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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

228: Shuttle Peptide Delivers Base Editor RNPs to Rhesus Monkey Airway Epithelial Cells In Vivo

Type: Oral Abstract Session

Presentation Details
Session Title: Gene Targeting and Gene Correction: New Technologies
Location: Room 515 AB
Start Time: 5/18/2023 15:45
End Time: 5/18/2023 16:00

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelia. Here, to improve base editor RNP delivery, we optimized S10 to derive the S315 shuttle peptide with improved Cas9 RNP delivery properties. Following intratracheal aerosolization of a Cy5-labeled peptide cargo with a nuclear localization signal in young rhesus monkeys, we confirmed delivery throughout the respiratory tract. A nuclear Cy5 signal was observed in luminal ciliated cells (acetylated tubulin+), secretory cells (SCGB1A1+), sporadically in basal cells (CK5+), and in alveolar type II (SP-C+) cells in the parenchymal regions. The delivery efficiency ranged between 0.5% - 20.8% and 1% - 17.8% in the large and small airways, respectively. Subsequently, we targeted the rhesus CCR5 locus with co-administration of ABE8e-Cas9 RNP and S315 peptide by intratracheal aerosol. We achieved editing efficiencies up to 5.3% in rhesus airway epithelia (range 0.2% - 5.3%). The magnitude of Cy5 delivery and CCR5 editing efficiency correlated with the anatomical distribution of deposited aerosolized materials as assessed by analysis of chest CT scans taken pre- and post-delivery. To investigate the long-term persistence of gene edited airway epithelial cells in vivo, we used the Ai9 ROSA26 tdTomato reporter mouse model. Following delivery of MAD7 nuclease RNP with the S10 shuttle, we documented persistence of edited tdTomato positive epithelial cells for up to 12 months (duration of the experiment). Finally, we examined the utility of the peptide-mediated base editor delivery to correct the CFTR function in human airway epithelia. We delivered ABE8e-Cas9 RNP with the S315 peptide to cultured human airway epithelial cells with the R553X mutation. We achieved 4.9% A•T to G•C editing at the target locus and restored CFTR-mediated anion secretion as measured in Ussing chambers. In summary, a single shuttle-peptide mediated delivery of ABE8e-Cas9 RNPs to the airways of the young rhesus monkeys, and in vitro delivery of the editor to human airway epithelia with the R553X mutation yielded similar editing efficiencies. In human CF epithelia adenine base editing restored CFTR anion channel function. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in human respiratory epithelia.

Katarina Kulhankova1, Soumba Traore1, Xue Cheng2, Hadrien Benk-Fortin2, Stéphanie Hallée2, Mario Harvey2, Joannie Roberge2, Frédéric Couture3, Thomas Gross4, Gregory A. Newby5, David R. Liu6, Alice F. Tarantal7, David Guay2, Paul B. McCray1

1Pediatrics, University of Iowa, Iowa City, IA,2Feldan Therapeutics, Québec, QC, Canada,3TransBIOtech, Lévis, QC, Canada,4Internal Medicine, University of Iowa, Iowa City, IA,5Broad Institute of MIT and Harvard, Cambridge, MA,6Harvard University and HHMI, Cambridge, MA,7University of California, Davis, Davis, CA
 K. Kulhankova: None.

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