standard Efficient Delivery of RNAi Prodrugs

RNAi as an insecticide.

RNAi as an insecticide.

Some insect scientists are thinking about dsRNA and siRNA as “insecticides” so it is not surprising that clinical medical researchers are thinking about them as “drugs”.

If RNAi kills insects then they are insecticides; if they eliminate or alter a disease state, they are drugs. Regardless of how one thinks about RNAi a common challenge is delivering RNAi triggers (dsRNA, siRNA, etc) to appropriate cells at appropriate times.

RNAi drug being delivered in a nanoparticle. from doi:10.1038/nrd2742

RNAi drug being delivered in a nanoparticle.
from doi:10.1038/nrd2742

The meme of RNAi as a drug has stimulated an enormous amount of research into the delivery of RNAi triggers. Insect scientists could not just learn a lot from that research but could greatly benefit from some of the technologies emerging from it.

siRNAs can not enter cells on their own, they are excessively large – somewhere around 14 kDa, and excessively charged – somewhere around 40 charged phosphates. As a result, delivering these molecules to cells can be very challenging.

Various RNAi triggers

Various RNAi triggers

Insects are small relative to humans so presenting cells in vivo with massive amounts of RNAi triggers by simply injecting the material into the hemolymph can work to achieve the desired effects on gene expression even if uptake is inefficient, but not always.

Potential solutions to this delivery problem abound – lipid nanoparticles, synthetic nanoparticles, specific molecular conjugates.

Meade et al. in Nature Biotechnology describe a very clever solution to the RNAi delivery problem. They describe the synthesis of what they call siRNNs – short interfering ribonucleic neutrals.  siRNNs are siRNAs that have all or some of their phosphates chemically neutralized . When siRNNs enter a cell the chemistry responsible for neutralizing the phosphates is reversed, resulting in an ‘active’ siRNA.

Basic idea of a prodrug

Basic idea of a prodrug

So, siRNNs are like prodrugs – therapeutically inactive chemicals that are chemically modified inside cells to be therapeutically active. For example, codeine (3-methylmorphine) is a prodrug which is converted to morphine through the action of a P450 upon entry into a cell.

Meade et al. neutralize the phosphates by joining various conjugating groups to them via a thioester bond. Extra-cellular esterases cannot cleave thioester bonds but cytoplasmic esterases can, resulting in loss of the conjugating group and restoration of charged phosphates

siRNN prodrug and siRNA 'drug' from: doi:10.1038/nbt.3078

siRNN prodrug and siRNA ‘drug’
from: doi:10.1038/nbt.3078

Meade et al. explore which conjugating groups are most effective, how many phosphates need to be neutralized and which phosphates in a siRNA need to be neutralized.

Phosphate charge neutralization strategies using thioesters. from: doi:10.1038/nbt.3078

Phosphate charge neutralization strategies using thioesters.
from: doi:10.1038/nbt.3078

Restoration of phosphates following cytoplasmic esterase cleavage of thioesters. from: doi:10.1038/nbt.3078

Restoration of phosphates following cytoplasmic esterase cleavage of thioesters.
from: doi:10.1038/nbt.3078

 

 

 

 

 

 

 

The results are interesting, even impressive. Silencing effectiveness was increased and the ‘doses’ required decreased.

siRNN synthesis is modular, ie makes use of modified nucleotides, so precise control of placement of neutralized phosphates is possible. Meade et al report synthesizing over 3000 RNN oligonucleotides during the course of their study – which they claim is evidence of the robustness of the chemistry.

The technology reported by Meade et al. has been licensed to Solstice Biologics, Inc.

Others are exploring charge neutralization of siRNA. For example, Triphos Therapeutics, Inc..

Here’s a slide presentation on siRNNs from the president of Triphos Therapeutics.  A little long and detailed but informative.

Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. 2014, Meade, BR. et al.  Nature Biotechnology, published online 17 November 2014; doi:10.1038/nbt.3078

 Recent Related Posts on Technology Topics:

Issues in RNAi Delivery and Development 
Tripodal RNA: An Enhanced RNAi Trigger  

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