SCGE ID:1069 - Submission Date: 2022-07-27

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Experiment Name	Type	Description
Delivery of Cas9 RNP using Shuttle peptide candidates to human airway epithelial cells cultured at the air liquid interface targeting CFTR locus	In Vitro	Delivery of Cas9 RNP using Shuttle peptides candidates to human airway epithelial cells cultured at the air liquid interface targeting CFTR locus. Editing efficiency was assessed using NGS.
Rhesus macaque CCR5 gRNA screening using ABE8e-Cas9	In Vitro	Electroporation of plasmids containing ABE8e-Cas9 with 10 different gRNAs targeting CCR5 into rhesus primary skin fibroblasts. Editing efficiency was assessed using Next Generation Sequencing.
Inhibitory effect of Cas9 RNP alone on S10- or FSDS315-mediated GFP protein delivery to HeLa cells	In Vitro	Inhibitory effect of Cas9 RNP alone on S10- or FSD315-mediated GFP protein delivery to HeLa cells. GFP protein (10 μM), S10 or FSDS315 (10 μM) with or without Cas9 RNP (containing 2.5 μM Cas9 and 2 μM gRNA) were added to HeLa cells and GFP delivery quantified by flow cytometry.
Rhesus macaque CCR5 gRNA screening using ABE8e-Cas12a	In Vitro	Electroporation of plasmids containing ABE8e-Cas12a with 11 different gRNAs targeting CCR5 into rhesus primary skin fibroblasts. Editing efficiency was assessed using high throughput DNA sequencing.
Delivery of ABE8e-Cas9 RNP using Shuttle peptide candidates to human airway epithelial cells cultured at the air liquid interface targeting B2M locus	In Vitro	Delivery of ABE8e-Cas9 RNP using Shuttle peptides candidates to human airway epithelial cells cultured at the air liquid interface targeting B2M locus. Editing efficiency was assessed using Sanger sequencing.
Delivery of ABE8e-Cas9 RNP using Shuttle peptide candidates to rhesus monkey airway epithelial cells cultured at the air liquid interface targeting CCR5 locus.	In Vitro	Delivery of ABE8e-Cas9 RNP using Shuttle peptides candidates to rhesus monkey airway epithelial cells cultured at the air liquid interface targeting CCR5 locus. Editing efficiency was assessed using Next Generation Sequencing.
Amphiphilic Peptides Deliver Base Editor RNPs to Rhesus Monkey Airway	In Vivo	We utilized novel amphiphilic shuttle peptides to deliver base editor ribonucleoprotein (RNP) into the airways to edit airway epithelial cells (CCR5 locus) of rhesus monkeys. The Cas9-ABE8e RNP and shuttle peptides S10 or FSD315 were aerosolized into the rhesus monkey trachea. 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 FSD315 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. Ongoing analyses will identify the NLS-Cy5-positive epithelial cell types using co-localization with fluorescently-labeled antibodies. 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.