The problem with unintentional silencing is going to be very real to the FDA and i think they will insist on safety studies.
But the sentence that i outlined from the paper holds a much more likely pitfall to this therapy.
The target message RNA has to be a very precise sequence for the silencing to work. Thus any mutation in this sequence will tender the drug ineffective and unresponsive in the cell containing the mutated cccDNA. We must assume such mutations preexist and will give the respective HBV genome a selective advantage. It will spread quickly and repopulate the liver with therapy resistent progeny. No more supression, a happy virus and a drop in stock prices. Since there are so many places where it can mutate it will likely do so.
To see problem number 2 you need to go the place where they discuss the magnitude of the surface antigen suppression that can be expected.
In this context it needs to be mentioned that the replicor drug suppresses the antigen particle production by a factor better than 10000! fold. It is likely that a very high degree of surface antigen removal is necessary to remove the tcell tolerizing effect.
The famous idea, that a reduction under 500 units is sufficient to rekindle very likely does NOT apply in this setting. But this is complicated topic in itself.
The next problem that looms is not so apparent.
Consider the reason, why the precore mutant with elimination of the e antigen reduces the chance that the infected cell can be eliminated dramatically. It is the lack of the cytosolic processing of the e antigen carrying also the class I core epitopes through the proteasome to the MHC CLASS I presenting molecules on the hepatocte surface where the cd8+ CTL engage with their Cognate T cell receptors.
What would happen if the surface antigen production is shut down due to messenger RNA silencing? How can the now available surface antigen epitope specific cd8+ T cells come and recognize the infected cell? It is as if the critical markers of infection are now turned off.
The Replicor drug does not touch the primary sythesis of the surface antigen, it only blocks the formation of the globular structure on the ER membrane. Thus proteasome processing remains fully intact and effective. All the lights are on and the infected cell is visible to the searching cd8+ Tcells, resulting in targeted lysis and hyperintensive localized IFN gamma bursts, that clean up the neigborhood by noncytolytic cccDNA elimination.
Ok , now for starters to focus on the critical problem areas of this approach:
Look at the high specificity and sequence sensitivity of the target area sequence in these RNAs, which is discussed at a specific place in the paper. They made a strong effort to identify genotype independent sequences, so that they will have a drug that is effective across all relevant genotypes.
Also, they deselected from all mRNA sequences that can be expected in the human genome, to reduce the risk of switching off one of our own gene products. Very good and thanks to the human genome project for making that possible.
So now what major problem could occur due to the sequence sensitivity of the target sequence as it is mentioned in quite detail?
We will, after discussing this question, move on to the next problem area.
Here is the sentence you need to focus on for this:
Canonical siRNAs are based on 19 nucleotides of complementary sequence, but mismatches at the terminal ends are generally well tolerated and have much less effect on the efficacy of the siRNA than internal mismatches.
After reading it a few times. It seems like the major problem or challenge could be targeting HBV effectively without interfering with our own gene products.
They seemed to have narrowed it down to over 90% accuracy but not 100%. So it seems like there could be very adverse effects if it hits and silences our own genes from the mismatch at the terminal ebd but they believe that it's well tolerated?
As I was reading the paper I got the feeling that this could be a very powerful yet dangerous therapy.
Another off topic thing I picked up on based on what you had brought up I believe about Myrcludex is the potential problen of developing antibodies to the therapy by our immune system. With ARC-250 they mentioned that they didn't see this or it didnt occur which is a good thing?
This is not adding to the discussion, just news released by Arrowhead.
