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Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to an accumulation of glycogen in lysosomes, and resulting in the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and has limitations such as poor penetration into skeletal muscle and both the central and peripheral nervous systems, a risk of immune responses against the recombinant enzyme, and the requirement 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 central nervous tissue samples to gain insights into the underlying mechanisms of phenotype correction., Competing Interests: Declaration of interests All authors were former employees of AVROBIO, Inc., Cambridge, MA, USA during the conception and writing of the manuscript, except V.M., M.R., and A.S. N.P.v.T. and C.M. are inventors on patents in the field of HSC gene therapy. AVROBIO, Inc., has a preclinical gene therapy program for Pompe disease (AVR-RD-03) based on a genetically modified HSPC platform using lentiviral vectors. Collection of data and analysis was performed as part of the program. This research received no external funding and was sponsored by AVROBIO, Inc., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation., Competing Interests: All authors are current or former employees of AVROBIO, Inc., Cambridge, MA, USA during the conception and writing of the manuscript, except A.S. AVROBIO, Inc. has a preclinical gene therapy program for Pompe disease (AVR-RD-03) based on a genetically modified HSPC platform using lentiviral vectors. Collection of data and analysis was performed as part of the program. This research received no external funding and was sponsored by AVROBIO, Inc., (© 2022 The Authors.)

Mutations in glucocerebrosidase (GBA1) cause Gaucher disease and also represent a common risk factor for Parkinson's disease and Dementia with Lewy bodies. Recently, new tool molecules were described which can increase turnover of an artificial substrate 4MUG when incubated with mutant N370S GBA1 from human spleen. Here we show that these compounds exert a similar effect on the wild-type enzyme in a cell-free system. In addition, these tool compounds robustly increase turnover of 4MUG by GBA1 derived from human cortex, despite substantially lower glycosylation of GBA1 in human brain, suggesting that the degree of glycosylation is not important for compound binding. Surprisingly, these tool compounds failed to robustly alter GBA1 turnover of 4MUG in the mouse brain homogenate. Our data raise the possibility that in vivo models with humanized glucocerebrosidase may be needed for efficacy assessments of such small molecules.