Saturday, May 17, 2008

RNAi Therapeutics versus Antisense- Where Delivery Makes a Difference

Regulus, the microRNA joint venture between Alnylam and ISIS Pharmaceuticals, is the most visible manifestation of the scientific overlap that exists between antisense and RNAi. The overlap, however, is not just limited to the science, but also extends into the capital markets. To better help the investor differentiate between the two technologies, I’d like to use this blog to provide an overview of some of the fundamental differences underlying the development of RNAi and antisense into therapeutics and their long-term prospects, with an emphasis on delivery. Needless to say, beware this discussion will be heavily biased in favor RNAi, but then again these are the reasons why I’ve been attracted to RNAi in the first place.

Certainly, progress in both areas in the last 3-5 years has mutually benefitted the investment climate for both technologies as it has heightened interest and increased confidence in RNA therapeutics in general. However, the two technologies also compete for investment dollars with many of the same investors, which are typically upbeat about the future of gene-based medicines but unsure where to place their bets, allocating their investments based on where they see most promise. One issue that often comes up in making this decision is the observation that while for systemic applications antisense, as practiced in the most advanced programs today, is typically administered without a particular delivery formulation, the development of specialized delivery technology is frequently cited as the key challenge for RNAi to realize its ultimate therapeutic potential.

Antisense for gene knockdown works largely by two mechanisms: interfering with translational initiation (e.g. AVI Biopharma's morpholinos) or through an RNase H-type mechanism (e.g. Santaris and ISIS Pharmaceuticals). For this, the key factor is to achieve efficient hybridization of a single-stranded oligonucleotide antisense with its target mRNA which either prevents productive ribosome association to the mRNA (inhibition of translation initiation) or may be recognized as a substrate for the RNase H enzyme which may degrade the RNA portion of the mRNA-DNA duplex, but normally functions in the degradation of the RNA primer during DNA replication.

Various oligonucleotide chemistries have been developed to optimize these processes for in vivo applications. Essentially all of these are single-stranded oligos of which the sugar phosphate backbone is heavily modified to a) increase their in vivo stability; b) improve their pharmacokinetics and avoid rapid renal excretion by promoting their association with components of the blood; c) similarly allows them to be retained in tissues; d) facilitate crossing of cell membranes; and finally e) increase their target mRNA binding. By contrast siRNAs, because of their charge and more rigid double-stranded nature and with apparently some exceptions that include mucosal epithelia, do not cross cell membranes efficiently on their own and therefore need to be specially formulated for most applications.

The use of unformulated antisense is consistent with their mechanism of action. Since antisense does not harness a naturally existing endogenous gene silencing pathway, it relies on achieving concentrations of oligonucleotides in the target tissue over a prolonged period of time that are high enough such that, as a result of the rules of thermodynamics, a sufficient fraction of target mRNA will be recognized. Similarly, unlike RNAi, the specificity of antisense is largely governed by biophysics and benefits only relatively little from biological proof-reading.

In practice, to achieve the necessary tissue concentrations, patients are typically dosed frequently at the initiation of therapy so that the tissue concentrations reach steady-state therapeutic levels. Targeted delivery of antisense into cells of interest may allow one to achieve a knockdown earlier, but any benefit would only be short-lived as antisense is not retained in specific gene silencing complexes but will soon redistribute according to their partition coefficient throughout the entire tissue so that ultimately similar amounts have to be administered and a formulation would only be a nuisance with little benefit.

By contrast, RNAi harnesses an endogenous and catalytic gene silencing mechanism, which means that once it has been delivered, either by conjugation or in nanoparticles into the cytosol, they are efficiently recognized and stably incorporated into the RiSC silencing complex to achieve prolonged gene silencing. In fact, measurable RNAi-mediated gene silencing can be observed at siRNA concentrations so low that it becomes difficult to detect them (e.g. fluorescently-tagged siRNAs by microscopy). This means that as the majority of siRNAs that do not reach the cytoplasm may disappear quite rapidly, the total exposure of the body to the nucleic acid can be much lower compared to antisense which should be beneficial both in terms of safety and pharmacodynamics (activity profile of drug over time).