Arrowhead Receives Notice of Patent Allowance for New Protease Sensitive Masking Chemistry for DPC siRNA Delivery System
PASADENA, Calif. - February 28, 2013 - Arrowhead Research Corporation, a targeted therapeutics company, today announced that it has received a Notice of Allowance from the U.S. Patent and Trademark Office for U.S. Patent Application Number 13/336,028 entitled, "In Vivo Polynucleotide Delivery Conjugates Having Enzyme Sensitive Linkages." This new IP expands the Dynamic Polyconjugate (DPC) platform, broadly protecting Arrowhead's next generation DPC polymer masking technology. Using this masking technology, DPCs can be engineered for long circulation times and improved tissue-targeting characteristics. In addition, hepatocyte-targeted DPCs formulated using this new masking technology have shown to be highly potent upon subcutaneous administration. The company has previously reported target gene knockdown of 99% in monkeys after a single subcutaneous injection of 1 mg/kg, with >80% knockdown for 3 months. Additional data will be reported at upcoming scientific conferences and through peer-reviewed publications.
"This patent protects a new masking chemistry that broadens the reach of DPC-enabled RNAi therapeutics. It enables efficient subcutaneous delivery of siRNA and opens up new therapeutic area targets including oncology," said Dr. Chris Anzalone, President and CEO of Arrowhead. "Arrowhead scientists continue to discover innovative solutions for siRNA delivery and we look forward to providing updates on how these technologies are being deployed to create new drug candidates."
The formation of drug effect blocking antibodies is always a concern with macromolecular therapeutics. With regard to Myrcludex, the doctoral thesis of Alexa Schieck has shown that in a rat study the existing antibodies, despite a high titer against Myrcludex, did not inhibit the liver uptake and distribution of the drug. Antibodies against not structurally stable peptides without a stable folding structure are likely to be weakly binding and to only a small portion of the injected peptide. Furthermore the almost complete and tight binding to Albumin and plasma HDL particles will shield the peptide from AB attack.
The two components of the arrowhead system, the cholesterol bound iRNAs and the NAG linked bee venom peptide will also hopefully not be neutralized by AB responses on their way to the liver. I consider this a low risk concern.
Thanks for explaining the antibodies.
I read the ARC-520 paper again and also the link to "reference 16". My current understanding is that mismatches could lead to "off-target" slicencing, this will be a challenge because the HBV geonome has overlaps with the human geonomes which they refer to as mRNA's. I believe this mismatch is at the terminal ends.
They say that even so based the article below that these are well tolerated.
16.Huang, H, Qiao, R, Zhao, D, Zhang, T, Li, Y, Yi, F et al. (2009). Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs. Nucleic Acids Res 37: 7560–7569
This is not related to ARC520, but to precore mutants. After natural HbeAg seroconversion, most patients will be in the used to be called "inactive" phase. However, some researchers argued that in this phase, the HBV is actually under intense immune control/pressure, leading finally to the immune escape phase (100% precore, no wildtype, according to Liaw). No-one actually stated what that immune pressure is. So can it be due to e-antigen specific CD8+ Tcells? Some papers described an increase in viral diversity both before and after e-antigen seroconversion. So it seems plausible that a gradual decrease in e-antigen production due to increase in precore mutants, finally leads to breaking of tolerance, some clearance of infected liver cells by e-antigen specific CD8+ Tcells? The loss of infected liver cells seems to be supported by the fact that serum HbsAg is lowest in this inactive phase. So is it true that in this phase, HBeAg is still being produced, therefore CLASS I MHC molecules with e-antigen epitopes are expressed on the surface of infected cells and can be seen by e-antigen specific CD8+ T cells? Thereby maintaining control and keeping replication to very very low? If all this is true, why don't the e-antigen specific CD8+ T cells completely clear the infection?
Finally, ARC520 does not seem to silence the gene for HBcAg(?) and so core antigen epitopes MHC Class1 molecules should still be expressed on the cell's surface.
You are raising some very important and fundamental questions, the answer to which explains the very existence of the e antigen negative stage and the subphases of low and then higher disease activity.
A few items for introductory consideration:
The e antigen, that in the immune tolerant phase tolerizes the class1 and likely the class2 Tcells is present in absolutely substantially lower amounts than the surface antigen in the circulation.This fact makes it easier for an antibody response to neutralize it when its production is slowed down in the liver by a cytokine milieu that reduces its expression intensity from the cccDNA.
