Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells
Alessandra Biffi, Michele De Palma, Angelo Quattrini, Ubaldo Del Carro, Stefano Amadio, Ilaria Visigalli, Maria Sessa, Stefania Fasano, Riccardo Brambilla, Sergio Marchesini, Claudio Bordignon, Luigi Naldini
Alessandra Biffi, Michele De Palma, Angelo Quattrini, Ubaldo Del Carro, Stefano Amadio, Ilaria Visigalli, Maria Sessa, Stefania Fasano, Riccardo Brambilla, Sergio Marchesini, Claudio Bordignon, Luigi Naldini
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Article Genetics

Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells

Abstract

Gene-based delivery can establish a sustained supply of therapeutic proteins within the nervous system. For diseases characterized by extensive CNS and peripheral nervous system (PNS) involvement, widespread distribution of the exogenous gene may be required, a challenge to in vivo gene transfer strategies. Here, using lentiviral vectors (LVs), we efficiently transduced hematopoietic stem cells (HSCs) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice and correct a neurodegenerative disorder, metachromatic leukodystrophy (MLD). We proved extensive repopulation of CNS microglia and PNS endoneurial macrophages by transgene-expressing cells. Intriguingly, recruitment of these HSC-derived cells was faster and more robust in MLD mice. By transplanting HSCs transduced with the arylsulfatase A gene, we fully reconstituted enzyme activity in the hematopoietic system of MLD mice and prevented the development of motor conduction impairment, learning and coordination deficits, and neuropathological abnormalities typical of the disease. Remarkably, ex vivo gene therapy had a significantly higher therapeutic impact than WT HSC transplantation, indicating a critical role for enzyme overexpression in the HSC progeny. These results indicate that transplantation of LV-transduced autologous HSCs represents a potentially efficacious therapeutic strategy for MLD and possibly other neurodegenerative disorders.

Authors

Alessandra Biffi, Michele De Palma, Angelo Quattrini, Ubaldo Del Carro, Stefano Amadio, Ilaria Visigalli, Maria Sessa, Stefania Fasano, Riccardo Brambilla, Sergio Marchesini, Claudio Bordignon, Luigi Naldini

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Identification of bone marrow–derived vector-expressing cells in the CNS...
Identification of bone marrow–derived vector-expressing cells in the CNS of transplanted mice. Immunofluorescence analysis of cryostatic sections from the brains of transplanted mice. Fluorescent signals from single optical sections were sequentially acquired and are shown individually and after merging (merge). Immunostaining for GFP (green), F4/80, NeuN, GFAP, or CD45.1 (red) are indicated. (A) Representative sections from the cerebellums of transplanted mice, analyzed at 3 months (left panel, scale bar: 200 μm) and 6 months (middle and right panels, scale bar: 300 μm) after BMT. Three months after BMT, few ramified cells were identified in the upper cortical layers near the meninges. By 6 months after BMT, several GFP+ cells were present in the cortex, and small clusters of ramified GFP+ cells were detected throughout the parenchyma. (B–E) Representative brain sections from transplanted mice 9 months after BMT, immunostained as indicated. (B) GFP+ cells showed a ramified, microglial morphology and F4/80 immunoreactivity. Scale bar: 100 μm. (C and D) Overlay of GFP staining with the neuronal-specific marker NeuN (C) and the astrocytic marker GFAP (D) demonstrated separate localization of the two signals, with GFP+ ramified cells found between neurons and astrocytes. Scale bar: 300 μm. (E) CD45.1 immunostaining identified GFP+ cells as donor-derived. Scale bar: 70 μm.