Monday, May 12, 2014

GalNAc 2.0 with Greatly Improved Single-dose Efficacy and Duration

Last night, Alnylam kicked off a week of what promises to be exciting disclosures about continued progress in therapeutic gene silencing of genes expressed in the liver.  In a presentation by oligonucleotide star chemist Mutiah Manoharan at TIDES, the company provided a more thorough chemistry and pharmacology background behind the apparent improvements of the GalNAc delivery platform (Enhanced Stabilization Chemistry).

Journey along a hostile environment

The improvements are based on the observation that the 5’ ends of both the guide and passenger strands are subject to degradation by 5’-3’ exonucleases.  These may act at various stages during the relatively long journey of a subcutaneously administered RNAi trigger-conjugate: in the subcutaneous space, the circulation and lymphatics, and finally along the nuclease-rich endosomal/lysosomal uptake pathway in the target cell itself. 

Accordingly, by adding undisclosed chemical modifications to the 5’ (but also 3’) termini of the RNAi trigger strands, ~5 times the amount of RNAi trigger reaches the liver, and 10-100x RNAi trigger is found in the liver over time compared to first-generation chemistry as exemplified by ALN-TTRsc.  This means that single digit microgram per gram liver tissue can now be achieved at steady-state.  For comparison, gen 2.0 and 2.5 RNaseH ASOs (--> ISIS) depend for activity on ~100-300 microgram per gram liver tissue steady-state concentrations of phosphorothioated oligonucleotides. 

There obviously is a balance between maintaining high drug concentrations for efficacy and avoiding excessive concentrations for fear of causing inflammation and subsequent tissue scarring.  In that regard, Alnylam reports a wide therapeutic index, including in non-human primates which, laudably, were generally extensively used in these studies.

Great benefit for single-dose efficacy and duration

The new pharmacological profile is somewhat counter to a critical advantage of the RNAi platform over single-strand RNaseH technology: achieving great and sustained efficacy with minimal tissue exposure.  

Mechanistically, this fundamental capacity is explained by the fact that once loaded onto the RNAi effector complex, RISC, the duration of RNAi trigger activity in non- or very slowly dividing tissues such as the liver is largely limited by the slow (weeks) natural turnover of RISC.  By contrast, although RNaseH is a catalytic mechanism, too, no such sustained holding on to the antisense oligonucleotide is known for RNaseH such that the guide oligonucleotide has to be constantly available.

According to this model, an important determinant for the efficacy and very feasibility of traditional RNAi approaches is the size of the unloaded pool of RISC during the short period of time that an otherwise unstable RNAi trigger is available.  By contrast, unstable RNAi triggers are ill suited to take advantage of newly synthesized RISC complexes as part of natural RISC protein turnover.   

This is where GalNAc2.0 comes in: by extending the presence of the RNAi trigger, RNAi triggers can now also be loaded into newly synthesized RISC, thus extending the duration of gene silencing by replenishing the pool of RISC that gets lost during its turnover.  As discussed last week, in the case of ALN-PCSsc for the treatment of hypercholesterolemia, GalNAC2.0 can achieve sustained potent gene silencing of PCSK9 for 2-3 months following a single dose compared to only days/weeks with the old chemistry.  Moreover, when it comes to single-dosing schedules, GalNAc2.0 is also vastly (~10x)  in terms of maximal knockdown potency compared to GalNAc1.0 which relies on a loading dose schedule (5x daily injections) for efficient loading of free RISC.

ESC less transformational in multi-dose regimens

Somewhat lost in Alnylam’s press release was the fact that for multi-dosing, the benefit of GalNAc2.0 is less dramatic in terms of the amount of RNAi trigger required to achieve say a 80% target gene knockdown.  For example, for TTR, the ED80 with weekly GalNAc1.0 in non-human primates was ~2.5mg/kg, the same as that now reported for the ED80 with a GalNAc2.0-chemistry improved version in rodents.

This confusion was not helped by the fact that direct comparisons between GalNAc1.0 and 2.0 were only shown for single-dose studies or by the fact the efficacy summary slide compares GalNAc1.0 for TTR with GalNAc2.0 for PCSK9.

