Long-term regulated expression of Epo using second-generation AAV vectors and rapamycin or rapamycin analogs. (A) High vector doses. Primate FTF was cotransduced with AAV-CMV-TF1Nc and AAV-Z12I-rhEpo-2 once at the beginning of the study and subsequently administered rapamycin (▵) or rapamycin analogs intravenously (AP22594, ▾ ; AP22660, ♦; AP21370, •; AP23054, ⊕), or AP22594 orally (⋄), at the indicated doses (mg/kg). Beginning at year 4.2, AP22594 was dosed intravenously weekly at 0.02 mg/kg 4 times, 0.01 mg/kg 2 times, and 0.015 mg/kg 7 times in consecutive weeks. Serum Epo levels (• with solid line) and hematocrit levels (⋄ with solid line) are also given. (B) Lower vector doses. Primate 97E099 was transduced similarly to FTF but using a 17-fold-lower total dose of vector (Table 1). Weekly dosing was performed as described for primate FTF (A), but beginning at year 3.2. Epo levels peaked at 747 mU/mL after the first induction as indicated. (C) Chemical structure of rapamycin analog AP22594. The arrow indicates the position of epimerization with respect to rapamycin (C28). (D) Reduced immunosuppressive activity of AP22594 in a mouse model of immune rejection. C57bl/6 mice were grafted with skin patches from Balb/c mice, and animals subsequently received daily subcutaneous administrations of rapamycin (▪) or AP22594 (▧) at the indicated dose, or vehicle (□). The time for complete graft rejection is plotted (mean ± SD).