Tuesday, March 11, 2008

Dharmacon’s Accell Delivery Technology Signals Increasing Focus on non-Endosomal siRNA Uptake Strategies

[Update May 8, 2008: Based on a recent patent application by Dharmacon, WO 2008/036825, it appears that Accell siRNAs are in fact very similar in principle to Alnylam's 2004 cholesterol-conjugated siRNA paper in Nature and for which the Australian patent office has now accepted a related patent application (Australian Patent Application No. 2004206255)].

Dharmacon (now part of Thermo Fisher Scientific), the company that rose to prominence as one of the first and trusted siRNA suppliers that understood to maintain their leading position by staying at the cutting-edge of RNAi research, just announced the introduction of Accell siRNAs.

Accell siRNAs may not be just yet another type of modified siRNA, but appear to be inherently cell membrane permeable allowing for straight-forward in vitro gene knockdown in all cell types tested, including the notoriously difficult-to-transfect primary cells, without the need for prior formulation with special transfection reagents. This is in contrast to conventional siRNAs that, due to charge and size, are not cell permeable and are therefore require formulation with delivery reagents such that they may gain access to the cytoplasm (the site of Ago2-mediated RNAi) following endosomal uptake and lysis of that compartment. This can be toxic, either because of the chemistry of the specific formulation or the triggering of cytokine responses by engaging TLR receptors in the endosomes.

It is also clear that although potent gene silencing may be achieved following endosomal uptake, typically only a fraction of the siRNA that had been delivered to the cell is actually active in gene silencing. In fact, the rate-limiting step in vivo in many instances may not be getting the siRNA to the target cell, but getting it into the target cells once in close proximity. In addition to better understanding the endosomal uptake-release mechanism, research into alternative technologies may therefore prove highly rewarding.

Although technical details were not disclosed, it is reasonable to assume that Accell siRNAs have been rendered more cell permeable either by the addition of a small lipophile to the siRNA duplex or by modifying the highly charged siRNA backbone without compromising siRNA silencing activity. Stanley Crooke, the CEO of ISIS, has e.g. noted before that it is the amphiphilic nature of single-stranded antisense nucleic acids facilitates their cellular uptake, so that it is conceivable that Accell siRNAs may have been engineered to be similarly amphiphilic.

The current Accell siRNA technology, however, has some drawbacks in that a special tissue culture media is required and therefore does not allow them to be directly translated to in vivo applications, although formulating them in ways that get them to their target cells should not represent an insurmountable challenge, with small conjugates such as cholesterol being likely candidates. The recommended concentration of 1 micromolar siRNA (about 50-fold higher of what is typically used in vitro) further indicates that the process needs to be optimized before Accell siRNAs will be widely used for large-scale and routine tissue culture experimentation. The immediate market potential should therefore largely come from work with primary cells.

Accell siRNAs reminds us that systemic RNAi delivery is a multi-step process, starting with extravasation of the siRNAs from the blood stream into the tissues to cell attachment and cellular entry, and the potential for improving each step is significant. It is fascinating to see how intensely each of these tasks are being addressed and the many creative solutions coming out of these efforts.

5 comments:

b_lo said...

I wonder when we'll find out what the exact mechanism is that Accell uses to cross the liposomes.

jpb said...

And if the Accell siRNA delivery medium that should always be used just contained some excess cationic lipid ? Did anyboby try & succeed without ?

Anonymous said...

I know this is a late post considering Accell has been on the market for some time now so my apologies. Upon hearing about Accell siRNA I immediately ordered some as I have been working on developing a similar technology that would allow for soluble siRNA delivery in the absence of standard transfection reagents. I have had success not only with the Accell siRNA delivery media but also with DMEM (minus serum & antibiotics) at siRNA concentrations greater than 250 nM. The major problem, as I see it, is the amount of time for treatment that is needed. In order to obtain a maximal response, times of greater than 36 hours were necessary. This was significantly impacted in the presence of 5% FBS (60% decrease in activity). This will be problematic for many applications as many cell types (especially primary) don't like serum-free conditions for an extended period of time.

Anonymous said...

Also, analysis of the Accell siRNA duplex by electrophoresis showed a similar migration rate to standard siRNA duplexes so extensive backbone modifications or conjugation to a large lipophile is unlikely. Accell is most likely a cholesterol-like conjugated siRNA that is effective mainly because of Dharmacon's prior proprietary modifications that have dropped the EC50 of siRNA duplexes making Accell much more effective than Alnylam's original cholesterol-siRNA conjugates.

Dirk Haussecker said...

Thanks. These are very interesting insights. I therefore assume that Accell siRNAs are linked to the following patent application by Dharmacon and are consequently very similar to Alnylam's 2004 Nature paper on cholesterol-conjugated siRNAs for in vivo RNAi, and for which the Australian patent office has just accepted a related patent application ("Lipophilic derivatives of double-stranded ribonucleic acid"):

(WO/2008/036825) DUPLEX OLIGONUCLEOTIDE COMPLEXES AND METHODS FOR GENE SILENCING BY RNA INTERFERENCE:

1. A duplex oligonucleotide complex comprising:

a. a sense strand that ranges in size from about 18 to about 30 nucleotides ; b. an antisense strand that ranges in size from about 18 to about 30 nucleotides, wherein the antisense strand has significant levels of complementarity to both the sense strand and a target gene, and wherein the sense strand and the antisense strand form a duplex; c. a conjugate moiety that facilitates cellular delivery; and d. a linker molecule that is from about 3 to about 9 atoms in length and attaches the conjugate moiety to said sense strand;

Priority data seems to be 2006.

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