title: Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases.
abstract: The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.
authors.affiliation: Laboratory for Cellular Medicine, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Lijing
authors.initials: L
authors.lastname: Li
authors.forename: Ludmila
authors.initials: L
authors.lastname: Krymskaya
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.forename: Jill
authors.initials: J
authors.lastname: Henley
authors.forename: Anitha
authors.initials: A
authors.lastname: Rao
authors.forename: Lan-Feng
authors.initials: LF
authors.lastname: Cao
authors.forename: Chy-Anh
authors.initials: CA
authors.lastname: Tran
authors.forename: Monica
authors.initials: M
authors.lastname: Torres-Coronado
authors.forename: Agnes
authors.initials: A
authors.lastname: Gardner
authors.forename: Nancy
authors.initials: N
authors.lastname: Gonzalez
authors.forename: Kenneth
authors.initials: K
authors.lastname: Kim
authors.forename: Pei-Qi
authors.initials: PQ
authors.lastname: Liu
authors.forename: Ursula
authors.initials: U
authors.lastname: Hofer
authors.forename: Evan
authors.initials: E
authors.lastname: Lopez
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Paula M
authors.initials: PM
authors.lastname: Cannon
authors.forename: John A
authors.initials: JA
authors.lastname: Zaia
authors.forename: David L
authors.initials: DL
authors.lastname: DiGiusto
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000628:therapy; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D015497:HIV-1; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D019713:Receptors, CCR5 / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; D019713:Receptors, CCR5; D016179:Granulocyte Colony-Stimulating Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D004720:Endonucleases