Posted 28 August 2006

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Live Chat held 20 September 2002, featuring Zuoshang Xu, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA.

Participants: Zuoshang Xu, University of Massachusetts Medical School, Worcester; Claudius Vincenz, University of Michigan; Dave Teplow, Brigham and Women's Hospital, Boston, Massachusetts; Natasha Caplen, Medical Genetics Branch, NHGRI, NIH; Victor Miller, University of Iowa; Gabrielle Strobel, Alzheimer Research Forum; Claudia Almeida, Cornell Medical College; Ramesh Tennore, ALS Therapy Development Foundation, Newton, Massachusetts; Jungsu Kim, Mayo Clinic; Amita, USF; Bert Tseng, UC Irvine

Note: The transcript has been edited for clarity and accuracy.

Dave Teplow
Hello, there!

Gabrielle Strobel
Hi Dave, hi Zuoshang! Welcome, everyone.

Claudius Vincenz
Is this discussion exclusively about the in-vivo application, or solving the nuts and bolts in cell culture first?

Gabrielle Strobel
Claudius, I think it is safe to say that all here probably feel a lot needs to be figured out in cell culture first. But work in animal models should go in parallel. Disagree, anyone?

Dave Teplow
So, Zuoshang, is RNAi the "cure" we've all been looking for in the Alzheimer's field?

Zuoshang Xu
It could be one of several. I think the key is the delivery.

Victor Miller
And safety: I don't know if long term triggering of the RNAi pathway will be tolerated.

Dave Teplow
Are you talking about tissue or intracellular or organellar delivery?

Zuoshang Xu
Right now, virus-mediated gene therapy appears to be the obvious way, but there is a long way to go before it can be applied.

Gabrielle Strobel
Presently, what seems like the best way to approach delivery?

Dave Teplow
How is this progressing with respect to delivery to hippocampal neurons, for example?

Zuoshang Xu
In cultured cells, the current protocols can mediate highly efficient transductions. But in vivo, how can we deliver widespread infection so that effective inhibition can be achieved in the CNS? I think that is one of the key obstacles.

Dave Teplow
What are some possibilities, Zuoshang?

Natasha Caplen
At present I don't think any of the in-vivo delivery vectors can mediate sufficient specificity to target a particular population of neurons.

Gabrielle Strobel
Zuoshang, based on current evidence, do you think lentiviruses are safe and effective?

Zuoshang Xu
Lentiviruses have potential, but controlling the integration site is the challenge. This is important because of the potential effects on surrounding gene expression.

Gabrielle Strobel
Zuoshang and Victor, and also Natasha, do you want to tell us briefly what you have done so far in animal models?

Victor Miller
We have so far done most of the work on specific disease genes in cell culture. We have shown that the viruses can knock out GFP in mouse brain. We are currently constructing viruses based on the cell culture work to test in SCA3 mouse models.

Ramesh Tennore
What applications of RNAi are closest to the clinic? I read that the first RNA-based molecule got FDA approval recently.

Gabrielle Strobel
Do you remember the application?

Ramesh Tennore
It's from ISIS. It's for CMV. Vitravene (fomivirsen).

Zuoshang Xu
Hi, Ramesh. I don't know the answer to your question.

Natasha Caplen
A number of molecules based on RNAs are undergoing clinical trials primarily using antisense approaches to block protein expression. The development of an RNAi-based drug for testing is likely to be some time away, but based on the experiences in the antisense field, and the current RNAi literature, it is most likely to be a cancer or virally related target.

Ramesh Tennore
Thanks for the summary.

Claudius Vincenz
What promoters seem to work in lenti?

Zuoshang Xu
Many promoters have been used. For the ubiquitous ones, CMV, ubiquitin. There are a few recently published.

Claudius Vincenz
These are polymerase II promoters, not U6 (polymerase III).

Zuoshang Xu
One use of lentiviruses is to make transgenic mice. This has been done by several groups. The promoters used for RNAi are U6 and H1. All show high efficiency in mediating RNAi in mice.

Zuoshang Xu
Is it possible to administer antisense or siRNA directly to CNS?

Gabrielle Strobel
Perhaps a look at recent history could be instructive, too. Antisense RNA also looked terribly promising, but then big efforts in biotech failed to make it work, as far as I can tell. Does anyone know if RNAi stands a better chance, or is the use of antisense RNA bearing fruit now?

Claudius Vincenz
At least in cell culture, the specificity of RNAi is remarkable.

Zuoshang Xu
One advantage RNAi has is that it can be put in viral vectors. I think this is a big advantage.

