Retrovirally transduced PPCA^−/− BM cells restore cathepsin A activity in transplanted PPCA^−/−recipients.

(A) Schematic diagram of the retroviral bicistronic construct encoding the human PPCA cDNA. In this vector, expression of both the human PPCA and the GFP marker is driven by the viral LTR. Translation of GFP is initiated at the internal ribosomal entry site (IRES). (B) Platelets, white blood cells (WBCs), and erythrocytes (RBCs), obtained from PPCA^−/−mice transplanted with either MSCV-GFP– or MSCV-PPCA-GFP–transduced PPCA^−/−BM, at different time points after treatment, were FACS sorted and analyzed for GFP expression; 1 month (1 month, 7 mice total); 3 months (3 months, 6 mice total); LT (long-term, ages 6, 8, 9, and 10 months, 5 mice total). (C) Cathepsin A (CA) activity was assayed in tissue-homogenates of MSCV-PPCA-GFP–treated mice, at different time points after treatment. Wild-type (WT, 4 mice total) and MSCV-GFP–treated PPCA^−/−mice (GFP, 4 mice total), ranging in age between 3 and 8 months, were used as positive and negative controls, respectively. The level of CA activity was independent from the age of the wild-type or MSCV-GFP–treated mice. The inset shows the CA activity in brain homogenates ofPPCA^−/−treated and untreated mice, as well as controls. For the CA activity in the thymus of 1-month–treated mice, only one tissue sample was collected and measured; 1 month (1 month, 4 mice total); 3 months (3 months, 3 mice total); LT (long-term, ages 6, 8, 9, and 10 months, 5 mice total). We observed considerable variation in the measured CA activity between mice of the same age group, likely due to differences in engraftment of the transplanted BM cells. The presented data are average values with a typical high and low limit of ± 25% to 50%, which is significantly above the CA activity of the GFP controls. The bars represent SDs.