title: Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation.
abstract: Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35) vectors for zinc-finger nuclease (ZFN)- or transcription activator-like effector nuclease (TALEN)-mediated genome editing in human CD34+ hematopoietic stem cells (HSCs) from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA)-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35.ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells). The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2) within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.
authors.affiliation: Division of Medical Genetics, University of Washington , Seattle, Washington, USA.
authors.forename: Kamola
authors.initials: K
authors.lastname: Saydaminova
authors.affiliation: Shanghai Jiao Tong University School of Medicine , Shanghai, PR China.
authors.forename: Xun
authors.initials: X
authors.lastname: Ye
authors.affiliation: Division of Medical Genetics, University of Washington , Seattle, Washington, USA.
authors.forename: Hongjie
authors.initials: H
authors.lastname: Wang
authors.affiliation: Division of Medical Genetics, University of Washington , Seattle, Washington, USA.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Richter
authors.affiliation: Division of Medical Genetics, University of Washington , Seattle, Washington, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Ho
authors.affiliation: Shanghai Jiao Tong University School of Medicine , Shanghai, PR China.
authors.forename: HongZhuan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Shanghai Jiao Tong University School of Medicine , Shanghai, PR China.
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
authors.affiliation: National Creative Initiatives Center for Genome Engineering, Department of Chemistry, Seoul National University , Seoul, Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Division of Hematology, Department of Medicine, University of Washington , Seattle, Washington, USA.
authors.forename: Eirini
authors.initials: E
authors.lastname: Papapetrou
authors.affiliation: Sangamo Biosciences , Richmond, Virginia, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.affiliation: Sangamo Biosciences , Richmond, Virginia, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.affiliation: Baylor College of Medicine , Houston, Texas, USA.
authors.forename: Donna
authors.initials: D
authors.lastname: Palmer
authors.affiliation: Baylor College of Medicine , Houston, Texas, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Ng
authors.affiliation: Universität Witten/Herdecke , Witten, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt
authors.affiliation: Division of Medical Genetics, University of Washington , Seattle, Washington, USA ; Department of Pathology, University of Washington , Seattle, Washington, USA.
authors.forename: André
authors.initials: A
authors.lastname: Lieber