Wednesday, August 20, 2014

Stabilizing RNAi Triggers against Cytoplasmic Degradation Pays Dividends

In describing the preliminary phase IIa results of ARC520 for HBV, Arrowhead Research noted that the duration of gene silencing (2 months and more) was surprisingly extended in Man compared to the preclinical experiences in rodents and non-human primates.  Alnylam hasnoticed the same with its GalNAc-siRNA conjugates, especially the highly modified ESC version. 

The extended gene silencing activities, of course, bode very well for RNAi Therapeutics in general when in the early days (~2002-2003) I was a bit apprehensive when gene silencing in my transfections of cancer cell lines persisted for only 2-3 days (as we now know largely due to their rapid cell division).  To maximize the duration of gene silencing, thereby opening up RNAi Therapeutics to new applications and increasing its competitive profile, it is important to understand the factors underlying it.

Alnylam explained the differences to the preclinical experience because rodent and monkey hepatocytes seem to have a more hostile, degradative cytosol compared to human hepatocytes (hypothesis 1).  In one experiment, only 6% full-length ESC-GalNAc-siRNA remained after a given time in rodent and monkey cytosolic extracts while in human liver cytosol extracts more than 60% persisted.

This, however, was only a correlation and I have considered it equally likely that the difference in gene silencing duration might be a function of more stable RISC complexes in humans (hypothesis 2) or increased stability in the endo-lysosomal compartment (hypothesis 3).  Especially for GalNAc-siRNAs, I would think that the reason that it works in the first place is due to them being able to accumulate in endo-lysosomes from which they only get released in the wake of natural vesicle membrane turnover.  So chemical stability here would be a critical factor since the endo-lysosomal compartment is known to be highly degradative.

DPC and SNALP: two endosomolytic technologies with different durations of gene silencing

While I still consider that endo-lysosomal stability of the naked RNAi trigger is critical for approaches like GalNAc-siRNA conjugates, the new DPC-enabled ARC520 results strongly indicate that another critical factor lies downstream of endo-lysosomes.  This is because in the DPC approach which involves strong endosomolytic activities that should activate soon after endocytic uptake, the risk of the RNAi trigger being degraded in the endo-lysosomes should be low.  Similarly, there should be little contribution to gene silencing from RNAi triggers that get released into the cytoplasm in a delayed fashion.

SNALP is another delivery technology where the RNAi triggers that become active in gene silencing get released into the cytoplasm soon after endocytic uptake.  However, while clinical data supporting 3-4 week dosing frequencies have been obtained with SNALPs (e.g. ALN-TTR02), the silencing does not appear to be as extended as with DPCs.  So given that one marked difference of the payloads used with SNALPs and DPCs is the modest degree of chemical modification historically used with SNALPs, this, too, points towards cytosolic stability of the RNAi trigger being important for the duration of gene silencing.  Parenthetically, it also suggests that Tekmira may want to similarly explore heavily modified RNAi triggers while being mindful not to step on the McSwiggen patent toes of Alnylam.


RISC-optimized ultra-stable single-strand RNAi triggers

In the case of traditional double-stranded RNAi triggers as e.g. used with DPCs, the stabilized RNAi triggers get used up over time as they are recruited into RNAi effector complex RISC.  Part of this process involves their unwinding into single-strand RNAs with the guide strand being retained.   It is known that once used, a 'normal' guide strand (or microRNA) is not recycled into another RISC complex and will likely suffer metabolic destruction once the protein components of RISC have become degraded as part of natural protein turnover.  And even if the guide strand had been stabilized, because a standard single-strand molecule that had relied on being part of a double-strand structure for RISC recognition, old age will eventually catch up here, too. 

What a waste after all this effort of getting the RNAi trigger into the cytoplasm.  So why not take a cue from the single-strand RNAi practitioners who optimize single-strand RNAi triggers also based on being able to be recognized by RISC?  If a corresponding dsRNA contained corresponding recognition elements, then the guide strand could contribute to another round of gene silencing, thus extending and enhancing knockdown.  On the other hand, the lessons learned from stabilized dsRNAi triggers should also benefit the single-strand RNAi approach as increased cytosolic stability should also increase their duration of activity: RISC-optimized ultra-stable single-strand RNAi triggers.

