A major problem with organ/cell transplantation is rejection of the donor graft by the recognition of the cell surface antigens (major histocompatibility complex (HLA) antigens) by the recipient's immune cells. Additional complications result from the immuno-suppressive treatment such as infection and neoplasia. To mitigate these deleterious effects and enhance transplanted graft survival we propose to suppress the expression of the HLA cell surface antigens through the use of oligonucleotides (oligos). We designed two oligos, anti-gene TS1 and antisense A1, to our first target, HLA Class II DR antigen. TS1 inhibits inducible HLA DR expression 100% and partially inhibits constitutive HLA DR expression. TS1 also demonstrates unique features, such as unusual prolonged bioactivity, that may be generalized as transferrable design concepts to the discovery and development of other oligo therapeutics. A1 partially suppresses constitutive HLA DR and has not been tested with inducible HLA DR. The two major challenges in oligo therapy is to enhance oligo stability and cellular uptake. The partial activity on constitutive HLA DR antigen may be attributed to lack of stability and reduced cell uptake. For the most effective therapy, complete suppression of both inducible and constitutive is desirable. To this end we propose to stabilize the oligos against nuclease degradation and enhance cellular uptake. TS1 has a putative quadruplex structure which may stable it against nuclease degradation. TS1 structure will be analyzed by electrophoresis, circular dichroism, thermal melts, and equilibrium sedimentation. We will determine whether structured oligos are more stable to cytoplasmic and purified nucleases by optical absorbance and electrophoretic/filter binding assays. Since cellular uptake may be limiting bioactivity, and since TS1 has unusual prolonged bioactivity, we will determine whether structured and unstructured oligos have different cellular uptake characterizations by assessing the contributions of surface-bound and internalized oligo. Use of reagents will enable assessment of energy, protein, endocytosis, diffusion, ions in cellular uptake. Confocal microscopy will verify subcellular localization patterns. Use of exogenous reagents such as cationic liposomes or cations (e.g. Ca++) will be evaluated for enhancement of cellular uptake. The prolonged TS1 activity may be due to a gene intron target. We propose to determine whether TS1 can form a triple helix with duplex target DNA using electrophoresis. Also, we will study whether TS1 can inhibit intracellular transcription elongation through binding its intron duplex DNA target using Northern blot and transcription run-on assays, and through binding a transfected target plasmid. We propose to determine whether A1 prevents translation or transcription using flow cytometry and Northern blot assays and to maximize the activity by stabilizing to oligo to nucleases by structuring it.

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