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.
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.
