title: Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells.
abstract: Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.
authors.affiliation: Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Republic of Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.|Current address: Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
authors.forename: Jaichandran
authors.initials: J
authors.lastname: Sivalingam
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
authors.forename: Dimitar
authors.initials: D
authors.lastname: Kenanov
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
authors.forename: Hao
authors.initials: H
authors.lastname: Han
authors.affiliation: Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Republic of Singapore.
authors.forename: Ajit Johnson
authors.initials: AJ
authors.lastname: Nirmal
authors.affiliation: Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Republic of Singapore.
authors.forename: Wai Har
authors.initials: WH
authors.lastname: Ng
authors.affiliation: Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Republic of Singapore.
authors.forename: Sze Sing
authors.initials: SS
authors.lastname: Lee
authors.affiliation: CellResearch Corporation, Singapore, Republic of Singapore.
authors.forename: Jeyakumar
authors.initials: J
authors.lastname: Masilamani
authors.affiliation: CellResearch Corporation, Singapore, Republic of Singapore.|Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Toan Thang
authors.initials: TT
authors.lastname: Phan
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, Republic of Singapore.|School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Maurer-Stroh
authors.affiliation: Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Republic of Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Oi Lian
authors.initials: OL
authors.lastname: Kon
mesh_terms: D001665:Binding Sites; D004720:Endonucleases / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D055106:Genome-Wide Association Study; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D020014:K562 Cells; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D019076:Transgenes; D016335:Zinc Fingers
chemical_list: D005169:Factor VIII; D004720:Endonucleases