Elongated polyglutamine stretch in mutant huntingtin (mhtt) correlates well with the pathology of Huntington's disease (HD). Inhibition of aggregation of mhtt is a promising strategy to arrest disease progression. In this work, specific, high-affinity RNA aptamers were selected against monomeric mhtt (51Q-htt). Some of them inhibited its aggregation in vitro by stabilizing the monomer. They also recognized 103Q-htt but not 20Q-htt (nonpathogenic length). Inhibition of aggregation corresponded with reduced leakage of a fluorescent probe from liposomes and diminished oxidative stress in RBCs. The presence of aptamers was able to rescue the sequestration of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by aggregated mhtt. Some of the aptamers were able to enhance the partitioning of mhtt in the soluble fraction in a yeast model of HD. They were also able to rescue endocytotic defect due to aggregation of mhtt. The beneficial effect of a combination of aptamers was enhanced with improvement in cell survival. Since HD is a monogenic autosomal dominant disorder, aptamers may be developed as a viable strategy to slow down the progress of the disease. Since they are nonimmunogenic and nontoxic, aptamers may emerge as strong candidates to reduce protein-protein interaction and hence protein aggregation in protein misfolding disorders in general.

Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.

No abstract