David Milich has shown, that an intense anti e antibody level is usually present even in patients whose e AB is negative by regular tests. He showed using an unusual detection antibody that it is present in high titers in the form of immune complexes. Thus if the absolute amount of e antigen produced becomes low it will also travel undetectable in the form of immune complexes, despite its ongoing production in some or all infected liver cells.
Here you have the paradox state of an e antigen neg hepatitis with ongoing low level e antigen production. In that phase you expect intense immune pressure and resulting HBV adaption over time. The immune pressure is likely resulting from the cd8+ Tcells recognizing e or core epitopes class1 on the surface of liver cells that still produce e antigen.
It is important to understand that the core protein, despite sharing the same epitopes with the e antigen DOES NOT PARTICIPATE in the proteasome processing towards class1 epitope peptides, therefore a precore mutant carrying cell shows no core/e Ag on its surface. The reason is likely that the core component polypeptides are very strongly bound to each other forming the globular core particle and the synthesis rate is also much less than the e antigen. This also points to the fact that the remaining core expression under the arrowhead iRNA treatment will not lead to epitope visibility.
The adaptive escape mutations against the e antigen recognizing class1 T cells are not limited to a shutdown of the incriminating remaining e antigen expression. There is another route of escape by mutating the single relevant class1 epitope itself, from lower proteasome processing to lower to no binding to the Tcell receptor. This is without question a critical component of the final scenarios that we see in the later stages of e antigen negative disease.
Thus the answer to the question why the class1 anti e Tcells do not typically wipe out the remaining infected cells is that too many precore mutants are already present and that in non precore mutant cells the epitopes themselves have mutated away from being recognizable.
The density of the remaining e Ag epitope class1 Tcell activation in the liver has to be high enough and the activation strong enough to produce the collateral exstinction of cccDNA by the gamma IFN bursts sufficiently for a clearance in a range that the surface antigen Tcell response can finally set in and allow permanent maintainance of the ultra low infected state.
It looks like you posted a response and it was removed?
No idea why my response was removed?
I wanted to ask...
TARGET MESSAGE RNA:
So the concern with this is two fold, 1 being the efficacy on mutated HBV geonomes, and 2. the inhibition resulting in mutations of the HBV geonome. Are the mismatches mainly due to their goal of a genotype independent therapy.
1. Would it make sense for them to limit mismatches by possibly coming up with Genotype dependent RNA's
2. Forgive me for sounding so naive, but in the likely hood of mutations being developed, can message RNA's be developed to rescue viral breakthrough?
REDUCTION IN SURFACE ANTIGEN:
It seems from what your saying blocking would be better than inhibition for the purpose of Immune Response. Arrowhead didn't mention or show any effect on immune recovery which is very important for the overall goal of the therapy. I hope Replicor continues to make progress, since they have even presented results on immune reocvery.
1. I've read about Surface Antigen mutations, maybe some of these drugs can make things worse by forcing the virus to mutate where it doesnt release surface antigen at all, or is this absolutely required for the virus to survive?
1. Can some of these problems be predicted well in non-human vivo trials?
2. Do these companies consult viral experts. I am sure they do, but some of these things should be of a big concern in therapy development and resources used. Or should we expect to see these type of things from drug ccompanies to boost stock values?
Forgive my ignorance, but I am still stunned that Replicor has come up with such an efficient method of fighting HBV ahead of other companies. I haven't seen any public papers, but do you know if they have any known viral experts working with them contributing to thier success?
Here is to your questions:
1. Would it make sense for them to limit mismatches by possibly coming up with Genotype dependent RNA's
2. Forgive me for sounding so naive, but in the likely hood of mutations being developed, can message RNA's be developed to rescue viral breakthrough?
A genotype specific siRNA would be better functionally and less mismatches would be present. But it would be too expensive and they might need a trial for every genotype.
