Changing therapeutic approaches: the RNA level

[Jon Moulton]

The way we design therapeutics for many conditions is set up to undergo a radical change. Traditionally, herbs and animal parts were used as sources of bioactive small molecules. Once we realized the small molecules were the useful part, we started to isolate them from the natural world and use them as purified medicines. Then came chemical synthesis of small molecule drugs. More recently, large molecules like monoclonal antibodies have been purified from living systems and used as medicines. Now we are synthesizing large molecules, such as synthetic oligonucleotide analogs. Using larger molecules means the molecules can specifically recognize larger molecular surfaces, presenting a new possibility -- manipulating RNA through molecules targeted by Watson-Crick pairing to specific RNA sequences.

The field of RNA therapeutics started with molecules such as phosphorothioate antisense and, with the discovery of the RNAi system in cells, has branched into siRNA/shRNA and their modifications. While the attention of pharmaceutical companies has shifted mostly toward siRNA, therapeutics based on siRNA struggle with a set of recalcitrant problems: innate immune activation, off-target gene modulation, and delivery into cells in vivo. Meanwhile, antisense structural types like Morpholino oligos have been developed which don't activate immune responses, are far more specific (affecting their targeted RNA with little to no off-target RNA interaction), and can be delivered into cells in vivo by attachment of small moieties such as cell-penetrating peptides or guanidinium dendrimers.

These "delivery-enabled" antisense molecules can be used to prevent expression of targeted genes or to alter splicing of pre-mRNA, clipping targeted exons out during formation of mature RNA. Potential applications include antiviral drugs and treatments for genetic diseases. Morpholino oligos are already in clinical trials for Duchenne Muscular Dystrophy. Here are a few citations to the literature showing the emergence of this new sort of medicine.

Open access paper:
Wu B, Li Y, Morcos PA, Doran TJ, Lu P, Lu QL. Octa-guanidine Morpholino Restores Dystrophin Expression in Cardiac and Skeletal Muscles and Ameliorates Pathology in Dystrophic mdx Mice. Mol Ther. 2009 May;17(5):864-71. Epub 2009 Mar 10.

Open access review:
Moulton JD, Jiang S. Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos. Molecules. 2009 Mar 25;14(3):1304-23.

Either of these can be accessed using PubMed at the National Center for Biotechnology Information: ncbi.nlm.nih.gov

 

[Hercules Rockefeller]

These "delivery-enabled" antisense molecules can be used to prevent expression of targeted genes or to alter splicing of pre-mRNA, clipping targeted exons out during formation of mature RNA.

Yes, morpholinos have an advantage over siRNA in that they can modulate RNA splicing. But siRNA has an advantage in basic research in that siRNA knockdown can be genetically encoded. You can make transgenic organisms with tissue-specific expression of hairpin siRNA under inducible control, whereas morpholinos are synthetic and cannot be made by cells. Tissue-specific RNAi helps to minimize the whole-organism effects of off-target modulations.

Genetically-encoded siRNA is of no relevance to human therapeutics, of course, but exogenous siRNA is certainly being investigated as a human therapeutic approach, as is morpholinos. Off-target effects are something to be wary off with both siRNA and morpholinos (from personal experience).

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Finally, so as to avoid any suggestion of impropriety or conflict of interest, I think it should be pointed out that Jon Moulton works for Gene Tools, a commercial company that supplies morpholino oligonucleotides.

 

[Jon Moulton]

Hi Hercules,

Thanks for pointing out I'm with Gene Tools -- I normally set up a signature file that identifies my work, but this board does not allow me to do so. I apologize that I overlooked adding a note by hand identifying my affiliation.

Regarding tissue specificity of oligo delivery, local injection of the new delivery-enabled oligos allows targeted delivery, unlike the whole-embryo injection techniques used over the past decade.

- Jon
Gene Tools, LLC
(it would be nice to automate this...)

 

[Idle Mind]

I wouldn't worry about putting that in every post, Jon. Most of the people that come into this thread will be able to remember who you are and what your affiliations are from this point forward.

 

[Hercules Rockefeller]

I normally set up a signature file that identifies my work, but this board does not allow me to do so.

No problems. :thumbsup: I don’t think automatic signatures have worked here for a long time, if ever. Don’t worry about a manual signature – I just wanted to establish this early so that you wouldn’t be confused with spammers trying to sell stuff (mostly Nike rip-offs for some reason ).

Regarding tissue specificity of oligo delivery, local injection of the new delivery-enabled oligos allows targeted delivery, unlike the whole-embryo injection techniques used over the past decade.

Indeed. I have a zebrafish background, so when I hear the word “morpholino” I automatically think of embryo injections. But in larger organisms localised injection would produce targeted delivery.

Here’s a morpholino question I’ve been wondering about. I know I can go to the Gene Tools site but I might as well ask this here now....

Can antisense morpholinos be targeted to the 3’ UTR in an attempt to block miRNA binding? If so, is it possible that morpholino delivery might actually up-regulate protein production by blocking miRNA-mediated degradation?

 

[Jon Moulton]

I moused over the edit button, which popped up "Edit/Delete". As I wanted to delete this post (I had duplicated the post during editing), I clicked the button and searched for a way to delete. No luck. Just erasing the message returned the notice that I must have at least three characters in my post. So, instead of deleting I am narrating the experience.

 

[Jon Moulton]

Hercules wrote:
"Can antisense morpholinos be targeted to the 3’ UTR in an attempt to block miRNA binding? If so, is it possible that morpholino delivery might actually up-regulate protein production by blocking miRNA-mediated degradation?"

Yes. It's a more specific approach than blocking the miRNA itself, which affects expression of all the genes influenced by that miRNA. There is plenty of variation in the microRNA response elements (MRE) bound by a single miRNA, so blocking one can have little effect on most of the others. However, there are some sequence constraints on the MREs for a specific miRNA so a given Morpholino may cause some some off-target MRE protection.

Choi WY, Giraldez AJ, Schier AF. Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430. Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30.

- Jon
Gene Tools, LLC
(it's better this way...)