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C4 - Targeted Gene Insertion (integrase mediated insertion -targeted or safe harbor)

1676: Low RT-Based Fidelity in Mouse Hepatocytes: Challenges and Solutions

Type: Poster Session

Poster Board Number: 1676
Presentation Details
Session Title: Friday Posters: Targeted Gene Insertion

Integrase-mediated programmable genomic integration (I-PGI) utilizes the DNA targeting properties of Cas9 and couples it with the efficient, size-independent DNA integration abilities of large serine integrases (LSIs). This method enables the integration of large pieces of DNA in a site-specific, directional manner that is not dependent on double stranded breaks (DSBs). I-PGI technology can be used to develop gene therapies for monogenic liver diseases with the potential of a single drug per disease for diseases with a heterogeneous genetic landscape. I-PGI uses a Cas9 nickase (nCas9) with a writing enzyme, such as reverse transcriptase (RT), to write an integrase target site (attB, here called a ‘beacon’) in a programmed location. Beacon placement (BP) requires a minimum insert length of 38bp which is significantly longer than the majority of edits currently being explored with base and prime editing (<3 bp). After multiple rounds of optimization, we were able to efficiently place beacons with good beacon fidelity (BF) in human cycling cells (HEK293), non-dividing primary human hepatocytes (PHH), and primary cynomolgus hepatocytes (PCH) using transient mRNA delivery and chemically modified attachment guide RNA (atgRNA) pairs.When translating our technology in vivo to mice, we observed a surprising result, that despite being able to achieve a high level of editing in mouse hepatocytes following lipid nanoparticle (LNP) delivery of the editing components, that nearly all observed edits were incorrect, leading to very low BF rates. We found that this fidelity issue was also observed in vitro in non-dividing primary mouse hepatocytes, was independent of mouse strain, and was also observed in primary rat hepatocytes.To address the mouse beacon fidelity issue, we took two approaches. First, we engineered transgenic mice which had intact beacons in specific locations, allowing us to optimize integrase and DNA template dosing and kinetics. Second, we hypothesized that beacon fidelity in mice is impacted by the non-dividing nature of hepatocytes and can be improved by utilizing cycling hepatocytes. Therefore, we utilized neonatal mice which have a much higher proportion of proliferating hepatocytes than adult mice. Using LNP delivery in neonatal mice, we were able to significantly increase BF. We subsequently have demonstrated that these beacons are functional and have demonstrated functional gene expression after I-PGI mediated targeted integration in a therapeutically relevant target site.In summary, we have identified a previously undescribed challenge when using RT-based editing to write long (~40 bp) sequences in non-dividing mouse hepatocytes. This issue is likely due to DNA repair pathway differences between cycling and non-cycling mouse cells and appears to be limited to rodents, as we can routinely achieve high levels of beacon placement and fidelity in both human and non-human primate hepatocytes.

Maike Thamsen Dunyak, Patrick Hanna, Angela Nan, Mollie O'Hara, Rejina Pokharel, Jenny Xie, Jonathan D. Finn

Tome Biosciences, Watertown, MA"

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