Homology Requirements for Efficient, Footprintless Gene Editing at the CFTR Locus in Human iPSCs with Helper-dependent Adenoviral Vectors

Abstract

Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb, helper-dependent adenoviral vectors with long homology arms are used for gene editing. However, this makes vector construction and recombinant analysis difficult. Conversely, insufficient homology may compromise targeting efficiency. Thus, we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology, the frequencies of targeted recombinants were 50–64.6% after positive selection for vector integration, and 97.4–100% after negative selection against random integrations. With 14.8 kb, the frequencies were 26.9–57.1% after positive selection and 87.5–100% after negative selection. With 9.6 kb, the frequencies were 21.4 and 75% after positive and negative selection, respectively. With only 5.6 kb, the frequencies were 5.6–16.7% after positive selection and 50% after negative selection, but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore, we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However, low frequencies (≤ 1 × 10−3) necessitated negative selection for piggyBac-excision product isolation.

Gene Editing with Helper-Dependent Adenovirus Can Efficiently Introduce Multiple Changes Simultaneously over a Large Genomic Region

“…These analyses revealed that of the 95 clones analyzed, 85 (89.5%) were correctly targeted, 2 (2.1%) were aberrantly targeted (1 with correct targeting only in the 5′ homology arm and the other with correct targeting only in the 3′ homology arm), and 8 (8.9%) had random vector integration. The Southern blots also revealed the continued presence of the 31 kb band when analyzed with the external 5′ and 3′ probes (Figure 1C), indicating that targeted vector integration had occurred in only one of the two CFTR alleles in all 85 targeted recombinants, a result consistent with our previous study 13 . The frequency of targeting with HD-23.8-CFTRm-PACTk-DTA was not reduced (and perhaps was even a little higher) compared to HD-23.8-CFTR-PACTk, 13 indicating that the twelve 2 bp insertions in the region of homology did not negatively affect targeting efficiency.…”

“…Altogether, these studies have consistently demonstrated that HDAd-mediated gene editing of iPSCs and ESCs is not associated with ectopic random HDAd integrations, does not affect the undifferentiated state and pluripotency, and maintains genetic and epigenetic integrity. One study found that targeted gene correction in iPSCs by HDAd minimally affects whole-genome mutational load, as determined by whole genome sequencing, 9 and we have demonstrated that footprintless gene editing can be efficiently achieved in human iPSCs with HDAd 13 …”

“…As mentioned, HDAds can accommodate long homology arms, which we have shown to enhance the efficiency of gene editing 13 . In addition, Liu et al 4 .…”

“…We and others have shown that helper-dependent adenoviral vectors (HDAds) can efficiently deliver donor DNA into human embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells to achieve high-efficiency gene editing by spontaneous homologous recombination (HR) to a variety of transcriptionally active and inactive loci and thus to achieve knockins, knockouts, and corrections 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. Altogether, these studies have consistently demonstrated that HDAd-mediated gene editing of iPSCs and ESCs is not associated with ectopic random HDAd integrations, does not affect the undifferentiated state and pluripotency, and maintains genetic and epigenetic integrity.…”

Transformative therapies for rare CFTR missense alleles

“…Basic and clinical investigations are in progress to explore feasibility of innovative genetic and genomic medicine technologies, including transfer of nucleic acids by airway stem/progenitor cells [41,42], zinc finger nuclease-or CRISPR/Cas9-edited human pluripotent stem cells [30,31,32], and nanoparticles [43,44,45], as well as protein replacement via mRNA transfer [46]. Recently, enhanced adenoviral and lentiviral vectors were used to show functional CFTR gene delivery to airways of the CF porcine model [47,48], and the first lentivirus-based clinical trial is scheduled for 2017 [49].…”