DNA-PKcs Inhibition Improves Sequential Gene Insertion of the Full-Length CFTR cDNA in Airway Stem Cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells.

Single-stranded DNA (ssDNA) templates along with Cas9 have been used for gene insertion but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 10-17% of internal bases (eDNA) is compatible with the homologous recombination machinery. Moreover, eDNA templates improve gene insertion by 2-3 fold compared to unmodified and end-modified ssDNA in airway basal stem cells (ABCs), hematopoietic stem and progenitor cells (HSPCs), T-cells and endothelial cells. Over 50% of alleles showed gene insertion in three clinically relevant loci (CFTR, HBB, and CCR5) in ABCs using eDNA and up to 70% of alleles showed gene insertion in the HBB locus in HSPCs. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. Knocking out TREX1 nuclease improved gene insertion using unmodified ssDNA but not eDNA suggesting that chemical modifications inhibit TREX1. This approach can be used for therapeutic applications and biological modeling.

Airway disease decreases the therapeutic potential of epithelial stem cells.

BACKGORUND: Tissue-engineered tracheal grafts (TETG) can be recellularized by the host or pre-seeded with host-derived cells. However, the impact of airway disease on the recellularization process is unknown. METHODS: In this study, we determined if airway disease alters the regenerative potential of the human tracheobronchial epithelium (hTBE) obtained by brushing the tracheal mucosa during clinically-indicated bronchoscopy from 48 pediatric and six adult patients. RESULTS: Our findings revealed that basal cell recovery and frequency did not vary by age or region. At passage 1, all samples produced enough cells to cellularize a 3.5 by 0.5 cm2 graft scaffold at low cell density (~ 7000 cells/cm2), and 43.75% could cellularize a scaffold at high cell density (~ 100,000 cells/cm2). At passage 2, all samples produced the number of cells required for both recellularization models. Further evaluation revealed that six pediatric samples (11%) and three (50%) adult samples contained basal cells with a squamous basal phenotype. These cells did not form a polarized epithelium or produce differentiated secretory or ciliated cells. In the pediatric population, the squamous basal cell phenotype was associated with degree of prematurity (< 28 weeks, 64% vs. 13%, p = 0.02), significant pulmonary history (83% vs. 34%, p = 0.02), specifically with bronchopulmonary dysplasia (67% vs. 19%, p = 0.01), and patients who underwent previous tracheostomy (67% vs. 23%, p = 0.03). CONCLUSIONS: In summary, screening high-risk pediatric or adult population based on clinical risk factors and laboratory findings could define appropriate candidates for airway reconstruction with tracheal scaffolds. LEVEL OF EVIDENCE: Level III Cohort study.

Regeneration of tracheal neotissue in partially decellularized scaffolds.

Extensive tracheal injury or disease can be life-threatening but there is currently no standard of care. Regenerative medicine offers a potential solution to long-segment tracheal defects through the creation of scaffolds that support the generation of healthy neotissue. We developed decellularized tracheal grafts (PDTG) by removing the cells of the epithelium and lamina propria while preserving donor cartilage. We previously demonstrated that PDTG support regeneration of host-derived neotissue. Here, we use a combination of microsurgical, immunofluorescent, and transcriptomic approaches to compare PDTG neotissue with the native airway and surgical controls. We report that PDTG neotissue is composed of native tracheal cell types and that the neoepithelium and microvasculature persisted for at least 6 months. Vascular perfusion of PDTG was established within 2 weeks and the graft recruited multipotential airway stem cells that exhibit normal proliferation and differentiation. Hence, PDTG neotissue recapitulates the structure and function of the host trachea and has the potential to regenerate.