While the CRISPR community celebrates the recent first FDA approval of a CRISPR-Cas9 therapy (Casgevy), new entities are making strides in their efforts to write perhaps the definitive new chapter in programmable genome editing.
Last week, Tome Biosciences—a developer of genome editing treatments based on an improved version of programmable addition via site-specific targeting elements (PASTE) technology—emerged from stealth mode with a very tidy $213 million in financing and plans to create curative cell and “integrative” gene therapies capable of correcting genes in vivo.
Co-founded by PASTE developers Omar Abudayyeh, PhD, and Jonathan Gootenberg, PhD, former graduate students with Feng Zhang, PhD (Broad Institute), Tome says its programmable genomic integration (PGI) platform is designed to enable the insertion of any DNA sequence of any size into any programmed genomic location. PGI encompasses a series of genome editing approaches, each representing enhancements over PASTE.
“Programmable genomic integration is this idea of leapfrogging to hundreds, if not thousands, if not tens of thousands of base pairs inserted or changed at a time,” Rahul Kakkar, MD, Tome’s president and CEO, told GEN Edge. “There is an entire generation of therapies we can create if we stay true to and focus on that concept of reprogramming blocks of code in the human software in the human genome.”
Tome’s initial plans call for developing integrative gene therapies for monogenic liver diseases and cell therapies for autoimmune diseases. The company plans to disclose more details in 2024 about the clinical indications for its first therapies, progress and potential partnerships.
“We wouldn’t be true to our commitment to medicine and to patients if we were not discussing partnerships. Those conversations continue,” Kakkar said. Tome’s PGI technologies can “absolutely” be used to treat other disorders.
“As I think about conversations I’ve had with patients about the medicines that I am recommending for a certain condition, the patients don’t generally care what the mechanism of action is. What they care about is, ‘How risky is it? And how much benefit am I going to get?’” Kakkar said.
“As we sat back and looked at PGI, we recognize that this technology has utility, not just for integrative gene therapies as the maturation of this ability to correct natural defects in genes. Our ability to flexibly recode and reprogram DNA like software allows us to create very advanced cell therapies,” Kakkar added.
“We recognize a profound opportunity. If you look at our two verticals—an integrative gene therapy vertical and a cell therapy vertical—nobody has a pipeline as diversified as ours. I think that derives fundamentally from how revolutionary this technology is.”
Tome says its most advanced form of PGI, integrase-mediated PGI (I-PGI), uses proprietary integrases and has shown its ability to insert more than 30 kilobases (kb) of DNA with site-specificity in various dividing and non-dividing cell types. Other PGI technologies use different enzymes, which Tome says are company-invented, evolved and proprietary versions of enzymes available in public domain.
“Some of it is improvements and inventions on some of the structural components like guide RNAs, for instance, that we’ve brought to bear,” Kakkar said. “The best way, in general, to describe our improvements are: they’re all driving towards integrative efficiency. The more alleles and the more cells you can edit in a given target organ, the more clinical impact you can have.”
PGI-based technology could also have other applications outside the clinic, although Kakkar emphasized that the core of the company’s business was developing human treatments. “We have been open to discussions with potential partners to explore the utility in for research use and synthetic biology. But the stage of those discussions currently is still largely strategic rather than operational.”
Based in Watertown, MA, Tome has quickly recruited almost 150 employees. “We have built all of the core capabilities we need between R&D and CMC [chemistry, manufacturing, and controls], and then a very lean kind of G&A [general and administrative] support function,” Kakkar said.
“There will be some growth, particularly as we think about building more manufacturing capability, and eventually, clinical operational capability. I think where we are today is in a very, very comfortable, lean, but comfortable place to execute in the preclinical arena, where we’re currently set.”
Tome was founded in 2021 by Abudayyeh and Gootenberg during their tenure as McGovern fellows at MIT’s McGovern Institute for Brain Research; the two discussed PASTE technology with GEN Edge earlier this year. The duo has since spread their wings south of the Charles River—Abudayyeh is now a lead investigator at Brigham and Women’s Hospital, while Gootenberg holds the same position at Beth Israel Deaconess Medical Center, both affiliated with Harvard Medical School.
PASTE entailed 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.
In their study detailing PASTE, published last year in Nature Biotechnology, Abudayyeh, Gootenberg, and more than two dozen co-authors detailed how they delivered genes ranging from 779 up to 36,000 base pairs to three human cell lines, primary human T cells, and liver cells, as well as to liver cells in mice: “We married advances in programmable CRISPR-based gene editing, such as prime editing, with precise site-specific integrases,” the co-authors wrote.
