title: Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis.
abstract: Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.
authors.affiliation: Department of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Chiriaco
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Giada
authors.initials: G
authors.lastname: Farinelli
authors.affiliation: Department of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Capo
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Erika
authors.initials: E
authors.lastname: Zonari
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Samantha
authors.initials: S
authors.lastname: Scaramuzza
authors.affiliation: Department of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome, Italy.
authors.forename: Gigliola
authors.initials: G
authors.lastname: Di Matteo
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Lucia Sergi
authors.initials: LS
authors.lastname: Sergi
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Migliavacca
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Raisa Jofra
authors.initials: RJ
authors.lastname: Hernandez
authors.affiliation: Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Bombelli
authors.affiliation: Laboratory of Flow Cytometry and B Cell Development, Children's Hospital Bambino Gesù, Rome, Italy.
authors.forename: Ezio
authors.initials: E
authors.lastname: Giorda
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Kajaste-Rudnitski
authors.affiliation: École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
authors.affiliation: Georg-Speyer Haus, Frankfurt, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Grez
authors.affiliation: Department of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.affiliation: Department of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Finocchi
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy; "Vita-Salute" S. Raffaele University, Milan, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gentner
authors.affiliation: Department of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome, Italy; San Raffaele Telethon Institute for Gene Therapy (TIGET), Scientific Institute HS Raffaele, Milan, Italy. Electronic address: aiuti@med.uniroma2.it.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aiuti
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000627:therapeutic use; D006105:Granulomatous Disease, Chronic / Q000473:pathology / Q000628:therapy*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000821:virology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D022423:Myeloid Cells / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*
chemical_list: D018952:Antigens, CD34; D008562:Membrane Glycoproteins; D035683:MicroRNAs; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases