RNAi - Wave of the future or baloney?

[Tezcatlipoca's Hat]

OK, so I was at home the other night, waiting for dinner to finish baking in my erratically (some might say malevolently) inconsistent oven, and NOVA was on, so I paused to see what new ways PBS has found to make me feel like a drooling idiot.

They didn't disappoint; part of the program concerned RNAi, which is apparently some sort of interior mechanism inherent to every living cell that allows for the detection and removal of anomalyous (and, by extension, harmful) instructions issued by the DNA in the nucleus. As it was explained, inserting a mirror image of a specific set of instructions into a given gene will cause the destruction of that set of instructions by the cell's defense mechanism (here called "The Cop") and therefore cause the trait that is manifested by the ersatz set of instructions to disappear from the organism. In their initial experiments, the scientists credited with discovering RNAi were attempting to significantly increase the pigmentation of a purple pansy, and in the course of this attempt doubled (or perhaps tripled) the "purple" instructions on the gene controlling pigmentation within the flower. The result was a colorless (i.e., white) flower, which is of course the very opposite of the desired effect. Further research uncovered the mechanism behind this phenomenon (RNAi) and led to theorizing about its use in controlling or even eliminating infectious diseases.

My questions are:

1) Did anyone who actually deals with DNA, RNA, RNAi or the DMV (ok, maybe not that last one) actually see this episode of NOVA?

2) Is the idea of using this discovery to sabotage viruses like HIV a plausible one, and in what way do you think this will be employed in future research?

3) Does anyone else ever watch something like this and think, "Wow, and I thought I was doing well when I figured out where that missing $1.37 was in my checking account. Guess I'm still an idiot after all!"

 

[Hercules Rockefeller]

I did not see NOVA but I am a developmental biologist who uses RNAi (semi)routinely. What you have described is all correct (in a broad general sense). Your description of the process leaves a little to be desired , but there is lots of info out there on the Web and I won’t say any more on the specifics unless asked.

RNAi is not hyperbole. On the contrary, it has become a routine and indispensable technique for knocking down gene function in cell culture as well as <I>in vivo</I>. To date, RNAi is used in basic science to investigate gene function. But the buzz now surrounding RNAi is whether or not it can be used therapeutically in humans.

Whilst RNAi is an established technique for basic science, it remains to be seen whether or not it can be developed into a therapeutic treatment. There are numerous studies at the moment going on all over the world attempting to do just this. I have spoken to a researcher who is attempting to use RNAi to treat macular degeneration. My feeling is that there may be some specific instances in which RNAi can be applied to humans as a treatment (maybe even a cure) but there are many caveats to the idea. There is a great deal of media exaggeration going on at the moment that gives the idea that RNAi will be the savior of mankind. Like the concept of gene therapy, I’ll wager that RNAi does not live up to all the media speculation.<P>

 

[Tezcatlipoca's Hat]

Thanks for the reply, Hercules. I am in no way even close to being a biologist, but I was intrigued by the possibilities presented by the folks on NOVA.

The segment mentioned a specific application - in fact, I believe it was treatment of macular degeneration - but I figured the hype was pretty exaggerated with regard to actual therapeutic use.

It's good to know that it's really used in a research capacity; I guess the days of turning off all our little (and not so little) genetic foibles are a ways off, but at least we're on the right track.

 

[Hercules Rockefeller]

It's good to know that it's really used in a research capacity

Oh, indeed! In this era of genomics we are sequencing whole genomes from all sorts of different organisms and identifying genes at a tremendous pace. However, assigning functions to all these genes is a much slower and more difficult thing to do. RNAi has become a valuable technique for determining the function of unknown genes. In fact, in the invertebrate model organisms (the nematode worm -- <I>C.elegans</I> -- and the fruitfly -- <I>Drosophila melanogaster</I>) there have been whole genome RNAi knockdown experiments performed. In other words, every gene identified in the sequencing of these genomes has been “switched off” to determine what function they perform in the context of a whole living organism. Tremendous stuff!<P>

 

[Billy T]

....In other words, every gene identified in the sequencing of these genomes {RNA sequences?} has been “switched off” to determine what function they perform in the context of a whole living organism. Tremendous stuff!<P>

I thought genes were in the DNA?

