Correction of Sanfilippo A Skin Fibroblasts by Retroviral Vector-Mediated Gene Transfer

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Abstract
The recent cloning of the sulfamidase gene has made possible the consideration of gene-based therapies for Sanfilippo A syndrome (mucopolysaccharidosis type IIIA), one of the most common of the mucopolysaccharidoses. In this paper, we present the construction of a retroviral vector in which a sulfamidase cDNA is under the transcriptional control of the Moloney murine leukemia virus long terminal repeat. This construct was used to make a high-titer (4 × 105 colony-forming units/ml) producer cell line, PA317/LNSSN#19, in the amphotropic packaging cell line PA317. This producer cell line was shown to be helper virus free using an assay for horizontal spread of virus. Virus supernatant from PA317/LNSSN#19 was used to transduce Sanfilippo A fibroblasts, resulting in complete correction of both the enzymatic defect and the storage phenotype as assessed by intracellular accumulation of 35SO4¯-labeled material. Phenotypic correction was seen even when the levels of viral transduction were low. These results show that gene therapy of the Sanfilippo A syndrome is practicable, although the nature of the disorder suggests that careful consideration needs to be given to the choice of the cellular target for gene transfer. Gene therapy appears to be one of the best long-term approaches to the treatment of the mucopolysaccharidoses. We present initial data supporting this contention for one of the most common of these disorders, Sanfilippo A syndrome. A retroviral construct that transduces the human sulfamidase gene was made and used to generate high-titer retroviral stocks. These were used to transfer the sulfamidase gene into Sanfilippo A fibroblasts, resulting in the enzymatic and biochemical correction of the cells. Efficient biochemical correction was seen even in cultures in which the level of transduction was low, presumably due to cross-correction of untransduced cells via secretion of enzyme into the culture media and subsequent endocytosis via the mannose-6-phosphate receptor.