Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo

Abstract

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 epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

Fig. 1. Identification of shuttle peptides with improved delivery of Cas9 RNPs to airway epithelia.

a Delivery of Cas9 RNP (ribonucleoprotein) targeting CFTR locus in human airway epithelial cells cultured at the air liquid interface using indicated shuttle peptide candidates. Y axis represents the frequency of indels (insertions, deletions, and substitutions in the quantification window) attained with the indicated peptide. Individual closed circles represent data from n = 6 independent samples. Blue and red color represent the peptides used further in the study (S10 and S315, respectively). Results plotted as mean + SEM. Statistics by unpaired two-tailed t test, *P = 0.01. b Comparison of the amino acid sequences of S10 and S315 peptides. c Inhibitory effect of Cas9 RNP on S10- or S315-mediated delivery of GFP to CFF-16HBEge cells. GFP (green fluorescent protein, 10 μM), S10 or S315 (10 μM) peptide with or without Cas9 RNP (containing 2.5 μM Cas9 and 2 μM gRNA) were added to cells and GFP delivery quantified by flow cytometry. The Y axis represents the relative delivery activity (%), calculated as the GFP delivery attained with or without Cas9 RNP addition. Results plotted as mean + SEM. Statistics by unpaired two-tailed t test, **P = 0.003. Individual circles represent average data from n = 20,000 cells examined over 4 independent experiments. Source data are provided as a Source Data File.

Fig. 2. Adenine base editing in human and rhesus monkey airway cells in vitro.

a Delivery of ABE8e-Cas9 RNP targeting B2M locus using S10 and S315 peptides in human airway epithelial cells cultured at the air liquid interface. Editing efficiency was assessed using HTS. Top panel shows the target DNA strands and PAM sites (blue font). Yellow highlight denotes the predicted 4–8 nt ABE editing window (numbered). Target A residue is in red font. Average frequency of desired product for human B2M locus with the indicated shuttle peptides. b Graphic representation of desired base editing efficiency at human B2M locus using S10 (blue column) and S315 (red column) peptides. Individual closed circles represent data from 4 technical replicates, and the data is plotted as mean ± SEM. Statistics by unpaired two-tailed t test, ***P = 0.0005. c Delivery of ABE8e-Cas9 RNP targeting CCR5 locus using S10 and S315 peptides in rhesus tracheal epithelial cells cultured at the air liquid interface. Editing efficiency quantified using HTS. Top panel shows the target DNA strands and PAM sites (blue text). Yellow highlight denotes the predicted 4–8 nt ABE editing window (numbered). Target A residue highlighted in red text. Target A residue values are inlarge font. Average frequency of desired product for rhesus CCR5 locus with the indicated shuttle peptides. d Graphic representation of desired base editing efficiency at rhesus CCR5 locus using S10 (blue) and S315 peptides (red). Individual closed circles represent data from 4 technical replicates, and the data are plotted as mean ± SEM. Statistics by unpaired two-tailed t test, ***P = 0.0006. Source data are provided as a Source Data File.

Fig. 3. Intratracheal in vivo delivery of DRI-NLS-Cy5 with S10 shuttle peptide results in widespread distribution and cell-type specific delivery in rhesus monkey airways.

a Representative epifluorescence microscopy images of the rhesus lung tissue sections demonstrating the DRI-NLS-Cy5 localization in the surface epithelial cells of airways of various sizes, ranging from trachea to small airways and alveolar regions (Fig. 3a, top panel). Control received DRI-NLS-Cy5 alone (Fig. 3a, bottom panel). Scale bar is 100 µm. b Confocal microscopy images documenting localization of DRI-NLS-Cy5 fluorescence (white) in ciliated cells (*, AcTub - red), secretory cells (#, SCGB1A1 – green, top panel), and very rarely to CK5⁺ basal cells (*, CK5 – green, middle panel), where DAPI is pseudo-colored blue. In the alveolar regions (Fig. 3b, lower panel), the DRI-NLS-Cy5 signal (white) localized to the nuclei of the surfactant protein C producing alveolar type II cells (#, SP-C - red) and alveolar macrophages (*, CD68 - green). Scale bar is 20 µm. For (a) and (b), the staining and microscopy was performed at least two times on tissue sections from different regions of the lungs and yielded similar results.