I did not see the NOVA show but want some clarifications. It is my understanding that RNA is long single strand molecules with the same informational content coded in (some different and some the same) lateral molecular attachments to the “backbone” of the long chain as DNA, or at least a segment of DNA. I don’t know what makes some of these sequences a “gene” but understood “gene knockout” to be a change in the DNA, not the RNA. If this is correct, then binding something on the RNA to change its shape (make it ineffective for its original purpose) would not constitute “gene Knockout.”

I tend to think of DNA as a library of information, RNA as a copied version of the information that is capable of being used by cells to control fabrication of proteins. Interference with the RNA does not destroy the information in the library (DNA) but can prevent the full production rate (Some RNA may escape the “grasp” of the interfering molecular agents). If you really want to destroy bad gene information, you would need to modify the DNA. - This may be hard to do as it is double stranded (already protectively bound to a compliment) and thus not readably available for new chemical reactions.

Please correct my errors and explain more why RNAi may (or may not) be the way to cure all diseases related to proteins in excess or of the wrong type, due to DNA errors..

 

[Idle Mind]

Gene transcription in a very general sense flows as follows:

DNA --> RNA --> protein

The gene on the DNA strand is transcribed into a complementary RNA molecule (which is called mRNA), which is translated into the protein end product. When you design an RNAi, it is complementary to the mRNA and will bind to it, blocking translation. Since no protein end product is formed, the gene is blocked, which is essentially the same as a knockout.

Having RNAi in cells where viral genes have to be transcribed then translated is potentially therapeutic since viral protein formation could be blocked.

 

[Hercules Rockefeller]

I thought genes were in the DNA?

Mr. T,

You are completely correct. RNAi works by utilizing an intrinsic cellular mechanism that degrades mRNA. In other words, the mRNA transcripts from the targeted gene are degraded and, thus, the protein corresponding to the mRNA is not made. But as you correctly point out, the gene itself (ie the DNA residing in the chromosome) is unaffected by the process.

You will notice that I use the term “<I>knock<B>down</B></I>” rather than “<I>knock<B>out</B></I>”. The term “knockout” and “knockin” nearly always refer to the targeted alteration of a genomic locus (ie. DNA) in murine ES cells and the subsequent generation of mouse lines carrying the knockout or knockin. The term “knockdown” refers to instances where gene activity is reduced by targeting the mRNA of that gene, such as RNAi or antisense oligonucleotides.

So what you are getting at is quite correct – RNAi is not a cure for mutations in genes (ie. for inherited genetic diseases) because the gene itself is unaffected. In such instances it may serve only as a treatment and will likely need to be applied continuously. And it will probably be feasible only in instances where the defect is monogenic which will limit the applicability to human diseases. Furthermore, using RNAi to degrade the transcripts of mutant genes is only half the story. In order to reverse a genetic disease it will be necessary (in many instances) to not only remove the ‘mutant’ transcripts but also provide ‘wildtype’ mRNA. This is a whole other story. However, as suggested above, for infections or other transient situations RNAi might be more feasible.

But as a human therapeutic treatment, RNAi suffers from the same major problem as gene therapy – how do you deliver the material (siRNA) to the cells that need it? Gene therapy has never reached anywhere near its hyped potential due to the lack of specific delivery systems, and I see exactly the same stumbling block developing for RNAi.<P>

 

[Billy T]

Thanks Hercules. I want to pick your brains some more. What s the "m" in mRNA? If it is for "murine ES cells" my question becomes: what aare these cells and where are they? I.e. are they cells floating around in the blood or a subsection of what I would call a "cell." (I know there are a lot of special subsections in cells.) Like wise, what is the "ES"?

Your "such as RNAi or antisense oligonucleotides" (with the "or" instead of "I.e." makes me think that RNAi may be something other than a set of molecules that "compliment" the set attached to "backbone" of the RNA one istrying to render nonfunctional.

If you will, I would appreciate a few words about what "antisense oligonucleotides" means if it is anything more that just the way workers in the field state what I stated by "a set of molecules that compliment or bind to the set attached to "backbone" of the RNA" (At least tell what a molecule needs to have/ be to be an "oligonucleotide." - I am traveling and don't have access to a good dictionary, and think your insites would be better.)

I have become very interested in this subject, in part because of investment potential. (My Ph.D. Is in physics and I retired before this field exploded with potential to change the world in this century as physics did in the past century.) I have listened to some on line discussion by drug research company presentations in meetings. Wonderful progress you guys are making.

