Chitosan Hydrogel-Delivered ABE8e Corrects PAX9 Mutant in Dental Pulp Stem Cells

Abstract

Hypodontia (dental agenesis) is a genetic disorder, and it has been identified that the mutation C175T in PAX9 could lead to hypodontia. Cas9 nickase (nCas9)-mediated homology-directed repair (HDR) and base editing were used for the correction of this mutated point. This study aimed to investigate the effect of HDR and the base editor ABE8e in editing PAX9 mutant. It was found that the chitosan hydrogel was efficient in delivering naked DNA into dental pulp stem cells (DPSCs). To explore the influence of the C175T mutation in PAX9 on the proliferation of DPSCs, hydrogel was employed to deliver PAX9 mutant vector into DPSCs, finding that the PAX9-containing C175T mutation failed to promote the proliferation of DPSCs. Firstly, DPSCs stably carrying PAX9 mutant were constructed. Either an HDR or ABE8e system was delivered into the above-mentioned stable DPSCs, and then the correction efficiency using Sanger sequencing and Western blotting was determined. Meanwhile, the ABE8e presented significantly higher efficiency in correcting C175T compared with HDR. Furthermore, the corrected PAX9 presented enhanced viability and differentiation capacity for osteogenic and neurogenic lineages; the corrected PAX9 even possessed extremely enhanced transcriptional activation ability. In summary, this study has powerful implications for studies into base editors, chitosan hydrogel, and DPSCs in treating hypodontia.

Keywords: ABE8e; PAX9; dental agenesis; dental pulp stem cells; hydrogel; hypodontia.

Figure 1

Physicochemical properties of chitosan hydrogel: (A) The preparation of chitosan-based hydrogel. SEM images of the chitosan-based hydrogel in (B) 50×, 100×, and 500×. (C) FTIR spectra of the chitosan-based hydrogel.

Figure 2

The influence of hydrogel on the delivery of DNA to DPSCs: (A) Flow cytometry assessment of mesenchymal markers CD90 and CD73 and hematopoietic markers CD45 and CD34 on DPSCs. GFP expression plasmids were delivered using Lipofectamine™ 3000 or hydrogel. (B) The fluorescence was measured using microscope. NC, negative control, no treatment. LV, lentiviral vector expressing GFP. (C) Representative histogram of flow cytometry assessment for the measurement of fluorescence intensity. (D) Mean fluorescence index of fluorescence intensity from flow cytometry assessment. (E) CCK-8 assays for evaluating the influence of hydrogel on DPSCs. The assay was conducted at 24, 48, 96, and 120 h post delivery. All data are from experiments performed in triplicates; ns, not significant; ** p < 0.01, *** p < 0.001.

Figure 3

The influence of C175T in PAX9 on the proliferation of DPSCs: (A) Construction of wild-type and mutated PAX9 plasmids. The mutated PAX9 contains the point mutation C175T. *, stop codon. Green rectangle indicates Exon 1, 3, 4. Orange rectangle indicates Exon 2. (B) Sequence of PAX9mut in vector. Green oscillogram indicates base A. Red oscillogram indicates base T. Blue oscillogram indicates base C. Black oscillogram indicates base G. (C) Western blotting assays for evaluating the expression of PAX9 in DPSCs. Mock vectors, PAX9mut vectors, and PAX9 vectors were delivered to DPSCs using hydrogel. (D) CCK-8 assays for evaluating the influence of C175T in PAX9 on the proliferation of DPSCs. Error bars represent mean ± SD (n = 3). ** p < 0.001, *** p < 0.0001.

Figure 4

Modification of PAX9 mutant using HDR: (A) Schematic of PAM-out gRNA design for HDR. (B) Schematic of PAM blocking and gRNA blocking in HDR template. The two PAMs are highlighted in yellow. The target sequence of gRNA (upper) and gRNA (lower) are underlined. The mutation for PAM blocking (upper) and gRNA blocking are in red. (C) Sanger sequencing of PAX9 72 h after HDR delivery into DPSCs by hydrogel. Green oscillogram indicates base A. Red oscillogram indicates base T. Blue oscillogram indicates base C. Black oscillogram indicates base G. (D) Western blotting assays for evaluating the expression of PAX9 72 h after HDR delivery into DPSCs by hydrogel in DPSCs.

Figure 5

Modification of PAX9 mutant using ABE8e: (A) Schematic of the ABE8e-mediated base editing for PAX9 mutant (PAX9mut). (B) Sanger sequencing of PAX9 72 h after ABE8e delivery into DPSCs by hydrogel. Green oscillogram indicates base A. Red oscillogram indicates base T. Blue oscillogram indicates base C. Black oscillogram indicates base G. (C) Western blotting assays for evaluating the expression of PAX9 72 h after ABE8e delivery into DPSCs by hydrogel.

Figure 6

Impact of hydrogel-delivered ABE8e on DPSCs functions: (A) Photos of proliferated DPSCs. (B) CCK-8 assays for evaluating the influence of modified PAX9 on DPSCs proliferation. The assay was conducted at 24, 48, 96, and 120 h post delivery. All data are from experiments performed in triplicates. (C) Alizarin red staining after osteogenic induction for 21 days. (D) Immunofluorescence staining after neurogenic induction for 7 days (green: TuJ1). (E) Dual luciferase assays for the evaluation of the influence of PAX9 on the transactivation of the BMP4 promoter. Error bars represent mean ± SD (n = 3); ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.