Viral breakthrough during therapy will occur, if a preexisting or de novo produced mutant has even a single internal difference in one nucleotide, for which a high chance exists. Remember that the hybridization sequence is only 19 nucleotides long, the match internally has to be perfect or it will fall off. That mutant virus will be unaffected by the siRNA therapy and start growing to the foreground, making the treatment ineffective.
A rescue siRNA could theoretically be developed, but it is not a feasable option.
I've read about Surface Antigen mutations, maybe some of these drugs can make things worse by forcing the virus to mutate where it doesnt release surface antigen at all, or is this absolutely required for the virus to survive?
A loss of suface antigen expression happens rarely or an invisibility on regular test platforms by mutations in detection Bcell epitopes. This situation, when combined with measurable HBV DNA and elevated liver enzymes is called occult Hepatitis B. It is remarkable, that the DNA never seems to rise high in these cases and normally the disease is mild.
Some remnant surface antigen expression is obviously needed to produce the viral envelope, but it is a comparably very small amount.
The hope of the company it seems is to suppress the virus production almost completely with monthly shots, realizing that it will not eliminate the cccDNA, but removes the disease symptoms. They carefully call it a "functional cure".
The expectation that the surface antigen specific Tcell response will be rekindled and a true elimination by CTLs will ensue is similar to the effects that the Replicor treatment has provided. But the limited diminishment of the surface antigen levels and the possible removal of cellular epitope targets by placing the block on the level of the messenger RNA might become a double stumbling block for that approach.
Do these companies consult viral experts?
Of course they do, but there is always healthy optimism present and the hope that things might work in spite of all the stumbling blocks ahead.
Replicor also cooperates with experts in molecular Biology of Hepatitis B, for example Jake Liang from NIH and Allyson Jilbert from Adelaide, Australia for their preclinical duck studies with the compound.
But their find of the particle secretion suppression in HBV is also an example of incredible good luck and great fortune, since they developed this class of compounds originally to treat influenca, HIV, HCV and other viral diseases under the concept of entry inhibitors. The effect on HBV surface antigen is kind of out of line and could not have been originally expected. But here it is and it represents a truly historical step forward in the possibility to fight and permanently suppress the HBV virus, even if this has to be achieved in combination with other treatment strategies in combination.
Thanks for the reply concerning e-antigen. It is certainly food for thought. Now I want to ask about how HBsAg inhibits the immune response. I have read articles by Locarnini that "circulating HBsAg subviral particles were shown to repress TLR9 signalling by enhancing SOCS expression in dendritic cells (pDCs)". I understand pDC to be a significant source of INF-alpha. On Replicor website, the Chinese page states that these SVP inhibits the proliferation of Tcells (my translation). I wonder can you explain how SVP actually inhibits our immune response to HBV.
Many thanks in advance.
The immunomodulating effects of the surface antigen that tend to protect the existence and spread of HBV can be divided into three categories.
1. The obvious direct effect is to remove any antibody that could coat the virions and protect the liver cells from reinfection. The large amounts of antigen in comparison with the available titers of antibody are completely eliminating the effectiveness of that antibody, that might exist in many cases at substantial titers, masked and undetectable by standard assays.
Thus the surface antigen ascertains HBVs capacity to constantly reinfect the liver.
2. There are numerous reports at conferences and some published papers that show that larger concentrations of surface antigen have the effect of deactivating, reactivity, have suppressing effect on dendritic cells and macrophages, reducing cytokine output, like Il18 and Il-12 and diminish activity in several other proinflammatory signaling pathways, especially NFKappaB.
In most cases however, the concentrations to demonstrate these effects are rather high and towards the high end of HBSAg concentations found in patients plasma.
Also, if the immunosuppressive effect is real, then it should effect not just the reactivity against HBV, but against other viral diseases as well and we should see a general immune weakness of HBV patients. While there are some claims in this direction, the general impression is that HBV patients have basically, aside from their viral infection with HBV, a normally reacting immune system as it is relevant to real life clinical performance.
