Distinctions without a Difference: The Puzzling Argument about PASSIGE vs PASTE

There is a puzzling controversy in the gene editing community whirling around a comparison of two relatively new, very promising gene editing technologies called PASSIGE and PASTE. Both of these technologies solve a fundamental challenge that has confronted the gene therapy field since its inception, namely: How do you put a large piece of DNA in a specific genomic location?

In my mind, what is puzzling is the need to compare one with the other. Because conceptually, they are the same. So, how did we get here?

At the 27^th Annual American Society for Gene and Cell Therapy (ASGCT) conference in Baltimore last month, one of the hot topics was large gene insertion, with many groups (including my colleagues at Tome Biosciences) presenting research progress. The majority focused on random, or near random, integration.

In his ASGCT Outstanding Achievement Award lecture, David Liu, PhD, an investigator at the Broad Institute, Howard Hughes Medical Institute, and co-founder of Prime Medicine, eloquently made the case that if we are going to bring genetic medicines to the majority of patients in need, the field needs to move beyond mutation-specific approaches that are not feasible to address most genetic diseases.

Rather, the ideal approach—at least for the many genes in which tens, hundreds, or even thousands of different mutations can give rise to a specific disorder—is to insert healthy genes into their endogenous location. (In this way, a single therapeutic process could potentially address patients with different specific mutations.)

Liu updated the audience on his group’s work developing PASSIGE (Prime Assisted Site Specific Integrase Gene Editing), a method for the site-specific integration of large DNA sequences, and its optimized version, eePASSIGE. After walking through some of the challenges his group had to overcome, he compared and contrasted PASSIGE and eePASSIGE with PASTE (Programmable Addition via Site specific Targeting Elements), a similar approach pioneered by Omar Abudayyeh, PhD, and Jonathan Gootenberg, PhD, formerly at MIT and now at Harvard Medical School. (Both are also co-founders of Tome Biosciences.)

This comparison led many people to ask the question, “What is the difference between PASSIGE and PASTE?” The purpose of this commentary is to answer that question. To do so, I first want to provide some background on these systems.

Drag and drop insertion

On November 1, 2021, Abudayyeh, Gootenberg (based at the time at the McGovern Institute for Brain Research at MIT), and their colleagues posted on bioRxiv a preprint of their manuscript, “Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases”, which described PASTE. This groundbreaking work detailed their efforts over several years to develop an editing system using large serine integrases (LSRs), also called recombinases, to enable the site-specific integration of large sequences of DNA into fixed locations.

PASTE uses the programmability of a Cas9 nickase, combined with a reverse transcriptase (RT) to first write a short DNA sequence (< 50 basepairs) at a specific location in the genome. This sequence is the recognition sequence (attB or attP) for a particular integrase. The authors then delivered a template DNA that contained the matching recognition sequence (attP or attB) as well as the integrase.

The integrase incorporates that DNA template at the target site in the genome. Importantly, they demonstrated that this integrase-based insertion approach was agnostic to the size of the DNA insert, meaning that as long as they could deliver the template DNA, the integrase efficiently inserts it. They showed that PASTE was functional with a range of enzymatic configurations (split vs fused) and with LSRs from diverse families, highlighting the flexibility of this platform.

This is a simple concept but the clever use of three enzymes (nCas9, RT, LSR) allowed for a solution to a problem that has hampered the gene therapy field for decades (see above): how to insert a large piece of DNA in a specific genomic location. Following peer review, the manuscript by Yarnall et al. was published a year later online in Nature Biotechnology in November 2022.¹

Programmable large DNA deletion, replacement, integration, and inversion

One day after Abudayyeh and Gootenberg posted their preprint, Liu, Andrew Anzalone, MD, PhD, and coworkers posted their own preprint on bioRxiv titled, “Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases.” This report described a guide architecture—“twin prime editing” (twinPE)—that could expand the range of edits possible with prime editing.

In the “twinPE” manuscript, the Liu group was able to use a Cas9 nickase with an RT enzyme to write the recognition site (attP or attB) of a specific LSR into the genome, and upon delivery of a template DNA that contains the matching recognition sequence (attP or attB) as well as the integrase, they could achieve targeted integration of that DNA template at the target site in the genome. (The Anzalone et al. paper was published in Nature Biotechnology in May 2022.²)

In his recent ASGCT lecture, Liu gave an update on this system (now dubbed PASSIGE) in which he described using directed evolution to evolve variants of an LSR called BxB1, which demonstrated increased potency and stated that this evolution was required to enable efficient levels of integration in HEK293 cells, a commonly used model cell system. This optimized version of PASSIGE was named “eePASSIGE.” (A paper on eePASSIGE is published in Nature Biomedical Engineering this week.³)

There do appear to be some nuanced differences between PASSIGE and PASTE. For example, one difference revolves around which half of the integrase landing site is written (attB vs attP). There are also differences in guide architecture and format. One purported difference Liu articulated in his ASGCT lecture was the incorrect assertion that the molecular components in PASTE are physically tethered rather than working independently as in PASSIGE. This is in fact not the case: PASTE as published operates in both tethered and non-tethered configurations. Regardless, these nuances are distinctions without a difference.

The fact is, both PASSIGE and PASTE enable efficient site-specific and directional integration of exogenous DNA into the genome of eukaryotic cells by employing a Cas9 nickase, RT enzyme, and an LSR. Clearly, these two groups independently arrived at the same solution to a challenging problem, specifically site-directed integration of large DNA sequences. Both groups should be commended for their vision and commitment to moving the field forward.

