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

Chao Li,Dewang Zhou,Erik Westin,Li-Chen Wu,Frederick D Goldman,Alireza Khodadadi-Jamayran,Kevin M Pawlik,Lei Ding,Tim M Townes,Chiao-Wang Sun

doi:10.1038/srep30422

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% (βA/[βS+βA]) 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.

Highlights

Following the seminal discovery of induced Pluripotent Stem Cells by Takahashi and Yamanaka[4], iPSCs have been used extensively as a tool for studying normal development and for developing new strategies for regenerative medicine and patient-specific cell therapy
The 6-factor HDAd/EBV vector that we report in this paper provides a foundation for production of additional vectors containing ten or more reprogramming factors that increase the efficiency and rapidity of iPSC production
We developed a novel hybrid HDAd/EBV reprogramming vector and proved the feasibility of using this vector to convert human sickle cell disease (SCD) patient keratinocytes to iPSCs with high efficiency

Summary

Introduction

Following the seminal discovery of induced Pluripotent Stem Cells (iPSCs) by Takahashi and Yamanaka[4], iPSCs have been used extensively as a tool for studying normal development and for developing new strategies for regenerative medicine and patient-specific cell therapy. Episomal reprogramming, which was first reported by the Thomson laboratory[5] and later improved by the Yamanaka laboratory[6,7], provides a method that avoids random chromosomal integration of exogenous reprogramming factors. These methods rely on electroporation of 3–4 plasmids to deliver 6–7 factors into target cells. We report the development of an efficient, rapid, and scarless CRISPR/Cas method to correct the sickle mutation in patient-derived iPSCs

Methods

Results

Conclusion