3.The third effect is the overwhelming presence of the surface antigen particles in high amounts distributed over the whole circulation and even in the extracellular space. Thus dendritic cells and macrophages will take them up in high numbers all the time, everywhere in the body. Consider the fact that large amounts are produced every day that also disappear in eual amounts in the steady state, thus those particles are basically phagocytosed and processed and presented in huge amounts in every dendritic cell and macrophage.
It is important at this point to consider the effect of dendritic cells phagocytosing antigens in the context of local noninflammation or non danger, as happens physiologically all the the time.
The lack of a danger signal to be recognized by the dendritic cell leads to a processing in the class1 and class II pathways with consequent engagement of the cognate Tcell receptors of patrolling naive or memory Tcells of both kinds THAT WILL BE FOLLOWED BY A LACK OF STIMULATION AND ACTIVATION, or a stimulation towards apoptosis of the engaged Tcell. This is an important way to physiologically avoid Tcell autoimmunity against "peaceful" antigens, a critical function of our immune system. What is rather stimulated are suppressor Tcells, to control the posssibly wrongly stimulated effector T cell that is CTLs or Thelper cells.
The surface antigen particle is seen as a peaceful antigen by the dendritic cells either for reasons analyzed in number 2 or because they lack the spiky ordered structure of the core and the intense danger signal that comes fromthe presence of DNA or RNA in a particle that is phagycytosed.
The HBV virions have all these features and are causing a danger signal activation.they also get caught first of all at their site of production in the liver and activate local macrophages and dentritic cells and then classII cognate T helper cells right on site, with resulting local proinflammatory cytokine production, but not of a very effective nature, hence the chronic hepatitis and hence the effectiveness of the antivirals to intensely reduce that inflammation by lowering virion production without reducing the number of infected cells, since the gamma IFN levels are typically not high enough to initiate cccDNA noncytolytic elimination.
Thus in summary, on a bodywide scale,the surface antigen eliminates Tcells principally active against its epitopes of both classes.
A further school of thought proposes that the activation away from the liver absorbs and activates surface antigen specific Tcells and that upon even stronger activation, the activated Tcell is far from the point of action and will have a limited life time as is typically the case for a stimulated cd8+ cell, since the body needs to recover from diseases quickly, they receive the order to apoptose even in the case of positive activation to a lytic, activated state with some delay after the activation took place.
All together these processes deplete the body from capable surface antigen specific Tcell activation and from accumulation and activation and action in the liver, rendering this antigen almost totally ineffective in the setting of chronic hepatitis.
It can thus easily be seen that removing the surface antigen particle from the circulation, while leaving its intrahepatocyte expression fully intact with intense epitope presentation due to the relative large intracellular amount will cause a game change in the bodys defense mechanisms against HBV.
I tried without success to send you a p.m.
I am a compensated cirrhotic; cleared HCV virus, sustained (SVR) for several years now - but left with damaged liver. Made worse by alcohol abuse (I no longer drink alcohol, however) -
I've seen you make references to HepTech products. Do you have experience with these products? And if so, then how long did you take them?..and are you still taking them?
What improvements have you seen?...symptomatically, any blood test improvements, liver numbers improved, etc??
i think you got confused by posts, it is me who used heptech to regress cirrhosis, it did work very very fast, about 2 years to reduce fibrosis and a fibroscan of 4.5kpa or maybe little less dont remember now at the end of 2012
i also changed my diet but made little exsercise and used black rice and bluberries every day for a long time to clear fatty liver too, just check all my older posts so you can see all the improvments
i also used gcmaf therapy,.....activation of macrophages because of ultra high nagalase in serum and a general immune deficency.we cannot say if this worked too on liver repair process with diet and life style, it did work on nagalase and general immune system which is very very strong since it lowered nagalase
Thank you so much for your response.
Ive been doing some reading on Virology and Immunology to understand more about how the immune system works so I can participate in other disucssions and prevent myself from posting elementry questions but I wanted to thank you for your answers you posted the other day.
1. I read about the two people you mentioned and they seem to be very reputable, its very encouraging to know that. The most encouraging factor is that you believe that Replicor has made a breaktrhough.
