Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications : Oligonucleotides

Gene Repair

Triplex Helix-Forming Oligonucleotides, Antisense-Induced Exon Skipping, Antisense-Induced Ribonucleoprotein Inhibition.



Triplex Helix-Forming Oligonucleotides


The triplex-forming ONs have been used for site-directed mutagenesis with or without the use of coupled mutagens, as well as homologous-site-spe-cific recombination using triplex-forming ONs alone, or in combination with a donor fragment to correct genetic disorders (Kalish and Glazer, 2005). Although the site-specificity is an important benefit for this technique, a complete understanding of the molecular mechanisms involved is still lacking, and rates of mutagenesis or gene correction are still too poor (£0.1%) to warrant clinical development.


Antisense-Induced Exon Skipping


The exon skipping technique tries to restore the reading frame by artificially removing one or more exons before or after the deletion or point mutation in the mRNA. The most popular disease target to work on is Duchenne’s muscular dystrophy which, using this technique could be changed into the much milder Becker ’s dystrophy (Wilton and Fletcher, 2005).


Exons can be skipped from the mRNA with antisense oligoribonucleotides. They attach inside the exon to be removed, or at its borders. The ONs interfere with the splicing machinery so that the targeted exons are no longer included in the mRNA. In Duchenne’s disease, if the missing amino acids are part of the central region of the dystrophin, they are often not essential, and the resulting shorter protein can still perform its stabilizing role of the muscle cell membrane. The technique is currently in clinical trials for Duchenne’s dystrophy, sponsored by the Imperial College London, UK, after demonstrating pre-clinical efficacy in mice and dogs (Fall et al., 2006; McClorey et al., 2006).


 Antisense-Induced Ribonucleoprotein Inhibition


Antisense ONs can be used to inhibit or alter the functions of ribonucleoproteins by specifically bind-ing to the RNA part of the ribonucleoprotein. For example, telomerase, the enzyme involved in pre-venting the shortening of telomere ends after each cell division, can be inhibited by using ONs directed against the hTERT domain (i.e., RNA binding do-main) (Folini et al., 2002). Antisense-induced telomer-ase inhibition resulted in progressive shortening of telomere ends and in some cases induction of apoptosis. As telomerase activity is found in many types of cancer, antisense inhibition of telomerase may be an effective approach for cancer treatment. Furthermore, antisense ONs have been used for programmed ribosomal frame-shifting to producedifferent proteins from a single open reading frame (Henderson et al., 2006).

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