Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Clchannel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the F508 mutation (found in ~70% of CF alleles and ~90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous (F508/F508) and compound heterozygous (F508/Other) CF patients, we achieved 28 5 % and 42 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Clcurrents relative to non-CF controls: 31 5 % in UABCs and 51 3 % in HBECs (both from subjects homozygous for F508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feederfree, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.
Colorectal cancer (CRC), a disease of high incidence and mortality, exhibits a large degree of inter- and intra-tumoral heterogeneity. The cellular etiology of this heterogeneity is poorly understood. Here, we generated and analyzed a single-cell transcriptome atlas of 49,859 CRC cells from 16 patients, validated with an additional 31,383 cells from an independent CRC patient cohort. We describe subclonal transcriptomic heterogeneity of CRC tumor epithelial cells, as well as discrete stromal populations of cancer-associated fibroblasts (CAFs). Within CRC CAFs, we identify the transcriptional signature of specific subtypes that significantly stratifies overall survival in more than 1,500 CRC patients with bulk transcriptomic data. We demonstrate that scRNA analysis of malignant, stromal, and immune cells exhibit a more complex picture than portrayed by bulk transcriptomic-based Consensus Molecular Subtypes (CMS) classification. By demonstrating an abundant degree of heterogeneity amongst these cell types, our work shows that CRC is best represented in a transcriptomic continuum crossing traditional classification systems boundaries. Overall, this CRC cell map provides a framework to re-evaluate CRC tumor biology with implications for clinical trial design and therapeutic development.
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