Fig. 4. Quantification of in vivo delivery of DRI-NLS-Cy5 and ABE8e-Cas9 RNP to rhesus respiratory epithelia.

a Diagram of rhesus monkey airway tree. The regions where airway tissue sections or cytology brushings were obtained are color coded and numbered as indicated. b Quantification of DRI-NLS-Cy5 delivery with S10 peptide in trachea (T), right mainstem bronchus (RMSB), and 7 lobar anatomical locations (RUL - right upper lobe, RML - right middle lobe, RLL - right lower lobe, AL - accessory lobe, LUL-CrP - left upper lobe, cranial part, LUL CaP - left upper lobe, caudal part, LLL - left lower lobe). Each circle represents one airway analyzed in a single tissue section from a given anatomical location, and columns represent mean ± SEM. Between 1 and 7 airways per section were analyzed, n = 1 animal. c Efficiency of shuttle peptide mediated Cas9-ABE8e RNP editing at CCR5 locus scored by airway region. Y axis indicates A to G editing efficiency. X axis denotes conditions including DRI-NLS-Cy5 alone (#3), S10 + DRI-NLS-Cy5 (#4), ABE8e-Cas9 RNP alone (#5), S10 + ABE8e-Cas9 RNP (#6), and S315 + ABE8e-Cas9 RNP (#7, 8). Each condition presents data from an individual animal. The animal numbers correspond to conditions described in Supplementary Table 2 (n = 6 animals). Source data are provided as a Source Data File.

Fig. 5. Application of chest CT scans to identify regions of deposited base editing reagents.

a Chest CT from monkey #7 (S315 + RNP) from (b) below. Arrows highlight areas of consolidation. b Correlation between regional editing efficiency or regional DRI-NLS-Cy5 nuclear localization and areas of consolidation on CT scan. Regions studied include RUL - right upper lobe, RML - right middle lobe, RLL - right lower lobe, LUL-Cr - left upper lobe, cranial part, LUL Ca - left upper lobe, caudal part, LLL - left lower lobe. The CT scans were scored in a blinded fashion for changes in aeration as follows: NC: no change from baseline; + subtle nonsegmental consolidation; ++ segmental consolidation; +++ dense consolidation. Filled circles represent editing efficiencies for indicated region as shown in Fig. 4c. Source data are provided as a Source Data File.

Fig. 6. Shuttle peptide delivery of ABE8e-Cas9 RNP to primary air liquid interface cultures of human airway epithelial cells (R553X/L671X) targeting R553X locus.

One week following the first application of ABE8e-Cas9 RNP delivery with shuttle peptides, Ussing chamber analysis was conducted, and DNA editing was analyzed by HTS. a Top panel shows the target DNA strands and PAM sites (blue text). Yellow highlight denotes the predicted 4–8 nt ABE editing window (numbered). Mutations highlighted in red text. b Average frequency of desired product and allelic editing efficiencies for R553X nonsense mutation with the indicated shuttle peptides. Percent allelic editing efficiencies calculated by (% base edited-50)/50 *100 and graphically represented in (c) and the values are highlighted inlarge font. Note that the cells were compound heterozygous for the CFTR R553X mutation. Thus, all cells contain “wild type” sequence for one allele and have the R553X mutation on the other allele. This mathematical adjustment provides a meaningful “per cell” estimate of correction. Statistics by ordinary one-way ANOVA without any adjustments, ***P < 0.0001. d, e CFTR-dependent anion channel activity summarized from short circuit current tracings across all treatment groups. Change in short circuit current (ΔIsc) in response to F&I (forskolin & IBMX) (d) and GlyH (e) in groups represented in (c) and non-CF donor cells. Statistics by ordinary one-way ANOVA without any adjustments, *P = 0.01, **P = 0.003. f Representative short circuit current tracings comparing mock, ABE8e-Cas9 RNP alone ABE8e-Cas9 RNP + S10 (blue), and ABE8e-Cas9 RNP + S315 (red) treated cells. For (c–e), the CF human airway epithelia are represented by 3 technical replicates (treatment goups) or 4 (Mock group) technical replicates, and non-CF human airway epithelia are represented by 12 biological replicates. Each data point represents one cell culture. Data are plotted as mean ± SEM. Source data are provided as a Source Data File.