Natasha Caplen
RNAi appears to be at least as specific as antisense, and probably much more so. With respect to delivery, antisense oligos have proven problematic because they have to be heavily modified to lengthen their half-life. Initial in-vivo experiments suggest that siRNA can be injected into the bloodstream, but the resulting RNAi is transient. This should be the advantage of viral delivery of siRNA.

Gabrielle Strobel
It seemed to me that specificity of integration and possible disruption of other gene expression was a problem. You think not, Claudius? I also found the specificity to single-nucleotide mutants, described by Zuoshang and Victor, to be remarkable....

Claudius Vincenz
I agree that is still potentially a problem; however, antisense was plagued by that and much more.

Zuoshang Xu
Both disruption or activation of a gene could be a problem. For example, if you disrupt a tumor suppressor or activate a cell proliferator, either could cause tumors.

Victor Miller
It is worth noting that the single nucleotide specificity took some "engineering" of the siRNAs to achieve. It may not always be sufficient to simply identify a mutation or SNP and then design an effective and specific siRNA. Several siRNA designs may need to be tried.

Claudius Vincenz
I'm having difficulties knocking down a new gene in cell culture. Does anybody have experience with the system that uses in-vitro Dicer digestion to produce 21bp oligos that cover the whole gene sequence?

Gabrielle Strobel
I am afraid some questions got drowned out. Let me repeat Zuoshang's: Is it possible to deliver antisense or siRNA directly to the CNS?

Zuoshang Xu
The answer to the question whether siRNA can be delivered directly is not known. It needs to be tested. Currently, it has been tested in liver, but I am not aware of it being tried on other cell types in vivo.

Ramesh Tennore
What lessons have we learned from the antisense RNA field that are applicable to RNAi regarding specificity and delivery? Sorry if this question is too broad.

Zuoshang Xu
One possible application is to use RNAi to generate "knockout" mice. It will be much easier to do than the conventional KO method. The question is whether it can be achieved. Does anyone know anything about using RNAi to achieve KO?

Claudius Vincenz
Already a couple of years ago there was a Nature Cell Biology paper showing knockout phenotypes with RNAi very early in embryogenesis (Wianny and Zernicka-Goetz, 2000).

Dave Teplow
Do these KO phenotypes last?

Victor Miller
Has anyone tried blood-brain barrier permeabilization to deliver duplexes directly?

Ramesh Tennore
Actually, I was wondering if anyone has created systems to allow these molecules to penetrate BBB.

Zuoshang Xu
Shall we discuss what are the possible targets for treatment of Alzheimer's disease?

Gabrielle Strobel
Thanks, Claudius, and yes, let's move to targets in Alzheimer's disease. Let me put out BACE as the obvious candidate. I am sure people are trying. Does anyone know how far that has come?

Dave Teplow
There are two obvious choices: the structural gene for Ab and the genes encoding the enzymes responsible for its metabolism.

Victor Miller
BACE, tau, AbPP, the familial presenilin mutations are all possibilities.

Dave Teplow
I think that g-secretase may be an even more important target than BACE.

Gabrielle Strobel
Why, Dave?

Amita
I am of that opinion, too.

Dave Teplow
Because the initial cleavage of AbPP does not lead to the production of the p3 and Ab peptides that appear to be linked to the formation of soluble and insoluble neurotoxic assemblies. Of course, you could argue that BACE is a prerequisite for the production of these peptides, and you would be right.

Zuoshang Xu
But is g-secretase inhibition safe?

Claudius Vincenz
So little is known about the function of AbPP. Presenilins seem to control maturation and cleavage of a whole bunch of proteins. Am I just a pessimist?

Claudius Vincenz
Conditional knockout of presenilin in adults would indicate that it is fairly safe.

Gabrielle Strobel
Even with "antitargets," i.e., presenilin-like enzymes such as signal peptide peptidases and other substrates for presenilins, my sense is that the pharmaceutical industry still views g-secretase inhibition as an opportunity. What advantage would a siRNA have over small molecule compounds?

Victor Miller
I think some apprehension about the safety of reducing expression of incompletely characterized proteins such as AbPP is a rational concern for RNAi therapeutics in general, and simultaneously, a question it can help us answer. Do animals tolerate manipulations of levels of potentially important proteins that we may wish to target to treat disease?

Amita
Maybe RNAi can be used to block AbPP expression in mice in order to assess AbPP's function.

Gabrielle Strobel
Regarding BACE, the structure of its active site makes it tough to find inhibitors for. Screens turn up g inhibitors easily, but never BACE inhibitors. Perhaps this is a reason to turn to RNAi in this case?