In January 2005 company "EyeTech" got FDA approval to market an agent that binds to only one of the 6 differetent types of VEGF proteins. They inject it directly into the eye ($1000/ shot) every 6 weeks. Company SRNAi is working on way to block the production of the VEGF proteins and plans at least initially to also target the eye directly. Probably for reasons you mention about the difficulty of delivery of an RNA interferring agent to the desired target. (Eye is a relatively closed/ separate part of the body - reason why cornea transplants are not rejected.) Other companies are working on specific delivery systems that will selectively target tissues. (Patches and implants of slow release capsules being the most obvious approaches.)

If you know of any people/ small companies that are in great positions to prosper (or at least be absorbed by the major drug companies) please send me PM about them. To get their reports etc. I bought a few shairs of Eyetech (now down greatly as larger company announced better results with modified vergion of their VEGF drug) and sRNAi (now more than doubled as very broad pattent was granted in England that effectively gives them complete control over all RNA interference agents drugs and same patent is pending in most of Europe)

I think you may be a little pesimestic about the practical application of these drugs, at least the VEGF blockers and the RNA interference agents that prevent the production of Vascular Endofelia Growth Factor. I like the RNAi approach more than EyeTech's, which only disable the proteins - one strand of RNA disabled can stop the production of many protein molecules. Perhaps RNAi drug is too powerful - hard to dose correctly? Comments.

Mr. T,

Thus I think you are completely correct. RNAi works by utilizing an intrinsic cellular mechanism that degrades mRNA. In other words, the mRNA transcripts from the targeted gene are degraded and, thus, the protein corresponding to the mRNA is not made. But as you correctly point out, the gene itself (ie the DNA residing in the chromosome) is unaffected by the process.

You will notice that I use the term “<I>knock<B>down</B></I>” rather than “<I>knock<B>out</B></I>”. The term “knockout” and “knockin” nearly always refer to the targeted alteration of a genomic locus (ie. DNA) in murine ES cells and the subsequent generation of mouse lines carrying the knockout or knockin. The term “knockdown” refers to instances where gene activity is reduced by targeting the mRNA of that gene, such as RNAi or antisense oligonucleotides.

So what you are getting at is quite correct – RNAi is not a cure for mutations in genes (ie. for inherited genetic diseases) because the gene itself is unaffected. In such instances it may serve only as a treatment and will likely need to be applied continuously. And it will probably be feasible only in instances where the defect is monogenic which will limit the applicability to human diseases. Furthermore, using RNAi to degrade the transcripts of mutant genes is only half the story. In order to reverse a genetic disease it will be necessary (in many instances) to not only remove the ‘mutant’ transcripts but also provide ‘wildtype’ mRNA. This is a whole other story. However, as suggested above, for infections or other transient situations RNAi might be more feasible.

But as a human therapeutic treatment, RNAi suffers from the same major problem as gene therapy – how do you deliver the material (siRNA) to the cells that need it? Gene therapy has never reached anywhere near its hyped potential due to the lack of specific delivery systems, and I see exactly the same stumbling block developing for RNAi.<P>

 

[Hercules Rockefeller]

What’s the "m" in mRNA?

mRNA = <B><U>m</U></B>essenger RNA

There are a variety of different types of RNA that perform a variety of different functions. Messenger RNA is the RNA that is transcribed from genes (ie. from a protein-encoding DNA) and translated into a polypeptide. It is the RNA that is referred to in the classic DNA-->RNA-->Protein dogma. For the majority of biological scientists, it is the only RNA that they will ever care about.

Messenger RNA is the target of RNAi – the targeted mRNA is degraded, so the corresponding protein is not made. Thus, the effect is as though the gene were “switched off”.

what aare these cells and where are they? Like wise, what is the "ES"?

ES cells = <B><U>E</U></B>mbryonic <B><U>S</U></B>tem cells.

The answer to this question is in my earlier post in this thread.

Your "such as RNAi or antisense oligonucleotides" (with the "or" instead of "I.e." makes me think that RNAi may be something other than a set of molecules that "compliment" the set attached to "backbone" of the RNA one is trying to render nonfunctional.

Sort of.

