Inosine Monophosphate Dehydrogenase II Mutant (Thr-333-Ile + Ser-351-Tyr) Does Not Confer Resistance to Mycophenolic Acid In Vivo.

Abstract Several immunosuppressive agents are currently being evaluated for their ability to inhibit host immune responses that prevent donor engraftment after reduced intensity allogeneic BMT. Unfortunately, these immunosuppressive agents have the potential to inhibit the beneficial graft-versus-infection and graft-versus-leukemia effects mediated by mature donor T cells that are present in the graft. One potential approach to retain donor T cell function in immunosuppressed recipients after allogeneic BMT is to genetically modify the donor T cells with a drug resistance gene that confers protection from the effects of the immunosuppressive agent. Mycophenolate mofetil (MMF), the morpholino-ethyl ester precursor of the active compound mycophenolic acid (MPA), is an immunosuppressive agent that prevents cell proliferation by inhibiting the enzyme inosine monophosphate dehydrogenase (IMPDH) II and has been used in conjunction with other immunosuppressants to promote donor engraftment and limit graft versus host disease after nonmyeloablative BMT. IMPDH II is the rate-limiting enzyme in de novo guanosine synthesis and is preferentially expressed in activated T and B cells. A number of investigators have identified IMPDH II mutants that have altered MPA binding capacity and normal guanosine synthesis in vitro (BBA 2002; 1594:27, JBC. 1997; 272:961, BBA. 1994; 1217:156). One prototypic mutant (IMPDH*; Thr-333-Ile, Ser-351-Tyr) has been used for in vivo selection of donor T-cells in a canine model (Blood 2003; 102:3696a) We hypothesized that over expression of IMPDH* in donor T cells would provide resistance to MPA in vitro and MMF in vivo, thus allowing MMF to be used in vivo to inhibit recipient immune responses while maintaining normal donor T cell function. In our initial studies, we transduced murine A20 cells (a B-lymphoblastic leukemia) with retroviral vectors encoding IMPDH or IMPDH* fused to the C-terminus of our previously described CD34/HSV-TK chimeric suicide gene. This strategy enabled us to rapidly and efficiently select genetically modified cells using a well-established CD34 immunoselection technique. Unfortunately, the CD34/TK/IMPDH* fusion protein failed to confer MPA resistance to A20 cells in vitro. To determine whether this failure to confer MPA resistance was caused by fusing IMPDH* to CD34/TK, we generated bicistronic retroviral vectors that expressed either IMPDH or IMPDH* and EGFP using an internal ribosome entry site (IRES). Human Jurkat T cells were efficiently transduced with the bicistronic vectors and GFP+ cells were selected using a MoFlo cell sorter. As before, forced expression of IMPDH* failed to confer MPA resistance to the transduced and selected cells in vitro (IC50 = 0.5uM). Interestingly, chronic selection of transduced Jurkat cells in 1 uM MPA for 2 weeks led to a 4 fold increase in the IC50 (IC50 = 2uM MPA). These studies suggest that the resistance to MPA observed in vivo by others for IMPDH* may not be derived from the aforementioned point mutations in IMPDH but rather to over expression or up-regulation of IMPDH II, which may act as a sink for MPA. Alternatively, differences between species and cell lines, introduction of novel mutations during selection, or alternative salvage pathways may be activated resulting in MPA resistance. We subsequently have generated six additional mutants of the MPA binding site based on in vitro data and x-ray crystallography of the MPA binding site and we have began expressing them in our in vivo model. Analysis of these mutants is ongoing.