Replies to post #26259 on Biotech Values

[poorgradstudent]

>One immediate question I have about the results described in the PR: mRNA expression for the protein in question (apoB) was reduced by 90% and the serum level of the protein was reduced by 75% -- why the discrepancy between the 90% and 75% figures?<

It is actually common place to observe the amount of protein decrease by a percentage that is lower than the accompanying mRNA transcript decrease. mRNA transcripts may be used more than once.

This type of disconnect between the mRNA level and protein level is seen very often in preclinical models. Mice that may be heterozygous for a specific gene deletion (so 1 of the 2 gene copies in the genome is lost) will often show levels of protein expression that are on par with mice that have both copies of the gene intact.

[aslan2772]

mRNA "dosage". Dew, PGS is right, there is often not a linear relationship between mRNA levels and levels of the protein that is encoded by the mRNA. A couple things to consider here. The cell uses feedback mechanisms to regulate protein production at the translational level, in addition to the transcriptional level. Mature, polyadenylated mRNA is quite stable in the cell and can be sequestered in the cytoplasm and used later for the rapid regulation of local protein synthesis through translational activation by biochemical signaling cascades. As PGS points out, a single copy of mRNA can be use to make multiple copies of a protein (a typical 50KD protein takes about 1 minute to translate). Furthermore, much of the mRNA in a cell normally never gets translated; its just there in case it is needed.

Another factor to consider is that mRNA is edited post-transcriptionally and thus there may be multiple forms of mRNA encoding the same or very similar isoform of protein that are resistant to the siRNA.

Finally there is the more obvious: in the tissue targeted by the siRNA, you probably will not achieve 100% "transfection" efficiency. Thus, the "untransfected" cells may work harder at protein production in an attempt to compensate for reduced serum levels. All these factors make siRNA dosing studies pretty complicated when you really think about it.

To my knowledge, there are no approved products for treating humans with siRNA. In fact, are there any approved drugs which target RNA other than antibiotics? Does anyone have a sense of the regulatory hurdles that will have to be overcome with the FDA for say, ocular delivery of siRNA therapeutics?

[jellybean]

<<<why the discrepancy between the 90% and 75% figures>>>>

This difference is irrelevant at a cellular level. There are so many independent variables controlling mRNA and protein levels that a 15% difference means little. What is important is that both drop. Remember that both mRNA and protein have half lives and, although they often reflect each other in trend, ie. rapid turnover or slow turnover, they are independent of each other. And then there are is the whole issue of whether that 15% difference is statistically significant. The PR does not tell us how reliable the measurements are.

Be happy that the levels are close and are moving in the same direction.

[aslan2772]

ALNY Liver/systemic delivery.

Dew, I don't know enough about the liver to comment meaningfully. I can say though, that Alnylam has demonstrated uptake of their "morpholinos" (with systemic administration in rodents) in several tissues other than liver, but not the brain. It's probably mostly systemic, but like most drugs, it may have tissue distribution biases, difficulty crossing the BBB or entering the prostate, etc.