title: 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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Ding
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Chiao-Wang
authors.initials: CW
authors.lastname: Sun
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Li-Chen
authors.initials: LC
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Dewang
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Pawlik
authors.affiliation: UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Alireza
authors.initials: A
authors.lastname: Khodadadi-Jamayran
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Westin
authors.affiliation: UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|Department of Pediatrics, Division of Hematology/Oncology, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Frederick D
authors.initials: FD
authors.lastname: Goldman
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.|UAB Stem Cell Institute, School of Medicine, University of Alabama at Birmingham, 1720 2nd Ave South, Birmingham, AL 35294, USA.
authors.forename: Tim M
authors.initials: TM
authors.lastname: Townes
mesh_terms: D000256:Adenoviridae / Q000378:metabolism*; D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000628:therapy*; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000378:metabolism*; D006378:Helper Viruses / Q000378:metabolism*; D004854:Herpesvirus 4, Human; D006720:Homozygote; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism