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C6 - Gene Targeting and Gene Correction New Technologies

1214: L-PGI: A DNA Ligase Mediated Gene Editing System for Versatile and Precise Genomic Integration

Type: Poster Session

Poster Board Number: 1214
Presentation Details
Session Title: Thursday Posters: Gene Targeting and Gene Correction New Technologies

The efficiency of genome modifications using CRISPR nucleases and reverse transcriptase (RT) is limited by RT processivity, template complexity, and intracellular dNTP levels, which can become rate limiting in non-dividing cells and tissues of therapeutic interest. To overcome these challenges, we present Ligase-mediated programmable genomic integration (L-PGI), a CRISPR-guided DNA ligase system for highly precise replacement/integration of a targeted sequence of pre-synthesized DNA containing desired edit. L-PGI uses 5 components: (1) Cas9 nickase, (2) DNA ligase, (3) pre-synthesized DNA donor template that contains the desired edit, (4) splint, and (5) ligase mediated guide RNA (lmgRNA). Once delivered to the cell, Cas9 nickase forms an RNP with the hybridized lmgRNA-splint-donor complex, binds to the genomic target, and nicks the DNA, which releases the genomic flap. The splint acts as a bridge between the lmgRNA, genomic target site, and donor DNA, precisely aligning all of the components in the correct spatial configuration, allowing the DNA ligase to ligate the genomic flap and donor. The ligated donor displaces the endogenous strand and results in incorporation of the edits encoded in the donor into the genome. L-PGI does not induce significant double stranded breaks, is not subject to RT limitations, and is efficient in both dividing and non-dividing cells. The single-step direct integration of pre-synthesized DNA can incorporate a variety of edits from small nucleotide conversions to deletions and insertions up to greater than a hundred base pairs, all with higher efficiency and lower incidence of unintended edits than existing gene editing platforms. Due to its versatility, L-PGI has the potential for broad therapeutics application in the treatment of a variety of genetic diseases, ranging from single mutation correction to whole gene replacement to exon displacement.

Jenny Xie1, Angela Nan1, Chris Bartolome1, Sandeep Kumar1, Shakked Halperin2, Michael Chickering2, Parbir Grewal2, Leonard Chavez2, Jonathan D. Finn1

1Tome Biosciences, Inc., Watertown, MA,2Replace Therapeutics, LLC, a direct and wholly owned subsidiary of Tome Biosciences, Inc., Watertown, MA"

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