Therefore, when the goal is to enhance the target product profile of your RNAi therapeutic by minimizing the frequency of subcutaneous administration (e.g. PCSK9 in light of the monoclonal antibody competition), then GalNAc2.0 certainly represents a very valuable advance, albeit at the cost of (still) relatively large injection volumes (10mg/kgà 4ml).  However, when it comes to the maximal potency against a given target gene, similar results may be obtained with GalNAc1.0 with possibly an improved safety profile.

Of course, more potent and at least equally sustained efficacy following subcutaneous administration may be achieved by Arrowhead's single-molecule DPCs.  If and when they can finally be translated into the clinic, is an important and open question.  I hope we see more data on that this week, also from the TIDES.


Latebloomer said...

I'd love to read your thoughts about how this news impacts the general move to take on HBV.

In particular, I'd be interested in your thoughts on how this news may impact how the medical/pharmaceutical field looks at knocking down s-antigen to affect change in the standard of care for HBV treatment...

In other words, what does this news do (and/or not do) to advance proof of concept regarding HBsAg KD for treatment of HBV?

Thanks in advance.


P.S. This kind of educating post (above) is indeed where you shine - a real service to all of us hoping to learn more about this field.

Latebloomer said...

Ah! Whoops! I see that this post is not yet about the ALNY news that came out this morning (Monday), but solely about the NR that came out yesterday, Sunday. (I had both NRs and your post in mind when I wrote my above comment.)

I'm guessing the next post about today's news regarding ALNY and HBV in particular is already forming for you and will be written up soon.

I'll be looking forward to it...


Anonymous said...

According to ALNY's pr, "The new ALN-HBV program derives from the company’s January 2014 acquisition of Merck’s RNAi assets, including their Sirna Therapeutics subsidiary."

I seem to recall that both MRK and ALNY have done a certain amount of due diligence into ARWR's technology. Given ALNY's history with TKMR (encroaching/infringing on TKMR's technology), I wonder whether ALNY might be doing something similar to ARWR.

What is known about the similarity/overlap between the ALN-HBV program and ARWR's technology? How secure in terms of patents and intellectual property rights is ARWR's RNAi technology obtained from Roche?

bikerieder said...

Not again the speculation mode please...
There is only one modus to show the world which candidate is the best for the very complex HBV.
Efficiency and safety in humans! And here we will see soon proof of concept from ARWR with ARC-520 in their ph2a data.
The others are far back. Just a few points to consider...
- ARWR uses 2 triggers to prevent mutations...
- ALNY and TKMR only 1 trigger...
- what is, when the virus is mutated...
- ARWR has a mellitin backbone...
- ALNY / TKM = more potency? If the case! more potency could get more tox...
- ARWR is very fast biodegradable, after 48 hours all has gone...
- No tox data from ALNY and specially no tox data, no data at all from TKM...
- the combos (ARWR with interferon / ALNY with ?? RT inhibitor?? maybe?? or maybe not??! are different...and for that, they will act differently....
- potency is not efficiency...
As you write above - the competitive landscape is very complex.
Not long ago, you doubted about HBsAg...
Let us cool down on speculation. Let ARWR / ALNY / TKM do their work.
We will see.

Dirk Haussecker said...

ALN-HBV-related blog went up at CureHBV site:

Bikerieder...TKMR will use more than 1 RNAi trigger. More potency/HBsAg knockdown, more tox...I don't buy that argument put forward by $ARWR's COO at recent Dt Bank conference.

Yes, by the end of 2015, ARWR will be half a year ahead of TKMR. Does this matter much?

Anonymous said...

Interested to hear any opinions on Arcturus press release today regarding Allele-Specific Silencing.


Dirk Haussecker said...

Arcturus may need some differentiation to break into the TTR market. I believe that is the motivation here. And who knows what the relative therapeutic profile will be for a broad vs allele-specific TTR RNAi Rx. The drawback is finding a sufficiently potent allele-selective RNAi trigger is more difficult if you are so constrained by sequence.

Anonymous said...

Thanks Dirk, I think you're right about Acrturus trying to differentiate itself. Is anyone else going after the Allele-Specific market? If they can do it and no one else is going after it, then they might be alone in a space that includes targets like Huntington's disease. I just haven't heard of anyone else yet and thought you or one of your readers might have.

Anonymous said...

Anonynous…regarding "anyone else going after the Allele-Specific Market?"

