Monday, August 23, 2010

A comparison of Tekmira’s and AVI Biopharma’s Candidates for the Treatment of Ebola Virus

I was quite amused today, while driving a car on Borneo, the local radio report the headline ‘US scientists said that they are ready to start clinical trials for Ebola’. It was yet another reminder that oligonucleotide therapeutics, including RNAi is firmly emerging from the research stage, entering the medical arena and gaining more widespread attention.

As you may also have heard by now, 3 months after the impressive publication in The Lancet by Tekmira and collaborators that liposomal siRNA essentially cured non-human primates from otherwise lethal infections of Ebola virus, morpholino antisense company AVI Biopharmaceuticals followed up with results from their own Ebola non-human primate treatment studies in the equally prestigious journal Nature Medicine (Warren et al.: Advanced antisense therapies for postexposure protection against filovirus infections). Since both companies were recently awarded highly lucrative government contracts from the US Department of Defense to further develop these two oligonucleotide therapeutic candidates for the treatment of Ebola and therefore have to be considered competitors for further contracts, I will briefly compare and contrast the results from the two studies.

One remarkable benefit of having both lines of research enjoy funding from the DoD is that they have been highly standardized which makes them very comparable to the extent that even the animal laboratory was shared, I believe. So no excuses with 'in our hands' or the likes. In each case the goal was to evaluate the RNAi or antisense in a post-infection model whereby the oligo Rx was first given once 30-60min following intramuscular injection of rhesus macaques with 1,000 plaque forming units of the Zaire strain of Ebola, an amount many orders of magnitudes higher than needed to kill such an animal if left untreated.

Just as a reminder, in this treatment regimen, Tekmira’s intravenously administered SNALP-RNAi when given 3 times every other day or daily for 6 days following viral exposure rescued 2 of 3 and 4 of 4 animals, respectively.

In the AVI study, morpholino antisense molecules which have been slightly altered compared to previous studies to be positively charged to facilitate enhanced cellular uptake and improved target RNA binding, were also given daily, although in this case the follow-up administration period lasted for 10-14 days and the dosage was 40mg/kg compared to 2mg/kg siRNA. In addition to intravenous administration which the investigators refer to as the preferred route of administration for needle stick scenarios and the like, some studies involved simultaneous intraperitoneal and subcutaneous administrations.

Route of administration, however, did not seem to make much of a difference since in both cases around 60% of the monkeys were rescued from near-certain death: 5 of 8 in the subQ+i.p. regimen, and 3 of 5 in the intravenous regimen.

Put differently, the risk of dying from Ebola infection was 14% in the Tekmira study (1 out of 7 animals; 0 of 4 with the optimized protocol) compared to 39% in the AVI studies. Although the numbers are still small, this indicates an almost 3-fold risk reduction with SNALP-RNAi compared to morpholino antisense.

So while Tekmira’s SNALP-RNAi may in fact now lead the race towards a drug that can treat Ebola virus infection, it is far from over. AVI is apparently ready to move into safety studies with human (healthy) volunteers. In addition to such clinical studies, both companies and their collaborators will have to test in further non-human primate studies how long they can delay treatment after the initial infection, as ‘in the field’, as opposed to laboratory accidents and first responder situations, treatment will only begin after first symptoms emerge.

However, with the current efficacy of TKM-Ebola and what appears to be an already extensive pre-clinical safety database, if the initial human studies are uneventful, the case could be made that a first-generation Ebola standby for needle pricks and first responders in local outbreaks may be quite close indeed. In fact, researchers exposed to these viruses should be pushing for it, and the fact that the current SNALP-RNAi has to be administered intravenously should not be a problem for such purposes.

AVI’s antisense morpholino though may have some advantages over the SNALP-RNAi for wider outbreaks in that it may be administered subQ, although a pure subQ protocol has not been tested yet in the post-infection model and I am not clear about the practicalities of administering 40mg/kg x70kg= 2.8 grams (!) of oligonucleotides subQ. With the more potent, second generation SNALP-RNAi formulations ('LNP' is the official designation now), subQ administration of liposomal siRNAs should become more practical and I would not be surprised if some of the future Ebola contract funds were to be spent on further developing subcutaneous LNP administration.

