title: Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.
abstract: Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV-resistant progeny.
  
  
  OBJECTIVE
  Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland Department of Immunology and Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
authors.forename: Renier
authors.initials: R
authors.lastname: Myburgh
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ivic
authors.affiliation: Department of Immunology and Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Pepper
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Gers-Huber
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Duo
authors.initials: D
authors.lastname: Li
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Annette
authors.initials: A
authors.lastname: Audigé
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland.
authors.forename: Mary-Aude
authors.initials: MA
authors.lastname: Rochat
authors.affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Vincent
authors.initials: V
authors.lastname: Jaquet
authors.affiliation: Clinic of Immunology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Regenass
authors.affiliation: Division of Hematology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Markus G
authors.initials: MG
authors.lastname: Manz
authors.affiliation: Department of Neurosciences, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Salmon
authors.affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Krause
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland roberto.speck@usz.ch.
authors.forename: Roberto F
authors.identifier: http://orcid.org/0000-0002-8453-1137
authors.initials: RF
authors.lastname: Speck
mesh_terms: D000818:Animals; D060467:Disease Resistance; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D015658:HIV Infections / Q000276:immunology* / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000821:virology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D016513:Mice, SCID; D019713:Receptors, CCR5 / Q000378:metabolism*; D015728:Receptors, HIV / Q000037:antagonists & inhibitors*; D014180:Transplantation; D019562:Viral Load; D053585:Virus Attachment
chemical_list: C584933:CCR5 protein, human; D019713:Receptors, CCR5; D015728:Receptors, HIV