Wednesday, December 7, 2011

SNALP RNAi versus RNaseH Antisense for Gene Knockdown in the Liver

Following recent phase I results from ISIS Pharmaceutical’s Factor XI (ASH abstract 12999; addendum: PR on phase I data reported on December 12) and Apo C III programs, there is little doubt left that phoshorothioate-based RNaseH antisense as developed by this company and Santaris can mediate target-specific gene knockdown in the liver in Man. These results confirm the clinical experience with the registrational hypercholesterolemia candidate mipomersen and are corroborated by the impressive HCV results obtained by Santaris’ with its anti-miR122 HCV candidate. Beyond the liver, RNaseH efficacy has been demonstrated for solid cancer (custirsen) and possibly Excaliard’s (now Pfizer’s) anti-scarring candidate. On the other hand, the recent trial termination(s) by Santaris, and the safety profiles of mipomersen and OncogeneX' custirsen highlight some of the challenges facing phosphorothioate antisense technology.

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Therefore, as RNAi Therapeutics have progressed from the basic discovery of its mechanism in mammals 10 years ago to solid proof-of-concept gene knockdown in the form of the ALN-TTR01 results 2 weeks ago, it may be a good time to compare and contrast these two technologies also in light of the fact that, after the RNAi Therapeutics backlash, there is a clear trend towards Big Pharma (and other pharmaceutical companies) opening themselves up again towards antisense, meaning that the 2 technologies are competing for precious non-dilutive funding. For this purpose, I will focus on the liver as the best developed target organ for both these technologies.

20 to 100-fold more antisense required

An obvious advantage of antisense, with about 3x the age of RNAi Therapeutics, is that more is known about its clinical pharmacology. As such, there is good visibility as to how much antisense will be needed to achieve the kind of 50-75% knockdown that will be required for therapeutic outcomes in most cases. Dose has important implications particularly in terms of safety and cost.

Current ‘2nd gen’ molecules (fully phosphorothioated gapmers) like mipomersen require 200mg oligonucleotide per week. Actually, if 300-400mg would have been better tolerated, the higher dosages would have enhanced the commercial profile of mipomersen considerably. Let’s therefore say 1000mg per month for 2nd gen RNaseH antisense.

With the higher-affinity ‘2.5 gen’ technologies that are starting to move into the clinic, best exemplified by Santaris’ LNAs which in fact may also symbolize the most potent version, it is expected that clinical dosages can be further reduced. Based on the non-human primate data and clinical dosage regimes, I expect that dosages of around 100mg/week or 500mg per month are feasible in the foreseeable future. Also because of the modifications involved, I would be therefore very surprised if the cost of oligonucleotides for treating a patient over a year would be below $15,000 even at commercial scale.

By contrast, it can be expected that it will take about 0.15mg/kg of siRNA formulated in the ‘2nd gen’ SNALPs that are now moving into the clinic to achieve the type of once-a-month pharmacology that Tekmira and its licensees are aiming for. If you do the math, that translates into about 10mg per month of siRNA oligonucleotides. Give and take the added costs of the lipids and formulation process, but cheaper nucleotide chemistries involved, this translates into a maybe 50-fold cost of goods difference alone. For some diseases and in some countries, this may be less of an issue, but it will be a factor for others.

Safety

The even larger implications of dosage is the related safety. Although clinical repeat-administration studies with SNALP have yet to be conducted, it seems that with the 2nd gen SNALP formulations, the main safety challenge with SNALP will be in managing acute hypersensitivity reactions around the time of drug administration. Based on similar issues with intravenously administered biologics such as monoclonal antibodies where e.g. transient immune suppression with steroids is routine (and widely accepted), I believe that infusion-related acute toxicities will be manageable.

What is nice with the pharmacology of SNALP RNAi Therapeutics is that the bulk of the drug that does not hit the target, i.e. gets incorporated into the RISC silencing complex, is rapidly turned over by the body, meaning that drug exposure levels between drug administrations will be extremely low. It is because of this that I am hopeful that the risk of causing liver toxicity, long believed to be the main toxicity challenge for SNALP, is quite limited at dosages of 0.15mg/kg/month.

By contrast, RNaseH antisense do not harness a natural gene silencing mechanism and, in the case of the phosphorothioate-based gen 2 and gen 2.5 antisense, work by saturating the target (and off-target) organs with high levels of the ‘sticky’ phosphorothioated oligonucleotides so that mass action carries enough of them into the cells. Consequently, the exposure of the body to the antisense drug is significantly higher compared to SNALP-delivered siRNAs. Assuming ~100-300mg of antisense oligonucleotide per kg of liver or kidney tissue (e.g. ISIS TTR patent application US 2011/0294868) and single-digit microgram siRNA oligonucleotide per kg of liver tissue (e.g. Landesman et al. 2010), you could argue that the real difference in bioburden between antisense and SNALP RNAi is about ten thousand fold. It also does not take into account that it is the sticky phosphorothioate chemistry that is thought to be responsible for much of the toxicity (interactions and turnover) whereas RNAi triggers employ more natural chemistries. On the other hand, double-strand RNAs are recognized by more innate immune receptors than highly modified, single-stranded oligonucleotides.