16 comments:

Anonymous said...

Great piece, as usual Dirk. Are you intimating that ALNY have an ace up their sleeve in this area?

Striking is the very last line.

"RISC-optimized ultra-stable single-strand RNAi triggers".

Another nail in the coffin for ddRNAi proponents.

Latebloomer said...

I rather liked the idea that maybe the very long duration of lowered s-antigen that ARWR has reported so far might have to do with some viral clearance beginning to happen (along with reinfection being greatly hampered due to entecavir keeping HBV-DNA in check)... less infected cells producing overall less s-antigen.

But this is a pretty good explanation too, ;-).

Wouldn't mind one bit, though, if BOTH reasons for prolonged lowered s-antigen levels were happening...

Linda

Anonymous said...

Does this suggest that the SS RNAI work started between Isis and alnylam should resume? Why did that work discontinue?

Anonymous said...

I am not so sure it ever stopped. It just looked like it did. But the proof would indicate otherwise.

Is it only through ALNY one can get exposure to ssRNAi?

This is what I found on Google:

Multiple US provisional patent applications have been filed covering the design, modification and delivery of single-stranded oligonucleotides which anneal intra-cellularly and then initiate RNAi.

But I can find no way of leveraging in to these patents on market.

Anonymous said...

Anonynous...I've pasted the ss-RNAi related information from Isis' 10-K for year ending 2013 starting with "Table of Contents".

Note the last paragraph, the we, meaning Isis..."retain...all rights to single-stranded RNAi, or ssRNAi, therapeutics".

Also, on a related note, Isis had published several ss-RNAi articles in collaboration with the Corey Lab at UT Southwestern. The most recent published article from Isis and the Corey lab can be accessed via the link below.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814390/pdf/gkt693.pdf

Table of Contents

Alnylam Pharmaceuticals, Inc.

In March 2004, we entered into a strategic alliance with Alnylam to develop and commercialize RNAi therapeutics. Under the terms of the agreement, we exclusively licensed to Alnylam our patent estate relating to antisense motifs and mechanisms and oligonucleotide chemistry for double-stranded RNAi therapeutics in exchange for a $5 million technology access fee, participation in fees from Alnylam’s partnering programs, as well as future milestone and royalty payments from Alnylam.

In August 2012, we expanded the license to include using the double-stranded RNAi technology for agricultural products. For each drug Alnylam develops under this alliance, we may receive up to $3.4 million in substantive milestone payments, including up to $1.1 million for the achievement of development milestones and $2.3 million for regulatory milestones. In 2013, we earned a $750,000 milestone payment when Alnylam initiated a Phase 3 study for a drug targeting TTR. We will earn the next milestone payment of $375,000 if Alnylam initiates a Phase 1 study for a drug in Alnylam’s pipeline.

We retained rights to a limited number of double-stranded RNAi therapeutic targets and all rights to single-stranded RNAi, or ssRNAi, therapeutics.

Anonymous said...

Does that mean ISIS invented/discovered ssRNAi and have proprietory rights which allows them to retain all rights?

I think the purpose of RGLS was to develop ssRNAi in partnership but I can't find anything that explicitly says that.

After a bad run of luck an inveterate gambler can be in hock so deep to his loan shark that sometimes its cheaper and easier to just get rid of the loan shark.

Anonymous said...

Thanks for pointing me to that. Based on listening to the old web casts when regulus and ss rnai joint venture was launched those arrangements were portrayed by management to be quite distinct.

Anonymous said...

This is what I found on Google:

Multiple US provisional patent applications have been filed covering the design, modification and delivery of single-stranded oligonucleotides which anneal intra-cellularly and then initiate RNAi.

Would you be so kind as to post a link to this quote?

Anonymous said...

This is the link. It is under the R&D tab.

http://www.genesis.co.nz/research-and-development

But this is what I can't understand. Alnylam returns the ssRNAi stuff to ISIS. ISIS says they retain all rights to it as if they own it. Then you have this bankrupt company in NZ saying they discovered it.

