Gene Therapy of the Human Erythrocyte Pyruvate Kinase Deficiency.

Abstract Human Erythrocyte Pyruvate Kinase (hRPK) deficiency, an autosomal recessive disorder produced by mutations in the PKLR gene, is known to be the most common cause of chronic non-spherocytic haemolityc anaemia (CNSHA). Patient survival and clinical severity have been associated with the type of mutation found and to a compensatory expression of the broadly tissue distributed M2PK isozyme in their erythrocytes. Mutations in the RPK gene with severe clinical symptoms have been described in our laboratory. The transduction of the wild-type RPK to hematopoietic stem cells from deficient patients may become an alternative therapy in those patients where the mutation cause clinical symptoms. In an attempt to address this hypothesis we constructed retroviral vectors based on the pSF11 backbone and expressing the hRPK and the green fluorescent protein cDNAs in a unique messenger RNA separated by an eukaryotic IRES (SF11RPKXEG). In a first step, murine and human cell lines were transduced with infective supernatants expressing this construct. FACS and western-blot analysis demonstred stable RPK expression in all transduced cell lines. In murine erithroleukemia cell line (MEL) the stability of the expression was screened for more than 20 weeks by EGFP flow cytometry evaluation. The percentage of transduced cells and the intensity of the expression, evaluated by quantification of the mean of fluorescence (MFI) remain unaltered over time. Moreover, the hexamethylene bisacetamide-induced differentiation of MEL cells neither produce any change in RPK or eGFP expression. Secondly, Lin−Sca-1+ murine hematopoietic stem cells were infected in vitro with SF11RPKXEG supernatants and transplanted into myeloablated recipients. Three months post-transplant, very high expression of both RPK and EGFP proteins was detected in mononuclear cells, platelets and, most importantly, mature erythrocytes, the cell target for the treatment of this disease. These findings show that retroviral vectors could be a successful system for RPK delivery and expression in erythroid cells and provide evidences of the gene therapy potential in the phenotypic correction of erythropoietic defects related to human RPK deficiency.