Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus

Abstract

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome

“…Current gene integration approaches primarily rely on nuclease-directed DNA double-stranded breaks (DSBs) to direct cellular DNA repair pathways, such as homologous recombination (HR). Despite important advances in optimizing HR in specific contexts 3,4 , these approaches generally suffer from low insertion efficiency, high indel rates and cargo size limitations, with limited success for cargoes larger than 1 kb [5][6][7] . Furthermore, HR-based gene editing is not feasible in post-mitotic cells, and formation of DSBs is toxic in many primary cell types, leading to undesired deletions, complex rearrangements 8 or activation of…”

Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

“…In fact, several studies reported the enrichment of p53-inactivating mutations following CRISPR-based editing in immortalized human cell lines with constitutive or inducibly expressed Cas9 when a subset of p53 mutant cells were spiked in 1,2 . In contrast, prior studies in human primary cells found that Cas9 RNP delivery did not introduce mutations in p53 or 129 other cancer-related genes (using the Stanford Solid Tumor Actionable Mutation Panel) 36,37 . Therefore, we developed a novel tumor suppressor/oncogene ultra-deep sequencing pipeline to determine whether editing and short-term ex vivo expansion leads to disruption and/or enrichment of cancer-associated variants when delivered in a clinically relevant context-i.e., when high-fidelity Cas9 15 is transiently delivered as RNP via electroporation to human primary HSPCs without subpopulations of cells with pre-existing tumorigenic variants.…”

CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome

“…This makes use of two consecutive HDR steps that first integrate one half of the gene and then the next half, hence overcoming the capacity limit for a single AAV, which potentially would enable a universal HDR approach to target all known DOCK8 mutations present in the reading frame ( Figure 5E ) ( Balakrishnan and Jayandharan, 2014 ). This approach has recently shown promise as a curative correction strategy for cystic fibrosis, which involves the large 4.4 kb CFTR ORF ( Vaidyanathan et al, 2021 ).…”