Isis has spoken of keeping the option open to perhaps pursue an allele-selective approach with their Huntington's Disease drug partnered with Roche (although the 1st drug will be a "total" knock down approach.

Studies investigating a SNP-based allele-specific approach for antisense is what Isis has published most (see 1st two reference articles below).

1) Carroll et al. Molecular Therapy vol. 19 no. 12, 2178–2185 dec. 2011

In this study, the authors note when using Isis ASOs for an allele-specific knock down approach (in this case they were targeting mutant hungtingtin mRNA (muHTT)),..

* ASOs can be designed to target SNPs ANYWHERE in the pre-mRNA, including introns, and so the number of potential SNP targets is more extensive than only being able to target SNPs in the protein-coding region.

Conversely, only 8% of the validated SNPs in the muHTT pre-mRNA in this study were found in the protein-coding region, and therefore the number of SNP targets accessible to siRNA is smaller.

I haven’t looked into Arcturus’ technology for allele-selective targeting with siRNA, but will be interested to take a closer look and compare to the allele-selective knock down approach studies already published from Isis & other ASO therapeutics companies.

2) Ostergaard et al. Nucleic Acids Research, 2013, Vol. 41, No. 21 pgs. 9634-9650

On page 9648, in the next to last paragraph of the article, the authors note that in vivo data from this study provides evidence that a SNP-targeting methodology can be used to design ASO drugs optimized for selective degradation of mutated huntingtin (muHTT) mRNA by….

a) First, screening the muHTT mRNA for SNPs that can be targeted with the ASO and then….

b) Second, finding the optimal position of that SNP present in the muHTT mRNA within the seven or eight base gapmer region of the Isis ASO.

Importantly, they go on to note the same strategy could be pursued in a broader context, to achieve efficient allele-selective inhibition for other gene targets.

The article closes by stating the work in the study “lays the foundation for the rational design of SNP targeting ASOs for treating autosomal dominant diseases.”

3) Hu et al. NUCLEIC ACID THERAPEUTICS Volume 00, Number 0, 2014

This study explores the effect of Isis single-stranded siRNA (ss-siRNA) sequence length and chemical modifications on allele-selective inhibition of human Huntingin expression.

In Figure 8 of this study, they take one of the ss-siRNA candidates that demonstrated superior allele-selectivity during in vitro testing and then determine the extent of allele-selectivity achieved in vivo by quantifying mutant and wild-type huntingtin protein reduction in various brain regions of an HD mouse model.

Anonymous said...

Thank you for the excellent reply, very much appreciated.

bikerieder said...

I tried to post on the curehbv.blogspot but it is immpossible for me. the profiles to select are not so easy as here on this side.
Maybe this is the reason there are not many messages at the curehbv.blogspot?

Dirk Haussecker said...

It is possible w/ both RNAi and ASOs to develop allele-specific knockdown. However, in both cases, this comes at the significant disadvantage of greatly limiting yourself in sequence space and you lose all the benefits of deep screening (particularly claimed by ISIS).

Having said this, in some cases you will just get lucky. In other cases, and this is where I believe companies like Arcturus could further differentiate itself, you might be able to rescue some less potent RNAi triggers through chemistry (--> UNAs e.g.) and design.

Anonymous said...

What happened to Alnylam's partnership with Medtronic to develop a treatment for Huntington's disease? has it been discontinued?

Anonymous said...

In the presentation Benitec's CEO made to AZ recently he stated TKMR was using BLT's proprietary ddRNAi technology.

If this is the case, why is it Dirk, you are so down on BLT but consistently upbeat on TKMR?

Anonymous said...

Dirk's Tweet: $TKMR back to 3/4 of old position in terms of share count. Low volume sell-off. TKM-HBV presentation after $ARWR pIIa data as major catalyst

And still no BLT position? BLT up over ten percent in Friday's trading in Australia. Similar in BNIKF.

TKMR uses ddRNAi according to BLT's CEO.

Dirk reloads on TKMR but holds nil BLT.

And you expect yourself to still be regarded as credible?

Anonymous said...

"In the presentation Benitec's CEO made to AZ recently he stated TKMR was using BLT's proprietary ddRNAi technology."

He did not actually "state" this, however, his slide was misleading because the line separating what was siRNA and ddRNAi was drawn in the wrong place. I minor technical glitch rather than a statement of fact, but, I agree, potentially misleading.

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