title: Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells.
abstract: Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinicalin vitroandin vivotesting to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potentialin vitroandin vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).
authors.affiliation: Laboratory of Cellular Medicine, City of Hope , Duarte, California, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: DiGiusto
authors.affiliation: Department of Molecular Microbiology & Immunology, University of Southern California's Keck School of Medicine , Los Angeles, California, USA.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Cannon
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.affiliation: Laboratory of Cellular Medicine, City of Hope , Duarte, California, USA.
authors.forename: Lijing
authors.initials: L
authors.lastname: Li
authors.affiliation: Laboratory of Cellular Medicine, City of Hope , Duarte, California, USA.
authors.forename: Anitha
authors.initials: A
authors.lastname: Rao
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Lee
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Kim
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Samuel B
authors.initials: SB
authors.lastname: Hayward
authors.affiliation: Sangamo BioSciences Inc , Richmond, California, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Meyer
authors.affiliation: Department of Molecular Microbiology & Immunology, University of Southern California's Keck School of Medicine , Los Angeles, California, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Exline
authors.affiliation: Department of Molecular Microbiology & Immunology, University of Southern California's Keck School of Medicine , Los Angeles, California, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: Lopez
authors.affiliation: Department of Molecular Microbiology & Immunology, University of Southern California's Keck School of Medicine , Los Angeles, California, USA.
authors.forename: Jill
authors.initials: J
authors.lastname: Henley
authors.affiliation: Laboratory of Cellular Medicine, City of Hope , Duarte, California, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Gonzalez
authors.affiliation: Cytogenetics Core Laboratory, City of Hope , Duarte, California, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Bedell
authors.affiliation: Center for Gene Therapy, Hematological Malignancies and Stem Cell Transplantation Institute, City of Hope , Duarte, California, USA.
authors.forename: Rodica
authors.initials: R
authors.lastname: Stan
authors.affiliation: Center for Gene Therapy, Hematological Malignancies and Stem Cell Transplantation Institute, City of Hope , Duarte, California, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Zaia