Acute and chronic transplant rejection episodes should decline and the probability of long-term graft survival should increase if the expression of heterologous major histocompatibility complex (MHC) antigens can be selectively blocked on pretransplants. To reach this goal we propose a strategy of treating tissues and organs ex vivo pretransplant with novel oligodeoxynucleotides (ODNs) that block transcription and thus prevent expression of MHC class II antigens by i) using transcription factor (TF) decoys to block MHC class II gene activation, and ii) antisense ODNs to block translation of CIITA, a factor that is necessary for class II gene expression. RFX, a TF that binds to class II gene promoters and is essential for class II gene activation, is one target. Decoys will be designed to mimic a region of the promoter and thus complex RFX. As a consequence transcription will be quenched. The transcription activator CIITA is also essential for class II gene expression. We will design and synthesize sequence specific antisense ODNs to selectively bind its mRNA, and consequently, inhibit CIITA translation. Agents generated by each approach are expected to block expression of MHC proteins in organs, tissues and cells intended for transplant. Specifically we will design and synthesize several decoys to mimic key promoter elements and selectively bind class II gene TFs. We will use a chloramphenicol acetyl- transferase expression and directly measure expressed MHC levels to quantify decoy activity. We will evaluate the effect(s) of making changes to the decoy structure. We will similarly quantify the activity, stability, cellular availability and duration of action of several antisense ODNs designed to selectively bind to CIITA mRNA and thereby inhibit its translation, and, as a consequence, class II gene expression. We will evaluate the immunomodulatory properties of the above antisense and decoy ODNs, and postulate structure/sequence/activity relationships. We will assess their duration of function and relate it to their structure and metabolic stability, and attach decoys to magnetic microspheres to isolate those TFs that bind to the decoy-microspheres. We will use Confocal Laser Microscopy to quantify the nonfacilitated cellular uptake of selected ODNs and decoys into human Raji, THP-1, and endothelial cells. We will also evaluate the ability of the chemical modification of selected antisense and decoy ODNs to produce superior cytoplasmic availability, as well as evaluate the ability of a cell membrane penetration enhancers to improve delivery of the above agents into the cytoplasm. Finally we will determine if two different decoys, each designed to mimic a different promoter element, will act synergistically or simply additively, when each has been confirmed to inhibit transcription of the same gene.
