Biology subjects

Dunyak, M. T.

Publications and source records attributed to Dunyak, M. T..

4 recordsLinked to original sources

bioRxiv2024

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

bioengineering↗

bioRxiv2024

Engineered Bxb1 variants improve integrase activity and fidelity

Many current genome editing technologies rely on the action of large serine integrases (LSIs) to insert gene-sized DNA sequences into the genome. Bxb1 is the most commonly used LSI for therapeutic efforts, including PASTE, PASSIGE and I-PGI. While Bxb1 demonstrated good activity in vitro in cycling cells, the activity in non-dividing hepatocytes was significantly less efficient. Further, wild-type Bxb1 is known to have detectable off-target activity at cryptic genomic sites, which presents a potential safety risk for therapeutic development. To address these issues, we developed Bxb1 variants that demonstrate increased specificity and potency in vitro and have engineered stabilized Bxb1 variants that increase in vivo activity over 25-fold enabling targeted integration at therapeutically relevant levels.

biochemistry↗

bioRxiv2024

Curative levels of endogenous gene replacement achieved in non-human primate liver using programmable genomic integration

The ability to efficiently place a large piece of DNA in a specific genomic location has been a goal for the gene therapy field since its inception; however, despite significant advances in gene editing technology, this had yet to be achieved. Here we describe two methods of programmable genomic integration (PGI) that overcome some of the limitations of current approaches. Using a combination of clinically validated delivery technologies (LNP, AAV), we demonstrate the ability to specifically integrate large (>2 kb) DNA sequences into endogenous introns in the liver of non-human primates (NHP). PGI was effective across multiple genomic locations and transgenes, and insertion led to expression from the endogenous promoter. PGI was highly efficient, achieving expression in >50% of liver cells after a single course of treatment, which would be curative for most monogenic recessive liver diseases. This is the first report of clinically curative level of gene insertion at endogenous loci in NHP.

bioengineering↗

bioRxiv2024

Ligase-mediated programmable genomic integration (L-PGI): an efficient site-specific gene editing system that overcomes the limitations of reverse transcriptase-based editing systems

Since their discovery, CRISPR/Cas9 systems have been repurposed for programmable targeted genomic editing. This has led to unprecedented advancement of gene editing for therapeutic benefit. Initial uses of CRISPR/Cas9 were focused on gene disruption via DNA cleavage, but significant engineering led to systems for single base editing as well as insertion, deletion and manipulation of short stretches of genomic sequences using nicking Cas9 and RT-based methods. These technologies allowed safer and more precise editing but were limited to small corrections and showed significantly reduced efficiencies in nondividing cells, presenting difficulty for translation to in vivo therapies. To find an alternate editing strategy that could address these shortcomings, we revisited the mechanism of DNA nicking by nCas9. nCas9 nicking creates a free 5 phosphate group and a 3 hydroxyl group on the complementary strand of the target sequence. Under ordinary conditions in the cell these ends are re-joined by endogenously expressed ligases to repair DNA back to wild-type. If, however, a DNA fragment containing the desired edit were present, ligation of the nicked genomic DNA with the delivered fragment could result in gene editing. We demonstrate that optimization of each component and introduction of a chemically modified high affinity splinting DNA allows a variety of ligase-based edits, including longer edits not efficient with RT-based systems, at high efficiencies and fidelities that minimize genomic byproducts in both dividing and nondividing cells as well as in vivo in adult mice. Here we present the first therapeutically relevant ligation-based programmable gene editing technology, L-PGI.

bioengineering↗