Claudius Vincenz
Knocking out AbPP alone conventionally yields no phenotype.

Amita
No abnormalities in vasculature, considering its role on platelets?

Gabrielle Strobel
Claudius, I seem to recall that knockouts of AbPP and APLPs did have a phenotype, but were not fully analyzed? Correct me.

Claudius Vincenz
RNAi targets genes; small molecules target activities. They are not necessarily the same. Presenilins are a good example.

Gabrielle Strobel
What do you mean by that, Claudius?

Claudius Vincenz
It is still disputed if presenilins are directly involved in the proteolytic process. So, by knocking out presenilin, you may affect protein maturation of multiple ER resident proteins. On the other hand, a small molecule will inhibit the cleavage of multiple presenilin substrates.

Dave Teplow
Claudius, I believe your comment is "semantic" in nature. There is no controversy as to whether PS is involved in a catalytic complex, only whether it itself performs the proteolysis. Therefore, controlling its expression is tantamount to controlling the enzyme.

Claudius Vincenz
But controlling its expression and inhibiting its proteolytic activity are not necessarily the same.

Dave Teplow
Yes, but it doesn't have to be, from a therapeutic perspective.

Zuoshang Xu
Enzymes are usually better targets for small chemicals. You could argue that RNAi can inhibit any molecules that you wish. The problem is still in delivery. It seems to me difficult to achieve inhibition in wide areas of CNS in order to inhibit production of AbPP.

Natasha Caplen
There is no spread of the gene-silencing effect mediated by siRNAs, so you will only induce downregulation in those cells directly transfected in the tissue of interest.

Victor Miller
RNAi may also allow us to titrate the degree of KO that we achieve rather than dealing with an all-or-none scenario, where complete absence of a given protein (or total inhibition of function by a drug) may be deleterious, whereas a partial knockdown is both therapeutic and tolerated.

Dave Teplow
Zuoshang's comment and Victor's response are very relevant. Because the protein assembly issues in Alzheimer's disease are concentration-dependent, one might not need an "all-or-none" effect to see a therapeutic benefit.

Gabrielle Strobel
Dave, that argument about a small inhibition/therapeutic index is what is said about g-secretase inhibitors, too, is it not? Knocking down the AbPP gene would abrogate the a cleavage, too, right? Do we want that?

Dave Teplow
Gabrielle, I think the only way to answer your question is to do the experiment.

Zuoshang Xu
RNAi may be used to selectively inhibit expression of mutant presenilins.

Gabrielle Strobel
How difficult would it be to test this in animal models?

Zuoshang Xu
It can be tested in transgenic presenilin mice by viral vectors or transgenic RNAi mice.

Gabrielle Strobel
I should have invited the creators of various AD mouse models.... Short of therapy, are there important functional questions that RNAi is particularly well-suited to address? Just as a guess, perhaps knock down gene modifiers to tease out pathways of degeneration?

Zuoshang Xu
To my knowledge, many new members of g-secretase have not been knocked out in mice. RNAi may be a fast way to accomplish this.

Gabrielle Strobel
Zuoshang, can you elaborate a bit?

Zuoshang Xu
If you make a transgenic mouse that expresses a specific shRNA, it may knock down or knock out these proteins.

Victor Miller
At least over short windows of time (three to seven days), reporter genes can be suppressed in mouse brain via viral delivery of shRNA.

Gabrielle Strobel
Victor, interesting. Can you target that to a specific brain region?

Dave Teplow
This could be a nice corollary to the work of Dave Holtzman and the effects he sees with acute passive administration of Ab-specific antibodies in transgenic mice.

Zuoshang Xu
Victor, what kind of virus? I thought adenoviruses or lentiviruses could mediate longer-term expression.

Victor Miller
We have used adenoviruses most extensively but are also going to use lentiviruses for further studies.

Victor Miller
The injection site can be controlled, of course, as well as the tropism of the virus and the promoter used to drive the shRNA. All have shortcomings for human therapy, but it is a start.

Gabrielle Strobel
We have reached the end of the hour. This is most interesting, and I encourage everyone to chat on as long as you like. Before people start dropping out, let me just thank you for coming and making this a fascinating discussion. I hope a lot of people will pick up all these questions we brought up and sort them out.

Dave, do you think a small reduction of Ab would have a clinically relevant impact on aggregation? Yes? How about a temporary reduction. No?

Dave Teplow
I think the answer to the first question is, yes, depending on how you define "small." The answer to the second question is probably, not much effect.