Speaking with GEN last year, Gootenberg said: “It’s the final frontier for therapeutics but also cell engineering and basic biology because we can craft the genome any way we want.
For the last two years, Kakkar said, Tome has been “improving, inventing, and growing beyond the initial embodiment” of PASTE.
Moving PGI beyond the initial PASTE platform has not included increasing the size of the DNA inserts. “Although there is certainly very interesting territory to potentially explore beyond the 30,000 base pair range, we just haven’t seen as yet the clinical need to do so. So we have not explored that,” Kakkar said.
“Although, broadly speaking, architecturally, PGI is very similar to PASTE, there are some differences that moved away from that initial invention and form the basis of much of our IP [intellectual property],” he added.
That IP, Kakkar continued, is more expansive than the three initial patents assigned to MIT for the core PASTE technology invented by Abudayyeh and Gootenberg—Nos. 11,572,556; 11,827,881; and 11,834,658, all titled “Systems, methods, and compositions for site-specific genetic engineering using PASTE.”
Tome has licensed from MIT all three core PASTE patents:
- U.S. Patent No. 11,572,556, published February 7, 2023, covers systems, methods, and compositions for PASTE—including methods of integrating an exogenous sequence into mammalian cell genome using a fusion nickase-RT protein, a guide RNA, an integrase and the exogenous sequence. The claims are limited to ex vivo methods and all editing components must be introduced into the cell concurrently.
- U.S. Patent No. 11,827,881, published November 28, is directed to systems for integrating an exogenous sequence in a mammalian cell genome, wherein the system comprises above elements (1 to 4) in a single composition.
- U.S. Patent No. 11,834,658, published December 5, is highly similar to the ‘556 patent except that it requires at least two guide RNAs, and limits its claims to ex vivo methods and concurrent introduction.
To license or not?
Speaking with GEN Edge earlier this year, Jacob S. Sherkow, a professor at the University of Illinois College of Law and The Carle Illinois College of Medicine, commented that while users of prime editing do not likely need a license covering PASTE, “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 [editing] patent license.”
How companies answer that question will depend on how they use both PASTE and prime technology, added Sherkow, an authority on the long-standing CRISPR-Cas9 patent standoff.
Tome insists it does not need to license prime editing.
“I think there is clinical room for CRISPR-based technologies, base editing, prime editing and PGI. We all serve different clinical indications, different patient populations, and I wish all of those companies well. I will not comment on their IP directly,” Kakkar said. “We continue to believe we sit on very firm IP grounds. And, at this point in time, we have not sought, nor do we intend to seek licensure from any third party.”
The original PASTE combined 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.
Carrying out genome editing without the need for double-stranded breaks in DNA was first laid out by David Liu, PhD, and colleagues at the Broad Institute. In a pair of landmark papers in Nature published between 2015–16, Liu’s lab developed base editing, work driven by two former postdocs, Alexis Komor, PhD (now of University of California, San Diego) and Nicole Gaudelli, PhD (now of Beam Therapeutics) (Hear this episode of The CRISPR Journal’s GuidePost podcast, available via SoundCloud and Spotify).
Later, in a 2019 paper published in Nature, Andrew Anzalone, MD, PhD, and colleagues in Liu’s lab described prime editing, which can introduce targeted insertions, deletions, and all 12 possible base-to-base substitutions. Liu told GEN at the time that of the roughly 75,000 cataloged pathogenic mutations in human genetic diseases, prime editing had the versatility and potential to correct the majority (89%) of them.
Liu co-founded Prime Medicine to commercialize prime editing based on Anzalone’s groundbreaking work when he was a postdoctoral fellow. Anzalone discussed the technology and the company on GEN’s “Close to the Edge” video interview series. (Anzalone is Prime’s lead developer of prime editing, and the company’s scientific co-founder.) Liu and Prime Medicine (which went public last year) have said they anticipate prime editing joining base editing in human clinical trials in less than a year.
Prime’s foundational patents
The third and most recently issued foundational U.S. patent covering prime editing methods and compositions—No. 11,795,452, dated October 24—includes issued claims to prime editing guide RNAs (pegRNAs) encoding recombinase landing site sequences, and to recombinases that recognize these sequences. That patent follows U.S. Patent Nos. 11,643,652, dated May 9, and 11,447,770, dated September 20, 2022, both of which cover compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis, All three patents have been assigned to the Broad Institute and Harvard College.
