title: Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy.
abstract: Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan 20132, Italy.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gentner
authors.forename: Ilaria
authors.initials: I
authors.lastname: Visigalli
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Hiramatsu
authors.forename: Eric
authors.initials: E
authors.lastname: Lechman
authors.forename: Silvia
authors.initials: S
authors.lastname: Ungari
authors.forename: Alice
authors.initials: A
authors.lastname: Giustacchini
authors.forename: Giulia
authors.initials: G
authors.lastname: Schira
authors.forename: Mario
authors.initials: M
authors.lastname: Amendola
authors.forename: Angelo
authors.initials: A
authors.lastname: Quattrini
authors.forename: Sabata
authors.initials: S
authors.lastname: Martino
authors.forename: Aldo
authors.initials: A
authors.lastname: Orlacchio
authors.forename: John E
authors.initials: JE
authors.lastname: Dick
authors.forename: Alessandra
authors.initials: A
authors.lastname: Biffi
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002469:Cell Separation; D019610:Cytoprotection; D005698:Galactosylceramidase / Q000378:metabolism; D005786:Gene Expression Regulation; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007965:Leukodystrophy, Globoid Cell / Q000235:genetics* / Q000628:therapy*; D051379:Mice; D016513:Mice, SCID; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity / Q000235:genetics
chemical_list: D035683:MicroRNAs; D005698:Galactosylceramidase