So then, what is the difference between PASSIGE and PASTE? The answer is quite simple: Conceptually, there is none.

The key question is not whether there is a difference between these two systems, developed in academic labs by leaders in the genome editing community, but whether any embodiment of their common concept can be used to usher in an era of gene therapy free from the editing limitations of systems that came before them. In short, can we make good on Liu’s assertion that for many genetic diseases, we need to move beyond mutation-specific approaches toward large gene insertion?

A gene editing technology is only as useful as the applications it can enable. While the foundational invention of PASTE and PASSIGE/eePASSIGE represents a true milestone in the field, translating these systems from their academic beginnings to therapeutically relevant embodiments with drug-like properties represents, in its purest form, the translation from science to medicine.

Such translation, in our opinion, requires unapologetic scrutiny of the system with a focus on potency (i.e., efficiency) and safety using scalable, manufacturable components to achieve performance characteristics that will enable best-in-class therapies for patients with a safety profile that will pass regulatory and patient-doctor scrutiny. These are the basic tenets of drug development.

From science to medicines

Tome Biosciences was founded with the ambition to translate a Cas-RT-LSI large gene integration system (PASTE) into a medicinal platform. Ultimately this ambition required ground-up re-engineering and optimization of almost every single component of the PASTE system. That is why, at ASGCT 2024, Tome shared the culmination of its optimization of PASTE to date. Given the scope of innovation required, our new approach has been named “Integrase-mediated Programmable Genomic Integration” (I-PGI).

Tome scientists introduced the concept of PGI at the ASGCT conference. Distilled into its simplest form, PGI is the ability to put any sequence of DNA of any size in any location in the genome, with complete control or programmability. There are clearly caveats associated with the concept of PGI, including dependency on efficient delivery of the template DNA and the requirement of Cas9 that can target efficient PAMs at the desired location. But when talking about PGI as a therapeutic platform, it has to have the following properties:

• It has to be programmable (i.e., site-specific, not random nor targeting a safe harbor).
• It has to be directional, where all of the integration events are in the same orientation.
• There cannot be an inherent restriction on the size of the insert.
• It has to be efficient enough to induce clinically relevant disease control or cure.
• It cannot be dependent on free double-stranded DNA breaks.

The journey from PASTE to PGI has certainly been more challenging than I anticipated, and has led my colleagues at Tome to uncover important new research findings and limitations of current gene editing systems (see Tome’s presentations at ASGCT 2024). But our re-engineering has also allowed Tome to be, as far as we are aware, the first company to achieve clinically relevant efficiencies of site-specific, large gene insertion in a living non-human primate (NHP) liver with commensurate protein expression using a Cas-RT-LSI system.

With a combination of drug-like reagents (lipid nanoparticles and adeno-associated virus), Tome was able to integrate promoter-less templates (hF9) into two genomic loci in NHPs—the cyno F9 locus, and the cyno PAH locus. For the PAH locus, which in humans is the gene mutated in phenylketonuria (PKU), Tome has achieved 8% PGI in bulk liver alleles, or approximately 10% of hepatocyte alleles (when normalized for the estimated percent of the NHP liver that is represented by hepatocytes) in initial experiments. With optimization in NHPs still in its early phases, this represents the floor, not the ceiling, of what I-PGI can ultimately do.

We believe this is a significant achievement given that, for PKU, preclinical data have consistently indicated that correction of 10% of hepatocytes is likely to normalize serum phenylalanine and, therefore, represent a clinical cure for patients with PKU.^4,5 (See also Tessera Therapeutics’ ASGCT 2024 poster, “RNA Gene Writers Drive Therapeutically Relevant Levels of Correction of Monogenic Disease Mutations Expressed in the Liver”.)

All of this is to say that asking, “What is the difference between PASSIGE and PASTE?” is the wrong question. The right question is, “How will we turn the academic invention of large gene insertion into genomic medicines for patients in need?”

This is the question we ask ourselves every day at Tome. In my opinion, our progress with PGI shows that we are getting closer to the answer every day.

John Finn, PhD, is the CSO at Tome Biosciences, a biotech company based in Cambridge, MA. Email: [email protected]

References:

1. Yarnell MTN, Ioannidi EI, Schmitt-Ulms C, et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nature Biotechnology 2023;41:500-12. Doi: 10.1038/s41587-022-01527-4
2. Anzalone AV, Gao XD, Podracky CJ, et al. Programmable deletion, replacement, integrations and inversion of large DNA sequences with twin prime editing. Nature Biotechnology 2022;40(5):731-740. doi: 1038/s41587-021-01133-w
3. Pandey S, Gao XD, et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nature Biomed Eng, June 10, 2024. DOI:10.1038/s41551-024-01227-1.
4. Hamman K, Clark H, Montini E, et al. Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria. Mol Therapy 2005;12(2):337-44. doi: 1016/j.ymthe.2005.03.025
5. Hamman KJ, Winn SR, Harding CO. Hepatocytes from wild-type or heterozygous donors are equally effective in achieving successful therapeutic liver repopulation in murine phenylketonuria (PKU). Mol Genet Metab. 2011;104(3):235-40. doi: 10.1016/j.ymgme.2011.07.027