Additional antigene agents RNA interference and ribozymes

RNAi and ribozymes represent two additional approaches to gene silencing/down-regulation with therapeutic potential. RNAi is an innate cellular process that achieves silencing of selected genes via an antisense mechanism. It shares many characteristics with the antisense-based approach described above, but also some important differences, e.g. in the exact mechanism by which the antisense effect is achieved.

RNAi probably evolved initially in primitive organisms in order to protect their genomes from viruses, transposons and additional insertable genetic elements, and to regulate gene expression. The RNAi pathway was first discovered in plants, but it is now known to function in most, if not all, eukaryotes.

RNAi represents a sequence-specific post-translational inhibition mechanism of gene expression, induced ultimately by dsRNA, be it produced naturally or synthesised in vitro and introduced into a cell. Entry of dsRNA triggers its cleavage into short (21-23 nucleotide long) sequences called short interfering RNAs (siRNAs). This cleavage is catalysed by a cellular nuclease enzyme called 'Dicer'. The siRNA is incorporated into a multi-subunit effector complex known as an RNA-induced silencing complex (RISC), which also contains several nucleic acid processing enzymes (a helicase, an endonuclease and an exonuclease). The double-stranded siRNA then unwinds (a process promoted by the helicase activity), and the 'sense' strand of the dsRNA is discarded. The remaining 'antisense' siRNA strand then facilitates RISC binding to a specific mRNA via Watson-Crick base complementarity, which is then degraded by RISC nuclease activity.

RNAi technology has obvious therapeutic potential as an antisense agent, and initial therapeutic targets of RNAi include viral infection, neurological diseases and cancer therapy. The synthesis of dsRNA displaying the desired nucleotide sequence is straightforward. However, as in the case of additional nucleic-acid-based therapeutic approaches, major technical hurdles remain to be overcome before RNAi becomes a therapeutic reality. Naked unmodified siRNAs for example display a serum half-life of less than 1 min, due to serum nuclease degradation. Approaches to improve the RNAi pharmacokinetic profile include chemical modification of the nucleotide backbone, to render it nuclease resistant, and the use of viral or non-viral vectors, to achieve safe product delivery to cells. As such, the jury remains out in terms of the development and approval of RNAi-based medicines, in the short to medium term at least.

Certain RNA sequences can function as catalysts. These so-called ribozymes function to catalyse cleavage at specific sequences in a specific mRNA substrate. Many ribozymes will cleave their target mRNA where there exists a particular triplet nucleotide sequence G-U-C. Statistically, it is likely that this triplet will occur at least once in most mRNAs.

Ribozymes can be directed to a specific mRNA by introducing short flanking oligonucleotides that are complementary to the target mRNA (Figure 14.16). The resultant cleavage of the target

Target mRNA

Cleaved mRNA

Nucleotide sequence at which — ribozyme can cleave

Ribozyme

A

U-

C

G

C

G

G

C

C

G

G

C

C

G

A

U

Flanking sequences which 'dock' ribozyme at the appropriate sequence of the appropriate mRNA via complementary base pairing

Free ribozyme

Figure 14.16 Outline of how ribozyme technology could prevent translation of specific mRNA, thus preventing synthesis of a specific target protein obviously prevents translation. One potential advantage of ribozymes is that, as catalytic agents, a single molecule could likely destroy thousands of copies of the target mRNA. Such a drug should, therefore, be very potent. Again, however, ribozymes suffer from similar complications to anti-sense-based products in terms of their development as biopharmaceuticals, and no such product is likely to gain approval for some time to come.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment