Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

Abstract

CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (β(A)/[β(S)+β(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications.

Figure 1. SCD patient-specific iPSCs derived with a novel 6-factor HDAd/EBV hybrid reprogramming vector.

(a) Schematic representation of two circularization strategies for HDAd/EBV hybrid vectors in transduced cells to produce episomes. The vector is efficiently circularized by recombinase systems, e.g. Flp/FRT. In the absence of recombinase, a small but significant percentage of HDAd/EBV vectors spontaneously circularize in transduced cells. (b) Timeline of SCD patient fibroblast reprogramming with pCLEB-R6 or keratinocytes with rCLAE-R6. (c) Alkaline phosphatase (AP) staining of reprogrammed SCD keratinocytes. Twenty-four days post-transduction with rCLAE-R6 (left) and rHDAdV-R6 (right). (d) PCR amplification of oriP in passage 3 (left) and passage 20 (right) SCD iPSCs to detect residual rCLAE-R6. We analyzed six colonies, and all six were free of rCLAE-R6 after passage 20. Only one iPS colony is shown in Fig. 1d (last lane in each panel). OriP/EBNA1 vectors replicate only once per cell cycle; therefore, in the absence of selection, episomes are lost at a rate of approximately 5% per cell generation due to defects in plasmid replication and partitioning. The second lane in each panel (pCLEB-R6) is a PCR control using untranduced plasmid DNA. (e)X-Gal staining of reprogrammed SCD keratinocytes 24 days post-transduction with rCLAE-R6, which contains LacZ. LacZ was only expressed in parental keratinocytes and not in iPSC clones. (f) Hematoxylin and eosin staining of teratoma sections derived from SCD iPSCs.

Figure 2. Rapid isolation of homozygously corrected SCD patient-derived iPSCs by sib-selection.

(a) Schematic representation of the strategy to rapidly isolate corrected iPSCs. (b) HBB corrected allele frequency as measured by ddPCR after the first sib-selection. Green bar: β^S-positive droplets; Blue bar: β^A-positive droplets. (c) HBB corrected allele frequency as measured by ddPCR after the second sib-selection. Green bar: β^S-positive droplets; Blue bar: β^A-positive droplets. (d) Representative Sanger sequencing results of PCR amplicons generated from an uncorrected SCD iPSC line, a heterozygously corrected line, a homozygously corrected line, and a singly-corrected allele/indel line.

Figure 3. Improved targeting efficiency with a CRISPR/Cas-expressing adenoviral vector.

(a) Schematic representation of the first generation adenoviral vector used to express CRISPR/Cas. (b) HBB corrected allele frequency as measured by ddPCR in pooled SCD iPSCs at 72 hr post-transduction of rAd-T2 + ssODN. Green bar: β^S-positive droplets; Blue bar: β^A-positive droplets. (c) Sanger sequencing results of PCR amplicons generated from pooled SCD iPSC lines transduced with rAd-T2 + ssODN.