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, to improve base editor RNP delivery, we optimized S10 to derive the S315 peptide. Following intratracheal aerosol of Cy5-labeled peptide cargo in rhesus macaques, we confirmed delivery throughout the respiratory tract. Subsequently, we targeted CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieved editing efficiencies of up to 5.3% in rhesus airway epithelia. Moreover, we documented persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restored anion channel function in cultured human airway epithelial cells. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

Keywords: adenine base editor; cystic fibrosis; ribonucleoprotein.

Figure 1

Identification of shuttle peptides with improved delivery of Cas9 RNPs to airway epithelia. a. Delivery of Cas9 RNP 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 attained with the indicated peptide. Individual closed circles represent averaged data from an individual donor. Results plotted as mean ± SEM, * P<0.05. 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 (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, ** P<0.005. Individual circles represent data from biological replicates.

Figure 2

Adenine base editing in human and rhesus monkey airway cells in vitro. a. Delivery of ABE8e-Cas9 RNP targeting B2Mlocus using S10 and S315 peptides in human airway epithelial cells cultured at the air liquid interface. Editing efficiency was assessed using HTS. Individual closed circles represent data from technical replicates. *** P< 0.001. b. 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. Individual closed circles represent data from technical replicates. *** P< 0.001.

Figure 3

Intratracheal in vivo delivery of DRI-NLS-Cy5 with S10 shuttle peptide results in widespread distribution and cell-type specific delivery in the 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, upper panels). Control received DRI-NLS-Cy5 alone (Fig. 3a, lower panels). b. Confocal microscopy image 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).

Figure 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 lobes (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, and columns represent mean ± SEM. 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.

Figure 5

Application of chest CT scans to identify regions of deposited base editing reagents. a. Chest CT images from monkey #7 (S315+RNP) from panel (b) below. Arrows highlight areas of consolidation. b. Correlation between regional editing efficiency or regional DRI-NLS-Cy5 nuclear localization and areas of ground glass opacity or 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 ground glass; ++ segmental ground glass; +++ dense consolidation. Filled circles represent editing efficiencies for indicated region as shown in Fig. 4c.

Figure 6

Persistence of gene editing in Ai9 mice following S10-mediated delivery of MAD7 nuclease. a.Schematic of reporter in Rosa26 locus of Ai9 mice. PAM and gRNA to target LoxP sites are shown. b. Ai9 mice received MAD7 RNP with the S10 shuttle as described in the Methods section. At the indicated intervals, editing was assessed in lung tissue sections using fluorescence microscopy. Nuclease dependent editing is signified by tdTomato expression. White scale bar indicates 200 μm. c. Quantification of tdTomato expression in airway epithelia at the indicated intervals. Each dot represents the counting of an individual mouse and columns represent ±SEM, *denotes P<0.05 by Kruskal-Wallis test.

Figure 7

Shuttle peptide delivery of ABE8e-Cas9 RNP to primary air liquid interface cultures of human airway epithelial cells (R553X/L671X) targeting R553Xlocus. 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 show 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 panel (c). Statistics by one-way ANOVA, ***P<0.001. 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 (d) and GlyH (e) in groups represented in (c) and non-CF donor cells. Statistics by one-way ANOVA, *P<0.05, **P<0.005. f. Representative short circuit current tracings comparing mock, ABE8e-Cas9 RNP alone ABE8e-Cas9 RNP + S10, and ABE8e-Cas9 RNP + S315 treated cells. Three technical replicates for each treatment group. Each data point represents one culture. (n=3 technical replicates).