Over-expression of human lysosomal alpha-mannosidase in mouse embryonic stem cells.
Alpha-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 alpha-mannosidase. One of the major features of alpha-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 alpha-mannosidase (rhalphaM). Two vectors (involving recombinant human alpha-mannosidase expression driven by either the chicken beta-actin promoter/CMV enhancer or by the elongation factor 1-alpha 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 alpha-mannosidase. Stem cell clones transfected with the chicken beta-actin promoter/CMV enhancer maintained rhalphaM expression levels throughout differentiation. This expression was not markedly elevated above background. In contrast, the vector incorporating the elongation factor 1-alpha promoter facilitated substantial over-expression of alpha-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 alpha-mannosidase that could mediate a reduction in the level of oligosaccharides stored by human alpha-mannosidosis skin fibroblasts. These results suggest potential for the use of this cell line for investigation of a stem cell therapy approach to treat alpha-mannosidosis.
Robinson
A J
AJ
Lysosomal Diseases Research Unit, Department of Genetic Medicine, Women's and Children's Hospital, 72 King William Road, North Adelaide, SA 5006, Australia. aaron.robinson@adelaide.edu.au
Crawley
A C
AC
Hopwood
J J
JJ
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology / Q000201:enzymology*; D014162:Transfection; D015854:Up-Regulation; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism*; D008363:alpha-Mannosidosis / Q000628:therapy
D008360:Mannosephosphates; D043323:alpha-Mannosidase