Abstract
Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life−long intravenous administration of recombinant glucocerebrosidase and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient’s own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebrosidase-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.
Highlights
Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity
The effectiveness of macrophage-targeted enzyme replacement (ERT) and allo-HSCT for treating GD type 1 (GD1) suggests that restoration of GCase function in macrophages alone is sufficient for phenotypic correction in GD1
The ribonucleoprotein complex (RNP) binds to the target sequence and Cas[9] catalyzes a double-stranded break, stimulating one of two repair pathways: (1) non-homologous end joining (NHEJ), in which broken ends are directly ligated, often producing small insertions and deletions; and (2) homology-directed repair (HDR), in which recombination with the supplied homologous repair template is used for precise sequence changes[21]
Summary
Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity. We report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells It constitutes an adaptable platform for other lysosomal enzyme deficiencies. Unlike ERT and the best tolerated SRT, it could provide enzyme reconstitution in the brain that could benefit neuronopathic forms of the disease[9] For these reasons, non-targeted gene addition into human hematopoietic stem and progenitor cells (HSPCs) have been explored, first using retroviruses[12,13,14,15] and later lentiviral vectors, and have yielded promising results in murine GD models[16,17,18]. This gene encodes the major co-receptor for HIV-1, and is considered a non-essential locus because of the high prevalence of healthy homozygous CCR5Δ32 individuals in European populations (>10%)[27] and the observation that homozygous carriers of the Δ32 mutation are resistant to HIV-1 infection[28]
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