I guess ISIS must've stolen it somehow. Probably with legal threats and patent challenges. Not hard to do against a company that is bankrupt.

"Genesis established a subsidiary company named Solirna Biosciences Ltd (soli: plural of solo) to develop the delivery of dual single strands of RNA to achieve gene inhibition through the RNAi mechanism. This gene silencing technology was invented by Genesis scientists. A Japanese investment group provided funding to Solirna, with Genesis retaining a substantial ownership position."

Anonymous said...

To Anonymous who posted August 22, 2104 at 7:20 pm

Per Lima et al. from Isis (Cell 150, 883–894, August 31, 2012) they reported…

• the ss-siRNA reduced the target mRNA both in in vitro and in vivo studies by recruiting the RISC endoribonuclease Ago2 to cleave the mRNA

• the 5’ phosphate is needed for ss-siRNA activity, but not for siRNA activity, and as a result, metabolically stable 5’ phosphate analogs were incorporated into the ss-siRNA for in vivo activity

• the SENSE strand found in double stranded siRNA is NOT required to activate RNAi in cells or animals with the ss-siRNA approach [i.e, only the single, ANTISENSE strand was required to activate RNAi]

Conversely, according to the summary on the Genesis “Research and Development” page that was referenced, “The technology is designed to deliver separate complementary strands of RNA and let them anneal [into double stranded molecules] inside the cell where they can induce gene specific RNAi,”

So not really sure where anyone can be coming from to claim the Isis approach “takes cues” from the Genesis approach, given that JUST a single-stranded RNA is interacting with components of the RNAi pathway inside the cell to reduce the expression of the target mRNA, as reported by Isis in Lima et al.;

Whereas conversely, the Genesis approach (at least from what is claimed on the website) states that TWO single stranded RNAs eventually form a double stranded RNA (dsRNA) once inside the cell, & THEN this dsRNA is able to interact with components of the RNAi pathway to reduce target mRNA expression.

Anonymous said...

Thank you for that, previous poster.

I guess to be sure, we need to validate the facts by checking patents not web page dialogue. Problem is the patents they talk about can't be found.

How do you explain that one?

Anonymous said...

"By checking patents not web page dialogue."

Exactly...I couldn't agree more, which is why I would have started by going straight to the source (I.e., contact Isis investor relations and ask them if they can direct you to appropriate patents covering Isis' ss-siRNA technology).

I cerainly wouldn't trust anyone's sole opinion on the matter from the blog comment section, so again, I encourage you to contact Isis IR in your search for "the facts".

Anonymous said...

Dirk, you're not censoring your website are you?

What happened to the post about GENs ssRNAi patents being covered by the Inventions Secrecy Act?

If you want to censor stuff, how about starting with those Dr So n So cured me with his hoodoo voodoo spell posts

Anonymous said...

Dirk please delete above post.
Regards,
Agent Brown.

Anonymous said...

To Anonymous August 22, 2014 at 5:26 PM, there may be some fire to this smoke.

If we look at the RGLS directors one of them used to wear brass at Genesis years ago. I guess the things learned then have been extrapolated up to RGLS.

The confusing element is Genesis saying they invented ssRNAi. But no patents recorded with their name in this space. ISIS saying, they retain all rights to it while ALNY returns the rights but continues over many years to ride rough over every other RNAi developing company in the field. All the while, side stepping around ssRNAi but taking an equity position in RGLS. Albeit a minority one.

The Roche/Santaris deal certainly indicates there is value in this area.

Pfizer has registered its interest through Mirna and Art Kriegs involvement in it.

Value yet to be accrued to RGLS however. Perhaps that will change shortly.


Anonymous said...

Interesting comments. A quick look at this failed company showed it to be under control of the Chinese.

Nothing to do with RGLS.

Definitely needs a transaction to be re-listed.

Don't think anything Chinese connected will have any attention til after Alibaba.

Once that happens, then markets are open for Chinese business.

Communists and Goldman Sachs, aka US Government, hand in hand. Who could ever have imagined that.

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