RNAi is achieved by introducing pieces of double stranded RNA into cells. Earlier protocols for targeting mRNA for degradation was to introduce single stranded DNA oligonucleotides (varying in length, generally ~25 nucleotides). The DNA oligos were the antisense sequence (ie. they were complementary to the mRNA) and would bind to the target mRNA and lead to its degradation through a similar pathway that is activated by RNAi. So both the old way (antisense oligonucleotides) and the new way (RNAi) involve introducing complementary pieces of nucleic acid.

Antisense oligonucleotides never worked particularly well – there was always non-specific mRNA degradation and they weren’t very stable once inside the cells – and was promptly dropped once RNAi came along. Interestingly, the antisense oligonucleotide approach has experienced a large resurgence of late due to a new type of synthetic backboned “DNA” that has been developed: morpholino-modified oligonucleotides (“morpholinos”). Morpholinos are highly specific for their target mRNA and are much more stable than previous DNA-based oligonucleotides. I mention morpholinos because there is some speculation that they might be useful for human therapeutic use.

I think you may be a little pesimestic about the practical application of these drugs, at least the VEGF blockers and the RNA interference agents that prevent the production of Vascular Endofelia Growth Factor. I like the RNAi approach more than EyeTech's, which only disable the proteins - one strand of RNA disabled can stop the production of many protein molecules. Perhaps RNAi drug is too powerful - hard to dose correctly? Comments.

Maybe I’m being too pessimistic. I said earlier that I thought that RNAi would likely be developed as a treatment for some specific areas. As for whether it’s better than targeting other areas of a pathway (eg. at the protein level), I don’t think a patient will care if whatever treatment they received actually works! We should investigate all options. VEGF is a secreted protein and is upregulated in tumors but not in the surrounding tissue, so it’s a great specific target for drug therapies.<P>

 

[Billy T]

...RNAi is achieved by introducing pieces of double stranded RNA into cells. Earlier protocols for targeting mRNA for degradation was to introduce single stranded DNA oligonucleotides (varying in length, generally ~25 nucleotides). ...

Thanks again. If you continue to help me I may understand more of this exciting field.

If doubled stranded RNAi is used there is someting I am missing or assuming incorrectly. I assume that each strand is the complimentary sequence of the other and that they must split to "kill" the target, but only one of the strands is ithe target's compliment. They other would be just like the target and now is its replacement. Thus nothing would be achieved. - Clearly I am wrong, but where?

 

[Billy T]

dear Herculus:
I understand that the genes are on the DNA, and that ES is ebrionic stem (cell), but don't know how "murine ES cells" differ from other ES or where they are in the body. I also know that most cells have double stranded DNA, but believe that cells produced for reproduction are only single stranded DNA. (Mom and Dad each give half to baby.) If this is correct, what keeps them stable? I.e. why do not some of the small molecules that can attach to RNA (the complements) attach to the single stranded DNA of the "germ cells"?

I am interested in how these RNAi agents are made. If I, who know next to nothing about it and very little organic chemistry, wanted to make some RNAi, I would grow it at one end. That is, I would put a particially completed RNAi molecule in solution that had the next molecule needed for the "backbone" and then into a solution that had the next attachment molecule desired.

These molcular solutions would have low molecular weight compared to the growing RNAi. Thus I would centrifuge the solution and collect off the growing RNAi for the next pair of "bath solutions" (more backbone and another attachment.)

I am reasonable confident this is naive and unworkable. I hope you can give me some idea as to the relative difficult of growing RNAi as constrasted with some, presumably more simple, agent that will bind to the active part of the protein that the RNA would fabricate. As you observed, either the protein or the RNA can be disabled and the patient does not care which. In some crude way, if RNAi is to compete economically, the cost of fabrication of one RNAi molecule can not exceed the cost of making one protein molecule "killer" (PMK) by more than the effectiveness ratio RNAi / PMK - which is roughly the number of protein molecules each RNA makes. This to is much too simple as one must consider (at least) how well PMK is bound to the protein before it "falls off," etc. (PMKs that fall off quickly or are themselfs not stable are not much use.) The stability of both the RNAi and PMK must be considered.

I realize this is getting too detailed for you to fully explain to one as ignorant of the field as me. Just make a guess as to how much more costly the manufacture of RNAi is compared to the production of the number of PMK agent molecules that would neutrlize the proteins the RNA would have produced. If you could point me to some papers available on line or other sources that discuss these production processes (probably details are carefully guarded secrets, but the general approach must be widely known.) Even a list of terms to google search would be a big help.

Thanks again and in advance. - Billy T