Biology subjects

Gatlin, J.

Publications and source records attributed to Gatlin, J..

3 recordsLinked to original sources

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↗

bioRxiv2018

Nucleo-cytoplasmic trafficking regulates nuclear surface area during nuclear organogenesis

Throughout development, nuclei must be assembled following every cell division to establish a functional organelle from compact, mitotic chromatin. During nuclear organogenesis, chromatin expands to establish a nucleus of a given size seperate from the cytoplasm. Determining how nuclear organogenesis is regulated is particularly significant in the context of certain cancers in which scaling relationships between cell and nuclear sizes are not maintained. Controlling cell size in vitro using a microfluidics approach, we determined that neither nuclear volume nor surface area scale directly with cell size. Looking to explain differential nuclear scaling relationships, we developed a simple mechano-chemical mathematical model. In simulating biological perturbations in silico, our model predicted crucial roles for nucleo-cytoplasmic trafficking in regulating nuclear expansion and in restricting the recruitment of a potential nuclear surface area factor. In mammalian tissue culture, inhibiting nuclear export increased nuclear expansion rates and reduced the amount of nuclear lamin, a candidate surface area factor, being recruited to assembling nuclei, supporting our models predictions. Targeting the principal nuclear export component in the Drosophila syncytial embryo, Embargoed, we show that nuclear expansion rates are also increased in this developmental context, consistent with our model. Using the MS2-reporter system in fly embryos, we demonstrate a role for nuclear export in regulating transcription activation timing and dynamics, suggesting that regulating nuclear assembly is crucial for downstream nuclear function. Taken together, we propose a simple model through which nuclear organogenesis is achieved and demonstrate a role for nuclear export in regulating nuclear assembly.

cell biology↗