Ex Vivo Lentiviral Engineering of Rapidly Expanding MSCs Enhances GALNS Expression, Preserves Chondrogenic Function, and Supports HSC Recovery in MPS IVA

Background/Objectives: Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal disorder characterized by severe skeletal dysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Although hematopoietic stem cell transplantation provides partial systemic benefit, its impact on skeletal disease remains limited. Rapidly expanding mesenchymal stem cell clones (REC-MSCs) may enhance skeletal-directed enzyme delivery while supporting hematopoietic cell recovery. This study evaluated lentiviral (LV) engineering of REC-MSCs and MPS IVA mouse hematopoietic stem cells (mHSCs) as an ex vivo strategy for enhancing GALNS expression. Methods: REC-MSCs were transduced with GALNS-expressing LV vectors driven by CBh, CD11b, COL2A1, or MND promoters and assessed for GALNS activity, vector copy number, viability, MSC phenotype, and chondrogenic differentiation potential. Functional interactions between engineered REC-MSCs and LV-transduced mHSCs were evaluated through enzyme transfer and co-culture recovery assays under regular, non-contact, and direct contact conditions. Results: LV engineering markedly increased intracellular and secreted GALNS activity in REC-MSCs, with CD11b and COL2A1 promoters providing the most favorable balance of expression and vector integration at the optimized MOI of 5. Transduced REC-MSCs maintained viability, MSC marker expression, colony-forming capacity, and early chondrogenic characteristics, including spheroid formation, Sox9 expression, and extracellular matrix deposition. GALNS activity and vector integration remained stable following chondrogenic differentiation. Conditioned media from engineered REC-MSCs enabled GALNS uptake by MPS IVA mHSCs, while direct contact REC-MSC/mHSC co-culture enhanced recovery, GALNS activity, and preservation of stem-like hematopoietic populations. Conclusions: These findings demonstrate that promoter-specific LV engineering of REC-MSCs enhances GALNS production while preserving fundamental MSC properties and early chondrogenic potential. In addition, REC-MSCs provide functional support for engineered mHSCs through enzyme transfer and direct stromal interactions. Together, these results establish a mechanistic foundation for future combined REC-MSC/HSC strategies targeting skeletal disease in MPS IVA.