While it was not obvious from the paper in The Lancet, reading the latest paper, it is quite clear that the 2 companies and probably even different groups within the Army that work on Ebola Rx are strongly competing with each other. It reminds me of the strategy of principal investigators of some ‘post-doc labs’ where at least 2 different post-docs are being competitively put on the same project. This way, the principal investigator increases his chances of timely success. Not being a military man myself, it would also make sense from a military point of view to even have 2 alternative Ebola Rx stockpiles, just in case one turns out to be more desirable than the other in a real emergency.

In the end, while the value of biodefense agents is often difficult to determine, the DoD Ebola program brings two significant benefits. One is that the program allows the involved companies plenty of room to develop their underlying platform technologies such as facilitating the scale-up of their manufacturing processes. The other is the images evoked by viruses such as Ebola and the attention it directs towards this rapidly growing area of oligonucleotide therapeutics. Even a remote island like Borneo is an escape from it no more.


John said...

Thanks for the entertaining article as always. Putting aside AVI's novel chemistry, it's difficult for me to see their approach winning over siRNA in the long run. Nevertheless, as you said it will be interesting to compare the progress of these two Ebola drugs. I am wondering if there is any evidence at all that antisense is recycled (with any chemistry).

Anonymous said...

Hi Dirk,

Wondering if you could comment on this...

It seems logical that the "many for one" action of siRNA molecule in a virus situation would be preferable to the "one for one" action of the PMOplus. Less drug needed.

Although how easily the two versions get inside the cell affects that comparison too. From Dirk's article it's a comparison of 2mg of siRNA to 40 PMO. But since we know a siRNA in effect will shut down something like a thousand RNAs each - and it's only outperforming the PMO here by a multiple of 20 - the siRNA is having a hard time getting into the cell. Still the few that get in make all the difference!

Another, more significant, thing I wonder about is does this siRNA - CAN a siRNA - handle the possible mutations that may arise, as the strategic positioning of the positive charges of the PMOplus can? Dirk doesn't explain the nature and function of that KEY modification of the basic PMO that allows the PMOplus to "override" those mutations when they happen!

Anyway, if the siRNA can be made to handle the mutation problem, then they do sound like a likely advancement if you only want to administer the drug intraveniously.

But if you want intra-nasal delivery of an anti-viral - which everuyone does! - then you'd be stuck with AVI's PMOplus. And the human dose for the influenza is expected to be just 2mg/kg. The same as Tekmira's Ebola dose here. So......

Dirk Haussecker said...

Dear Anonymous,

One of the reasons why Tekmira and AVI have used cocktails of oligonucleotides is that targeting multiple viral genes at the same time limits the ability for the (RNA) virus to mutate around the therapeutic. If PMO+ is less affected by viral mutations, you have to wonder about their inherent specificity.

Personally, if a mutation problem really emerged, then (IMO) the best approach would be to stick to the cocktail approach and find out where the mutations have occurred in the monkeys (easily done by sequencing) and avoid those regions and instead try targeting other conserved regions.

I don't think we need to discuss much whether morpholino or LNP-delivered siRNAs are more potent for gene knockdown in the liver: single-digit micrograms per kg (LNP-siRNAs) against 1-40mg/kg for the various antisense approaches. I agree, however, that for other organs, the difference with current technologies may not be that great yet.

bion4maticist said...

you said:
"Although the numbers are still small, this indicates an almost 3-fold reduction in risk with SNALP-RNAi compared to morpholino antisense."

Got a p value for that? I ran it and got a chi2 that made it seem more likely than not that this was a chance variation. Don't forget Yates' correction for continuity. Did I do that right?

you said:
"...I am not clear about the practicalities of administering 40mg/kg x70kg= 2.8 grams (!) of oligonucleotides."

Wow. We're scaling up the size of the organism by an order of magnitude, and you think the dose is going to scale linearly? Hmmm. You may want to consult some expert advice on that one. I'll bet that the exponent is something quite a bit less than 1.

John said:
"I am wondering if there is any evidence at all that antisense is recycled (with any chemistry)."

Are you going to let that go? Or point to the copious literature on RNAseH-mediated antisense as another type of catalytic antisense, along with siRNA?

I'm not really differing from your conclusions here: I don't really think that highly of AVI and their morpholino chemistry, either.

But it seems to me that you're being a little bit casual with the facts, here. Do you think I have a point, or no?


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