“We recognized the usefulness of using prime editing to install a recombinase landing site into a target site in genomic DNA in 2018 while we were developing prime editing. This is why our original 2019 prime editing paper and U.S. patents included examples of installing site-specific recombinase landing sites into genomic DNA in human cells using prime editing,” Liu told GEN Edge.
It is also, Liu said, why a 2021 paper published by his lab showed that prime editing and Bxb1 could be combined to install cargo genes into specified locations of the genome in human cells. A more recent paper published August 31 showed that prime editing could efficiently install recombinase landing sites into targeted genomic sites in vivo in animals.
“Across many months of experiments, we have consistently observed PASTE yielding lower integration efficiencies than simple untethered prime editing + recombinase systems,” said Liu, the director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad and a Howard Hughes Medical Institute investigator.
One possible reason: PASTE fuses the prime editor and the integrase enzyme into a single protein chain, while Prime’s PASSIGE™ (prime-assisted site-specific integrase gene editing) approach typically uses them as two separate proteins.
“We have never observed any benefit from fusing the prime editor and integrase,” Liu told GEN in March. He said fusing the two proteins together 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, fusing the DNA-nicking Cas protein and the engineered reverse transcriptase together into a prime editor protein 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.
Site-specific recombinases have been used to edit genomes for several decades, starting with classic enzymes such as Cre recombinase, Flp recombinase, and Bxb1 integrase. Recombinase landing sites are essential in order for site-specific recombinases to work. The human genome has no such landing sites for useful, well-characterized recombinases.
Before prime editing, a recombinase landing site could not be efficiently installed at a specified target position in the genomes of most types of living cells, including non-dividing mammalian cells and in vivo in animals.
“Broadly speaking, from an architectural standpoint, PASTE is a complex drug product. It is appropriate when you’re installing or integrating thousands, if not tens of thousands of base pairs or multiplexing thousands of base pairs in different areas of the genome,” Tome’s Kakkar explained. “It may not be the best tool if you want to address a single exon, for instance, or a promoter region, or you want to go after something that’s completely non-viral.”
“Tome is really committed to this idea that the future of genomic therapies rests upon our ability to recode the human genome in blocks of code: Exons in some cases, regulatory elements in other cases, full genes or multiple logic circuits of genes in other cases. It’s a horses-for-courses approach where integrated mediated PGI or I-PGI derived from PASTE as a kernel or as a core is one technology we’re working on. But there are other technologies that we hope to be able to announce that show the full suite of technologies at our disposal.”
Tome raised its total $213 million in Series A and B rounds from investors that included Andreessen Horowitz (a16z) Bio + Health, ARCH Venture Partners, GV, Longwood Fund, Polaris Partners, Bruker Corporation, FUJIFILM Corporation, Alexandria Venture Investments, and others.
Dan Lynch, executive venture partner with GV, chairs Tome’s board, which also includes Jorge Conde, a General Partner on the Bio + Health team at Andreessen Horowitz (a16z); Alan Crane, Entrepreneur Partner with Polaris Partners; and Jay Markowitz, MD, a Senior Partner with ARCH Venture Partners, in addition to Abudayyeh, Gootenberg, and Kakkar.
Abudayyeh and Gootenberg “have been a phenomenal pair of partners to the company,” said Kakkar, adding that both he and chief scientific officer John Finn, PhD, are in regular contact with them.
“Once or twice a year, we hold an internal styled very much like an academic scientific symposium with poster presentations and internal competitions for oral presentations. They give the keynote, so they are involved both at the board level and operationally, as a source of counsel and a sounding board to myself and the CSO,” Kakkar explained. “They are truly inspiring to our employees, including our scientific employees.”
Tome’s executive team also includes Matt Barrows, chief technology officer; Edward Freedman, chief operating officer; and Diane Wong, chief, people & culture.
Before helming Tome, Kakkar was CEO of Pandion Therapeutics, a developer of Treg-targeting treatments for autoimmune and inflammatory diseases, which he led from Series A financing through its acquisition by Merck & Co. for $1.85 billion. Earlier, Kakkar founded Corvidia Therapeutics, a precision cardiovascular company, where he served as chief medical officer and chief strategy officer until that company was acquired by Novo Nordisk for $2.1 billion.
“This is not my first rodeo. This is my third company,” Kakkar said. “My personal ambition is to usher in the mature era of genomic therapies, and to really develop both integrative gene therapies and cell therapies across the entire textbook of Harrison’s [Principles of Internal] Medicine for every therapeutic area.”
Alex Philippidis is Senior Business Editor of GEN.