for one or the other—based on the diseases they are working to treat. “Unlike the CRISPR 1.0 situation, where who are we going to take a license to turns on, who do we think is going to win the patent fight, taking a license for prime [editing] or PASTE turns on what is our actual technology, which to be blunt is how we want the licensing system to work,” Sherkow said.“We don't want the licensing system to work where people are placing horse bets on winners,” says Sherkow, “while others are just losing their shirts.”A version of this story was originally published online by GEN.