Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models

Abstract

Leukodystrophies are rare diseases caused by defects in the genes coding for lysosomal enzymes that degrade several glycosphingolipids. Gene therapy for leukodystrophies requires efficient distribution of the missing enzymes in CNS tissues to prevent demyelination and neurodegeneration. In this work, we targeted the external capsule (EC), a white matter region enriched in neuronal projections, with the aim of obtaining maximal protein distribution from a single injection site. We used bidirectional (bd) lentiviral vectors (LV) (bdLV) to ensure coordinate expression of a therapeutic gene (beta-galactocerebrosidase, GALC; arylsulfatase A, ARSA) and of a reporter gene, thus monitoring simultaneously transgene distribution and enzyme reconstitution. A single EC injection of bdLV.GALC in early symptomatic twitcher mice (a murine model of globoid cell leukodystrophy) resulted in rapid and robust expression of a functional GALC protein in the telencephalon, cerebellum, brainstem and spinal cord. This led to global rescue of enzymatic activity, significant reduction of tissue storage and decrease of activated astroglia and microglia. Widespread protein distribution and complete metabolic correction were also observed after EC injection of bdLV.ARSA in a mouse model of metachromatic leukodystrophy. Our data indicated axonal transport, distribution through cerebrospinal fluid flow and cross-correction as the mechanisms contributing to widespread bioavailability of GALC and ARSA proteins in CNS tissues. LV-mediated gene delivery of lysosomal enzymes by targeting highly interconnected CNS regions is a potentially effective strategy that, combined with a treatment able to target the PNS and peripheral organs, may provide significant therapeutic benefit to patients affected by leukodystrophies.

Normalization and Improvement of CNS Deficits in Mice With Hurler Syndrome After Long-term Peripheral Delivery of BBB-targeted Iduronidase

“…Several therapeutic strategies have been explored using viral vectors to treat neurological lysosomal storage disorders in adult animal models, including direct in vivo injection of adeno-associated virus or LV by routes of intracranial, [25][26][27] intrathecal, 28,29 and/or systemic delivery, [30][31][32] and LV-mediated HSC gene therapy using either myeloid 33 or erythroid/megakaryocyte 15 restricted or ubiquitous overexpression with strong viral promoter. 9 Correction of CNS abnormalities is commonly associated with high levels of enzyme activities in the brain (17-250% of normal) because the main enzyme-generating source is either within the CNS or on the BBB-forming brain endothelial cells, or it requires 1.2-12 VCN/genome in cases of HSC-mediated approaches.…”

Experimental Therapies in the Murine Model of Globoid Cell Leukodystrophy

“…Of the viruses that have been tested in the treatment of murine GLD 5, 34–38 , recombinant adeno-associated viruses (AAVs) have shown the most potential for the treatment of the neurological sequelae.…”

Recent Advances in RNA Interference Therapeutics for CNS Diseases

“…Both viruses are minimally immunogenic and can transduce a number of CNS cell types. Recombinant lentiviruses are pseudotyped with various glycoproteins that can impart different tropisms after directed delivery into brain [47,48], and they have been used successfully in gain-of-function [49] and loss of function studies [50][51][52][53].One difference between lentivirus and AAV or adenovirus-based systems is the level of expression.This is due, in part, because LV-mediated transduction often results in low copy numbers of transgene/cell. Also, the placement of the expression cassette in the LV genome can affect expression levels [54].Most LV vectors integrate unless the integrase activity has been inactivated.As integrase-deficient vectors often have low titers compared with their integrase competent counterparts, their production for use for therapeutic applications is impractical.Integration competency for CNS applications may be less of an issue than in the setting of stem cell transduction (most cells in the CNS are not dividing), where integration and activation of an oncogenic gene provides a growth advantage for the transformed cell [55,56].…”