2. It seems like Replicor has a good solution that could really benefit. Have they been making waves at the conferences? (I am sure this will lead to a good partnership opportunities for them and also good offers which will help speed up the development of the drug).
3. Do you think that Arrowhead's delivery model could be used to effieciently deliver other treaments more effectively to the liver such as TDF, Replicor or TDF?
4. Have you been presented with details at conferences on how Replicor works on the Surface Antigen in detail such as to the degree of detail that Arrowhead posted. If yes, does it make sense to you as for the unintended effect of Replicor on Surface Antigen?
5. Lastly since you have more exposure to development of HBV treatments. Do you think Replicor is moving at a decent pace to bring the drug to market? (I understand this is very legnthy process)
Thank you in advance.
At the conferences, the replicor presentations and posters are well attended, but clinical hepatologists have a hard time to believe the data sometimes, since the numbers are so low.
As for company personnel, I am sure they show some interest, but it is not at a stage for them to buy, I would guess, since the application per infusion is very hard to sell and they would also be afraid that unexpected side effects might pop up later, that could terminate the development as it happens so often in the world of drug development.
All in all replicor seems to move as fast as they can to arrive at the final formula and at a combo with immunmodulators that will generate a high chance of truly stable svr.
While the effect of surface antigen particle formation blockage was initially a pleasant surprise and serendipitous find, it makes sense to experts in the field of molecular morphogenesis of HBV, who understand the delicate and unique interaction of the touching points of the surface antigens transmembrane helices that lead to a closed protein sphere embedded in a membrane. This interaction is easily disturbed by mutation experiments as Volker Bruss, the worlds leading expert in this field has convincingly shown at the molecular biology meeting of hepatitis B in Orlando. Thus the disturbance of this interaction by the replicor drugs is a plausible mechanism.
Thanks Stefan. Of course I remember you, but I'm reasonably sure that Studyforhope had also made a reference to HepTech some time ago.
HT is not regressing my cirrhosis, so perhaps there is something else that I need to add. I have been on HT for several months, and only a month ago began developing palmar erythema and joint pains..possibly signs of decompensation. As I recall your cirrhosis was totally asymptomatic...is that correct? If so, then perhaps it was in much earlier stage than mine. Stefan, do you know of anyone with symptomatic cirrhosis that has benefitted from HT (HepTech) products??
I have seen several people regress their cirrhosis on the HT protocol. But it might take at least two years and has to be combined with a healthy lifestyle.
It is of course possible that it does not work in your case, but you seem to be using it for only a short time. The products are unfortunately way to expensive. You also need to use a pre and probiotic.
more information on HT protocol and antifibrotic study you can found on their website (was a antifibrtic study that was presented some time ago):
Study was conducted at the University of Alberta under the direction of Dr. Lorne Tyrrell, (co-investigators Dr. Peter Schmid and Dr. Michael Joyce).
Thanks - I appreciate your reply. I agree w/you that the HT products are way too expensive. I do have healthy diet and lifestyle - and also take a daily probiotic. As for HepTech (HT) efficacy, I am committed to a minimum of one year..to see if there is some benefit for me.
Stefan told me that his was fully compensated cirrhosis - no symptoms whatsoever.
Do you know of ANYone with symptomatic cirrhosis who has benefitted from the HepTech products? If so, I would be very interested in any information or experience that you could share with me.
Again, sincere thanks for your response -
I am not aware of a case of decompensated cirrhosis that has tried any of the heptech products. Once a patient is at this stage, bad episodes of encephalopathy or varicosis bleeding can occur easily and any recommendation must be made very carefully and best be done by an experienced hepatologist. However, the use of regular lactulose and acetylcysteine 3 x 600mg plus about 1000mg of TMG is most likely very beneficial. As for the antioxidants and antifibrotics in the HT products, if somebody decides to use them, i would use a dosing of only one third, with food, to lower possible spikes in uptake and exposure.