Preclinical development of hematopoietic stem cell-gene therapy for Mucopolysaccharidosis type IVB using a GLB1 transgene with enhanced therapeutic potential.

Mucopolysaccharidosis type IVB (MPSIVB) is a lysosomal storage disorder caused by β-galactosidase (β-GAL) deficiency characterized by severe skeletal and neurological alterations without approved treatments. To develop hematopoietic stem progenitor cell-gene therapy (HSPC-GT) for MPSIVB, we designed lentiviral vectors (LVs) encoding human β-GAL to achieve supraphysiological release of the therapeutic enzyme in human HSPCs and metabolic correction of diseased cells. Transduced HSPCs displayed proper colony formation, proliferation, and differentiation capacity, but their progeny failed to release the enzyme at supraphysiological levels. Therefore, we tested alternative LVs to overexpress an enhanced β-GAL deriving from murine (LV-enhGLB1) and human selectively mutated GLB1 sequences (LV-mutGLB1). Only human HSPCs transduced with LV-enhGLB1 overexpressed β-GAL in vitro and in vivo without evidence of overexpression-related toxicity. Their hematopoietic progeny efficiently released β-GAL, allowing the cross-correction of defective cells, including skeletal cells. We found that the low levels of human GLB1 mRNA in human hematopoietic cells and the improved stability of the enhanced β-GAL contribute to the increased efficacy of LV-enhGLB1. Importantly, the enhanced β-GAL enzyme showed physiological lysosomal trafficking in human cells and was not associated with increased immunogenicity in vitro. These results support the use of LV-enhGLB1 for further HSPC-GT development and future clinical translation to treat MPSIVB multisystem disease.