This is not to say that chemical modification is not practiced in RNAi. However, unlike in antisense, the purpose of modification in RNAi is mainly to avoid triggering innate immune responses, making the siRNA sufficiently stable so that they survive their journey into their target cells, and also to stabilize them as part of RiSC (Merck has been talking about that concept on several occasions); and as we learn more about the biochemistry of endogenous RNA silencing pathways, modification is also increasingly used to increase the inherent biological specificity of RNAi. Unfortunately, it is surprising to me that compared to RNAi only very little, if at all, is reported about the specificity of antisense and I would be grateful if somebody here could point out pertinent studies that I should be aware of.

Targeted delivery may also avoid unnecessary drug exposure of non-target tissues. For unformulated antisense, no matter what the indication and target tissue, the biodistribution is essentially the same, and toxicities of the liver and kidney due to extended exposure to large amounts of the heavily modified antisense compounds is well known.

Certainly, improving the therapeutic index is an important issue for RNAi Therapeutics, too, but as the many transgenic mouse models which express ample and highly efficient RNAi throughout their life without causing overt toxicity attest, ultimately the improvement in the therapeutic index of RNAi is not limited by its very mechanism of action.

While it is a certainty that antisense companies will come out with 4th and 5th generation antisense technology, advances after decades of antisense research aiming to improve target mRNA recognition will only be marginal and based on trying out yet more nucleic acid modifications, although it appears to be a challenge to improve upon the efficacy of probably the most potent antisense modification that have now been known for a while, namely LNAs and their derivatives.

While RNAi efficacy in animals has already surpassed that of antisense for applications of the liver and lung as well as other tissues, I am confident that future advancements in RNAi will be more than marginal. For example, even as recent liposomal formulations achieve 90% gene knockdown in the liver at 1mg/kg, this still means that only about 1 in 10,000 siRNAs that have reached the liver makes it into the cytoplasm (assuming it takes about 1000 cytosolic siRNAs to achieve that level of knockdown according to a recent presentation by Phil Sharp). Alone a better understanding of the endosomal uptake of these nanoparticles, which is only in its infancy and starting to be explored, should allow for more than incremental improvements in the therapeutic index of RNAi Therapeutics.

And if you are still undecided on where the future is heading, numerous transfection studies in vitro where it can be assumed that equal amounts of antisense and siRNAs are present in cells, have shown that RNAi is quite a bit more potent on a mole-by-mole basis comnpared to antisense.

I am aware that some in the antisense community, including investors, may take offense with this blog, but since I am often asked about this issue, I think a more straightforward approach is better than to keep beating about the bush. And, of course, there is always the comment section.


Anonymous said...

Well I have to at least give you credit for telling it like it is in the first paragraph, "heavily biased."

Enumerate said...

There is, of course, hybrid approaches ...

John Rossi's rHIV7-shl-TAR-CCR5RZ suppresses HIV by expressing three therapeutic nucleic acids that are directed against key steps in HIV replication.

Dirk Haussecker said...

Enumerate- the rHIV7-shl-TAR-CCR5RZ is indeed a hybrid approach, but does not make use of antisense as used in this context. "TAR" is a TAR decoy, and "CCR5 RZ" is a ribozyme against a viral entry receptor. It should also be kept in mind that both are expressed here in addition to an shRNA from a DNA template, and to my knowledge, while DNA-directed RNAi can be extremely potent, expressing antisense from DNA has never been met with much success.

Anonymous said...

Thanks for the entry, Dirk.

As always, very clear and right to the point.


Anonymous said...

Excellent write-up but what about Sangamo's ZFP technology? I think that's a more compelling alternative to RNAi than antisense. How about a blog sometime comparing RNAi to ZFP-based gene therapy?

Jon said...

Here is a paper comparing the mechanisms of action and specificity of siRNA and several forms of antisense.
Summerton J. Morpholino, siRNA, and S-DNA Compared: Impact of Structure and Mechanism of Action on Off-Target Effects and Sequence Specificity. Med Chem. 2007;7(7):651-660

Jon said...

Regarding delivery formulations, recent work with Morpholinos and PNAs conjugated with cell-penetrating peptides have shown impressive uptake in adult animal systems.

Lebleu B, Moulton HM, Abes R, Ivanova GD, Abes S, Stein DA, Iversen PL, Arzumanov AA, Gait MJ. Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev. 2008 Mar 1;60(4-5):517-29. Epub 2007 Oct 22.

