RNAi - transcriptional level knockdown of pharmacologically relevant genes

[mitogen]

[the following is from a review on the therapeutic implications of RNAi: Leiberman et al. "Interfering with disease: opportunities and roadblocks to harnessing RNA interference," Trends in Molecular Medicine, Vol.9 No.9, September 2003]

"RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA transcripts. Short double-stranded RNAs known as small interfering RNAs (siRNA) are incorporated into an RNA-induced silencing complex (RISC) that directs degradation of RNA containing a homologous sequence. RNAi has been shown to work in mammalian cells, and can inhibit viral infection and control tumor growth in vitro. Recently, it has been shown that intravenous injection of siRNA or of plasmids expressing sequences preocessed to siRNA [by an enzyme called Dicer or Dcr which belongs to the riboendonuclease III family of enzymes] can protect mice from autoimmune and viral hepatitis. RNAi could provide an exciting new therapeutic modality for treating infection, cancer, neurodegenerative disease of other illnesses."

That review is real good. I have a bunch of other refs, but you can pull up heaps of good stuff on pubmed if you want so i wont bother listing em.

But the point is...
you could do things like... find a specific isoform of something like beta-arrestin that is involved in opioid tolerance and use siRNA as a drug to reverse tolerance by silencing the gene. This kind of treatment will usually last a couple weeks or so. Maybe silence the DAT gene or something and see how a rat handles no dopamine transporters for a couple weeks
Also, you can use gene therapy combined with a specific inducer, such as tetracycline. Turn on the promoter for your insert that codes for your siRNA by giving tetracycline, and silence your gene of choice until your body gets rid of the tetracycline. In fact, you could choose any drug (usually an antibiotic or similar since they have little to no effects in mammalian cells) whose pharmacokinetics you like (hopefully that crosses the BBB) and use that.

anyway, this is some really interesting shit which is going to hit the clinical trials real soon. the main problem is the actual administration. in rats they use hydrodynamic injection, which is dependent on creating a very high intravascular pressure, and typically involves injecting 10-20% of the actual blood volume of the animal into it. This causes heart attacks more often than not...

comments?

 

[BilZ0r]

Well I don't think you could reverse tolerance, but you could stop tolerance from developing...

Still, if we're going to go that crazy, I'm sure we could do all kinds of genetic changes, like opioid overexpression that could be a lot easier...

..meanwhile, are you even gonna write that section in the textbook on down-regulation?

 

[fastandbulbous]

Always wary when it comes to inhibiting mRNA transcriptase, you never know what else it'll effect, like finding that you've also inhibited a gene expression that's needed as a component of say T lymphocytes, so you're left with a fucked immune system for the same period of time. The idea seems good in principal though.

Maybe a better approach would be to produce enzyme inhibitors for the enzymes that produce the end protein from the precursor that's expressed directly from the mRNA ,like say with the digestive enzyme trypsin, it has to be activated from a precursor by the action of another enzymr. Some proteins like insulin are expressed as pre-proinsulin, which is then modified to proinsulin, and finally to insulin. Say, for your example of the DAT, you could inhibit the enzyme that trims the amino acid chain product of transcription, thereby inhibiting production of a protein subunit.

They'd both achive the same end result

 

[mitogen]

its not inhibiting 'mRNA transcriptase' (whatever that is)
this is direct, specific, programmed destruction of mRNA's

 

[fastandbulbous]

Right, these siRNA double chains end up being part of a larger molecule that has the complimentary base pair sequence for the specific strands of mRNA, so that the RNA gets 'chopped up' the same way that restriction endonucleases act on DNA?

Thing is, once the DAT protein has been made, inhibiting the gene that codes for that protein isn't going to be much use - you need something that has to be manufactured on a large basis, like peptide neurotransmitters, or some other protein with a high turnover rate, for stopping the manufacture to have a significant effect. Even then, it's going to be a pretty blunt tool, as stopping all the activity of a certain protein is going to have effects in a lot more than just one or two areas


PS
mRNA transcriptase is the enzyme that takes a strand of DNA then uses it as a template to build a complimentary sequence, only using RNA bases (uracil replases one of the DNA bases - cant remember which one though) to build the strand of mRNA, Stopping it working has the same end result as destroying it once made - no protein production.

 

[phase_dancer]

Uracil normally replaces Thymine as a Pyrimidine nucleoside in RNA. But very rarely Thymine occurs in RNA and Uracil in DNA

 

[BilZ0r]

^Fastandfabulous. A 7 day treatment would be enough to seriously knockdown transporter density... should stop the possibility of addiction.