Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native "wildtype" DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach. Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells. Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Targeted Gene Insertion for Functional CFTR Restoration in Airway Epithelium.

Cystic Fibrosis (CF) is caused by a diverse set of mutations distributed across the approximately 250 thousand base pairs of the CFTR gene locus, of which at least 382 are disease-causing (CFTR2.org). Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach, in principle capable of correcting virtually all CFTR mutations. Provided that such a methodology is capable of efficiently correcting relevant stem cells of the airway epithelium, this could potentially provide life-long correction for the lung. In this Perspective we highlight several requirements for efficient gene insertion into airway epithelial stem cells. In addition, we focus on specific features of the transgene construct and the endogenous CFTR locus that influence whether the inserted gene sequences will give rise to robust and physiologically relevant levels of CFTR function in airway epithelium. Finally, we consider how in vitro gene insertion methodologies may be adapted for direct in vivo editing.

Differentiation of human pluripotent stem cells into functional airway basal stem cells.

Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ''induced basal cells (iBCs)'' from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Correction of Airway Stem Cells: Genome Editing Approaches for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF affects multiple organs, the primary cause of mortality is respiratory failure resulting from poor clearance of hyperviscous secretions and subsequent airway infection. Recently developed CFTR modulators provide significant therapeutic benefit to the majority of CF individuals. However, treatments directed at the underlying cause are needed for the ∼7% of CF patients who are not expected to be responsive to these modulators. Genome editing can restore the native CFTR genetic sequence and function to mutant cells, representing an approach to establish durable physiologic CFTR correction. Although editing the CFTR gene in various airway cell types may transiently restore CFTR activity, effort is focused on editing airway basal stem/progenitor cells, since their correction would allow appropriate and durable expression of CFTR in stem cell-derived epithelial cell types. Substantial progress has been made to directly correct airway basal cells in vitro, theoretically enabling transplantation of autologous corrected cells to regenerate an airway with CFTR functional cells. Another approach to create autologous, gene-edited airway basal cells is derivation of CF donor-specific induced pluripotent stem cells, correction of the CFTR gene, and subsequent directed differentiation to airway basal cells. Further work is needed to translate these advances by developing effective transplantation methods. Alternatively, gene editing in vivo may enable CFTR correction. However, this approach will require robust delivery methods ensuring that basal cells are efficiently targeted and corrected. Recent advances in gene editing-based therapies provide hope that the genetic underpinning of CF can be durably corrected in airway epithelial stem cells, thereby preventing or treating lung disease in all people with CF.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.