Dr. Neugenes said...

I enjoyed your piece, but it is a major inaccuracy to lump antisense into a single bucket. Most of the pitfall and challenges of antisense that you speak of relate to the negatively charged chemistries of Isis and Genta. AVI's chemistry features a neutrally charged backbone. As Jon already posted, the morpholino is superior in many regards to other antisense approaches, including the PNA and LNA competitors now on the scene.

There are multiple papers confirming naked morpholinos restoring dystrophin in animal models. That work is ready to take a giant leap forward based on a portfolio of peptide conjugates developed by AVI Biopharma. Systemic delivery, something that RNAi is very far away from, will be seen in 2009 in DMD and it has already been achieved this year with morpholinos modifed with a positive charge and/or a peptide conjugage. USAMRIID has run multiple primate models using 1,000 times lethal dose of Ebola and achieved 100% survival.

Too many in the RNAi crowd are moving too fast to bury antisense because they are so excited by the natural elegance of siRNA. When they come off the clouds once these mega rich deals slow down and reality sets in, they will be faced with some facts.

Fact 1 - siRNA has a moloculer weight nearly 10 times that of most antisense chemistries and a negative charge, so delivery is and will always be an issue.

Fact 2 - Manufacturing siRNA is more complex and more expensive than manufacturing and materials for antisense chemistires (particularly the morpholino, which can be produced cheaper than the other antisense molocules).

Fact 3 - While antisense players using PNA and morpholinos have found, have published and our now taking peptide conjugated antisense into the clinic, these "delivery assistants" are nowhere near as costly as the various lipo or lipid carriers being promoted and developed by RNAi companies. In the end, costs may limit areas in which these RNAi drugs can effectively compete.

Fact 4 - In terms of specifity, its not even a competition versus the morpholino when compared to siRNA. Increasing specifity comes with other trade offs in RNAi and other factors will play into this including the delivery agent used. Antisense and particularly morpholinos are amazingly specific and that specifity holds up with peptide conjugates. This potential future problem for RNAi won't be as focused on until after the delivery problem is a commercially viable way.

Fact 5 - There are key areas of high theraputic value, such as pre-rna splicing where antisense chemistires are simply much better suited, again, particularly the morpholino. You will see that Ryzard Kole, the founder of Ercole and the holder of the fundamental patents on pre-RNA splicing sold his company last month to AVI BioPharma for stock. He dis this despite a deal with Isis using their chemistry and Santaris on their LNA chemistry. Ryzard is a VERY smart guy and mRNA scientific heavyweight. siRNA will not play in this area...and one more point, the effects of a single dose of morpolinos in vivo restored dystrohpin in dogs for "months" based on study to be presented on Friday.

Too many people are painting with broad strokes when it comes to antisense and they are too caught up in the RNAi hype and exciting search for delivery agents and optimization that they are missing the major progress that is being made in the best 3rd generation antisense chemistry, which is the PMO. Pay attention to upcoming papers from USAMRIID and maybe even check out the presentations that will happen this Friday at the American Society of Gene Therapy. Just punch in "morpholino" and see what 20 years of work is about to unleash. Morpholinos are going to shake things up in RNA land shortly and it will be interesting to see how the folks now looking to develop microRNA are going to realize another area of antisense/morpholino value.

Dirk Haussecker said...

Dear Dr. Neugene,

How is the weather in Oregon?
I think it would be inappropriate for me to claim that RNAi will solve all ills. Unlike RNAi, antisense certainly has potential for therapeutically modulating splicing, inhibiting microRNAs, even to knock down mRNAs, and generally influence gene regulation whenever there is a biological single-stranded nucleic acid intermediate involved.

As for mRNA knockdown, I view RNase H as quite a bit more potent compared to the inhibition of translation initiation by morpholinos. It's maybe therefore not too surprising that with the acquisition of Ercole the focus of AVI has now been shifted towards splice modulation, but you are probably more privvy to this information and may like to comment on this.


Anonymous said...

Four years on from your original post do you see that there have been any changes in either field to cause you to think of either of the two technologies in a new light? How would you compare the two technologies these days?

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