Preclinical lentiviral vector-mediated hematopoietic stem and progenitor cell gene therapy corrects Pompe disease-related muscle and neurological manifestations.

Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to the accumulation of glycogen in lysosomes and the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and demonstrates poor penetration into skeletal muscle and the central and peripheral nervous systems, susceptibility to immune responses against the recombinant enzyme, and the need for high doses and frequent infusions. To overcome these limitations, lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy has been proposed as a next-generation approach for treating Pompe disease. This study demonstrates the potential of lentiviral HSPC gene therapy to reverse the pathological effects of Pompe disease in a preclinical mouse model. It includes a comprehensive safety assessment via integration site analysis, along with single-cell RNA sequencing analysis of CNS samples to gain insights into the underlying mechanisms of phenotype correction. In order to evaluate the extent of phenotypic correction of microglia in the brain, spleen focus forming virus (SFFV) promoter driven lentiviral vectors SFFV.GILT or SFFV.GAAco vector were used to transduce Lin- cells, which were subsequently infused in Busulfex-treated mice.At 32-weeks after infusion, FACS-sorted CD45+CD11b+CXCR3+ microglia cells from the brain of the GILT vs GAAco treated groups (2 mice per group, 2 samples per mouse) were analyzed by single-cell(sc)RNA-seq (10x genomics). We wanted to formally confirm that the cells isolated from the GILT mice carried a transcriptional profile more similar to normal microglia when compared to the GAAco group, as a result of a more efficient genetic correction driven by the GILT-vector. To this aim, we also analyzed by scRNA-seq the microglia isolated from untreated wild type (WT) C57BL/6 mixed background control mice or Pompe mice (2 mice per group).