Route of administration

Although the subcutaneous administration of SNALPs has been demonstrated (see e.g. Tekmira's ApoB patent) and may become practical with the higher potencies of SNALPs and extracellular matrix-degrading technologies as developed e.g. by Halozyme, antisense is currently more amenable to subcutaneous administration whereas SNALP have to be infused in an institutional setting. This means that, as is the case for essentially all monoclonal antibody drugs, SNALP drugs have to address diseases of considerable unmet medical needs where patients do not perceive a once-a-month trip to the infusion center a huge burden. Maybe Pfizer can't, but I can think of many such diseases. Infusion in an institutional setting also has the advantage that acute toxicities, the main safety challenge for SNALPs, can be well managed through professional supervision, whereas patients that inject themselves with antisense at home may be slightly panicked on seeing redness develop at the injection site or on experiencing ‘flu-like symptoms’ that have been reported to occur at high frequency with antisense (often 1/3 to 1/2 of patients), but has surprisingly been little discussed by ISIS Pharmaceuticals.

Manufacturing

Like route of administration, manufacturing is considered to be a practical advantage of antisense over SNALP RNAi. I agree…in purely practical terms. What is, however, entirely forgotten is that as long as you can deal with the manufacturing complexities, it suddenly gives you an invaluable competitive advantage. How about unlimited market exclusivity? Isn’t one of the lessons that Big Pharma should have learned from the current patent cliff that simple small molecule chemistries are highly vulnerable to generic competition? Isn't this also a major reason for why everybody obsesses about monoclonal antibodies these days, yet is often strangely held against SNALP RNAi? To my knowledge, there are no generics of a nanoparticle-formulated drug.

So in summary, as antisense has reached an inflection point as a slew of clinical data is confirming the early clinical results with mipomersen from 6-7 years ago which demonstrated gene knockdown in the liver, SNALP RNAi is making even faster progress with many of its theorized advantages, especially related to the amount of oligonucleotide required and pharmacology, turning into clinical reality quickly. The race is on. The most likely winners meanwhile are the patients.

8 comments:

Anonymous said...

ddRNAi potentially has even greater potencies and convenience, with the possibility of "one shot" cure. Many new non-viral delivery methods are now being studied which should help with safety and side effects.

Dirk Haussecker said...

Have you watched the movie 'The Rise of the Apes' recently? I find it amusing the images that 'viral gene therapy' evoke in the general public. What is sad, however, is that in the real world, decision makers are still swayed by these stereotypes. For the purposes of delivering large DNAs as in ddRNAi, I personally would much prefer to be treated with current viral rather than non-viral delivery methods based on safety alone.

Anonymous said...

No, I've not seen that yet! Forgive me, I'm only a medical practitioner and not a research scientist, but I thought new methods like minicircle vectors avoided immune reactions that one can sometimes get with viral vectors.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163107/?tool=pubmed

http://circ.ahajournals.org/content/124/11_suppl_1/S46.long

Anonymous said...

Human body is designed to detect and react against foreign invasions such as virus. Innate immunity react quickly makes repeat administration more difficult. If immune reactions can be avoided....... Immune reactions on viral vectors have dampened their clinical use, there were many trials over the decade demonstrate so. But on the other hand, deliver pharmacological signigicant doses of RNAi effector molecules without immune responses reactions is not easy. Any new development?

Anonymous said...

yes much progress in the science reported in last 12months with AAV viral vectors (for shRNA delivery)

Anonymous said...

Have you checked SNALP dose and cost?

Dirk Haussecker said...

Oligo providers are very coy about their prices and oligo Rx companies are equally coy about their manufacturing cost structures. The absolute numbers are therefore ballpark figures; the relative numbers, which are more relevant to the SNALP vs antisense comparison, however, are more reliable.

Having said that, the siRNAs determine the cost of goods of a SNALP and that of other lipid-based formulations. On a nucleotide basis, simple 2'-o-methyl modified siRNAs with otherwise standard phosphate-sugar backbones are much cheaper than nucleotides with cyclic sugars and phosphorothioates. If you go to the websites of standard oligo suppliers you will see e.g. that the price for an LNA nucleotide is several times that of an 2'-o-methyl.

I have yet to fully understand the potential of reducing price through scale-up to commercial scale, but exemplary numbers that I have heard from the industry of an siRNA synthesis run for a phase I study involving maybe 10-50g siRNA is half a million dollar, or 100k per 10g- which is the amount of mipo oligo needed per patient year.

To get then from 100k to 15k with 2'-MOE phosphorothioate chemistry assumes some confidence in gaining economics through scale-up. These economies, however, would equally apply to an (SNALP) RNAi Rx so the relative cost of difference remains more or less the same.

Anonymous said...

the haemophilia trial breakthrough is an early crystalization of the big leaps we're seeing in AAV vector delivery for gene therapy, mentioned above. exciting times ahead for gene therapy applications

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