Antifungal Oligonucleotides

Targeting RNA with oligonucleotides is emerging as an important therapeutic strategy to treat certain forms of cancer and some infectious states (Disney et al., 2001; Testa et al., 1999). The antisense oligonucleotide Vitravene (Galderisi et al., 1999), has proven to be effective in treating cytomegalovirus retinitis in AIDS patients intolerant, unresponsive or have contraindications to other treatment(s) of the infection. Gentasense, anti-bcl-2 anti-sense messenger RNA construct, and other oligonucleotide preparations (Childs et al., 2002) are proven potent against acute myeloid leukemia. Features that influence oligonucleotide affinity to bind target RNA and its nuclease stability can be accommodated into the sequence design (Freier & Altman, 1997). Recent evidence suggest that C. albicans takes up significant quantities of oligonucleotides in an energy-dependent manner, taken up oligonucleotides remain stable for >12 h (Disney et al., 2003). A 19-mer with a 2'-O-methyl backbone (19-mer2'-OMe) hairpin oligonucleotide can inhibit the growth of C. albicans in culture at pH < 4.0 which is easily tolerated in anatomic sites subject to candidiasis (Disney et al., 2003). Take up of the oligonucleotide by COS-7 mammalian cells in culture was reported to be tenfold lower than that of C. albicans; oligonucleotide stability inside COS-7 cells was minimal. The oligo-nucleotide failed to inhibit the growth of COS-7 cells suggesting some sort of selectivity in oligonucleotide uptake and stability in favor of fungal cells over mammalian cells (Disney et al., 2003). In vivo dimethylsulfate modifications of rRNA and the decreased rate of protein synthesis suggest that the hairpin oligonucleotides alter ribosomal activity independent of base pairing with target RNA.

RNA targets like rRNA, RNase, P RNA, group I and group II introns and mRNAs with untranslated regulatory sequences require secondary or tertiary folding to function properly (Testa et al., 1999; Disney et al., 2001). Generation of potential oligonucleotides is now achievable by oligonucleotide directed misfold-ing of RNA (ODMiR), which uses short oligonucleotides to stabilize the inactive form of target RNA (Childs et al., 2002). The oligonucleotides L(TACCTTTC) and TLCTLACLGALCGLGCLC that target group I introns of C. albicans were generated by ODMiR and tested on C. albicans in culture. Both oligonucleotides induced mis-folds in group I introns and inhibited 50% of its splicing in transcription mixtures 150 and 30 nM, respectively (Childs et al., 2002). Antisense repression of key genes in pathogenic fungi has been shown to alter the growth and reproduction of fungi (Gorlach et al., 2002). Serotype D yeast transformed with a plasmid containing the calcineurin A (CNA1) cDNA in an antisense orientation under the control of the inducible GAL7 promoter demonstrated a temperature-sensitive phenotype only when grown on galactose, which was shown to be associated with decreased native CNA1 transcript levels. Furthermore, it was possible to modestly impair the growth of C. neoformans at 37°C by a 30 bp antisense oligonucleotide targeting CNA1 (Gorlach et al., 2002).

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