Over-expression of human lysosomal α-mannosidase in mouse embryonic stem cells

α-Mannosidosis is a lysosomal storage disorder characterised by the lysosomal accumulation of mannose-containing oligosaccharides and a range of pathological consequences, caused by a deficiency of the lysosomal enzyme α-mannosidase. One of the major features of α-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment. Implantation of stem cells into the central nervous system has been proposed as a potential therapy for these disorders. We report the construction and characterisation of mouse embryonic stem cell lines for the sustained over-expression of recombinant human lysosomal α-mannosidase (rhαM). Two vectors (involving recombinant human α-mannosidase expression driven by either the chicken β-actin promoter/CMV enhancer or by the elongation factor 1-α promoter) were constructed and used to transfect mouse D3 embryonic stem cells. Selected clonal cell lines were isolated and tested to evaluate their expression of recombinant human α-mannosidase. Stem cell clones transfected with the chicken β-actin promoter/CMV enhancer maintained rhαM expression levels throughout differentiation. This expression was not markedly elevated above background. In contrast, the vector incorporating the elongation factor 1-α promoter facilitated substantial over-expression of α-mannosidase when analysed out to 21 days of differentiation in stably transfected cell lines. The highest expressing cell line was found to qualitatively retain a similar differentiation potential to untransfected cells, and to secrete α-mannosidase that could mediate a reduction in the level of oligosaccharides stored by human α-mannosidosis skin fibroblasts. These results suggest potential for the use of this cell line for investigation of a stem cell therapy approach to treat α-mannosidosis.