(617) Peptide-Mediated Delivery of Adenine Base Editors to Rhesus Monkey Airway Epithelia

Background: The development of safe and effective gene editing strategies for cystic fibrosis and other lung diseases is challenged by the complex barrier properties of airway epithelial cells. To overcome this limitation, we utilized novel amphiphilic shuttle peptides to deliver base editor ribonucleoprotein (RNP) into the airways to edit airway epithelial cells of rhesus monkeys. We previously reported that non-covalently combining shuttle peptides in solution with protein cargoes (CRISPR-Cas nucleases, GFP) facilitates rapid protein delivery into human and mouse airway epithelia.

Methods: We used a rhesus macaque model to document the efficacy of amphiphilic peptides to deliver adenine base editors to the airway epithelial cells and mediate editing of the CCR5 locus.

Results: Using primary air liquid interface cultures of rhesus tracheal epithelial cells, we demonstrated that the shuttle peptides S10 and S315 delivered Cas9-ABE8e RNP and achieved editing at the CCR5 safe harbor locus (editing efficiency 3-9%). To investigate the delivery efficiency in vivo, the Cas9-ABE8e RNP and shuttle peptides S10 or S315 were aerosolized into the trachea of sedated young rhesus monkeys during tidal breathing. Seven days later, tissues were obtained and dissected, and airway epithelia collected from the trachea, mainstem, and segmental bronchi using cytology brushes. DNA was extracted from epithelial cells and subjected to high-throughput sequencing. Using the S315 shuttle peptide and Cas9-ABE8e, we achieved a mean editing efficiency of 2.8% at the CCR5 locus in airway epithelial cells (range 0.02 – 5.3%) depending on the anatomic region sampled. To visualize the biodistribution of the RNPs within the respiratory tract and in specific cell types, we delivered a Cy5-fluorescent peptide fused to a nuclear localization signal (NLS-Cy5) using the S10 peptide. The lungs were obtained 1 and 2 hours post-delivery, fixed, and examined by microscopy. Epifluorescence and confocal microscopy documented an effective intra-nuclear delivery of NLS-Cy5 into epithelial cells throughout the respiratory tract, including large, medium, and small airways, and alveolar regions. NLS-Cy5-localized primarily to ciliated epithelial cells, and also was observed in the cells with the progenitor capacity, specifically CK5-positive basal cells and SPC-positive alveolar type II cells.

Conclusions: In summary, using a rhesus monkey model, following a single delivery of adenine base editor RNPs to the airways in a clinically relevant manner we achieved up to 5.3% editing efficiency of the CCR5 locus in airway epithelia, a level considered therapeutically relevant in cystic fibrosis.

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