Higher efficiency of TA-clamp modified single stranded oligonucleotides in targeted nucleotide sequence correction is not correlated to a lower intracellular degradation

Abstract

Specific single-stranded oligonucleotides can induce targeted nucleotide sequence correction in eukaryotic genes in vitro and in vivo. Our model for investigating the reasons for the low correction rates achieved by this method is the correction of a point mutation in the hypoxanthine-guanine phosphoribosyltransferase gene (hprt) in the cell line V79-151. Using single-stranded phosphorothioate-modified oligonucleotides, the correction rates of this hprt mutation were low but always reproducible. One reason for low exchange rates may be fast intracellular degradation of the oligonucleotides. Therefore we compared the exchange rates of different 3' and 5' end-modified oligonucleotides with their degradation rates. Thymine-adenine (TA) repeat (clamp)-modified oligonucleotides showed higher correction rates than those with a guanine-cytosine (GC) clamp and 5' clamps induced higher correction rates than clamps at the 3' end. Experiments on the stability of the most effective 5'-TA and 3'-TA clamp-modified oligonucleotide indicated rapid cleavage and the occurrence of shortened oligonucleotides in the presence of cytoplasmic and nuclear extracts. The phosphorothioate-modified oligonucleotides were more stable, but their correction rates were lower. We suggest that there is no direct correlation between the biological stability of the full-length oligonucleotides and the exchange rates achieved.

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

“…3). SSOs were initially evaluated using plasmids carrying reporter genes (Campbell et al, 1989) or in the targeting of the HPRT1 gene in lymphoblasts (HungerBertling et al, 1990;Kenner et al, 2002Kenner et al, , 2004Hegele et al, 2008;Wuepping et al, 2009). Although SSOs have been synthesized with phosphorothioate backbones (Gamper et al, 2000b), 2 0 -O-methyl uracil (Igoucheva et al, 2001) or with 5 0 or 3 0 thymidine clamps (Hegele et al, 2008) to inhibit degradation, they have also been used without modification to facilitate homologous exchange.…”

“…Further, an examination of the sequences, that is, base composition, could be important in establishing reproducibility. Along those lines, one study indicated that a TA repeat at the 5 0 end of an SSO is more effective at facilitating homologous exchange (Wuepping et al, 2009). Sequence is particularly important for TFO recognition and sequence-specific modification (Fox and Brown, 2005).…”