Following major IP battles with Benitec and tensions high within the company, DNA-directed RNAi Therapeutics company Nucleonics became the first major RNAi Therapeutics company to go out of business in 2008. During the liquidation process, Alnylam surprised observers by scooping up Nucleonics’ IP assets. Adding to the confusion, Nucleonics had just initiated dosing in a phase I study of NucB1000, a candidate for the treatment of Hepatitis B Viral infection. The fate of this trial has been a mystery.
Now, after three years, scientists and clinicians involved in the study broke their silence and reported data from the study in the journal Antiviral Therapy (Gish et al., 2011). The results from the prematurely terminated study that had enrolled only three patients in the first dose cohort were largely in line with (at least my) expectations. More surprising, however, were some disclosures related to Alnylam’s apparent interest in NucB1000 and DNA-directed RNAi Therapeutics.
The active ingredient of NucB1000 is a plasmid DNA that encodes for 4 shRNAi triggers targeting various regions of hepatitis B virus. The ability to easily accommodate a number of RNAi trigger sequences in one clinical candidate is one of the advantages of ddRNAi Therapeutics and particularly useful for antiviral applications as it should minimize viral escape. Using ddRNAi as opposed to a synthetic siRNA for HepB infection is also reasonable given the chronic nature of the disease.
This, however, is about it in terms of the positive attributes of NucB1000. The reason why I never thought this candidate had even a remote chance of clinical success is the fact that it uses a cationic lipid formulation to deliver the DNA to affected hepatocytes. Unlike the efficient liposomal delivery of small, synthetic siRNAs to the cytoplasm of hepatocytes, the field of gene therapy is still far away from using non-viral means to efficiently deliver large plasmid DNA- about 100-200x the molecular weight of siRNAs- to the nuclei of hepatocytes where the DNA can serve as the template for shRNAi trigger transcription. It is not just the size that complicates the cellular uptake and release of the nucleic acid, but the requirement for getting the DNA from the cytoplasm to the nucleus is the major rate-limiting step differentiating it from synthetic siRNA delivery.
The authors addressed this deficiency by stating that even if uptake efficiency was limited per given plasmid infusion, the long-lived expression from the plasmids means that it may be possible to achieve ddRNAi activity in sufficient numbers of hepatocytes simultaneously by multiple administrations. While I can follow the logic, in the absence of convincing pre-clinical data of this concept, it remains just that, a concept.
Of equal concern is that cationic lipid-formulated large plasmid DNA carries a high risk of triggering innate immune stimulations following intravenous administration. This has already been a challenge for lipid-formulated small siRNAi triggers, but it is less of a fundamental challenge there because of the limited sequence information in a given siRNA. This means that any inherent innate immune stimulatory potential can be relatively easily controlled by chemistry. The much larger plasmid DNA, which typically is of bacterial origin, does not allow for such control.
I should mention that in light of these concerns the authors state that no apparent immune stimulations were observed in the mouse and dog studies at much higher dosages than were anticipated for the trial.
Nevertheless, it is surprising to me that the FDA allowed this trial to go ahead based on pre-clinical co-transfection efficacy results that appeared to me to be glorified in vitro, and not in vivo models of HepB infections. It may be for this reason that the study authors made the point that they saw antiviral efficacy in a model that did not involve co-transfection:
‘Preliminary data showed that 1% of mouse hepatocytes were transfected. Subsequent experiments in which spike recovery was performed demonstrated that nearly 10% of cells were transfected; actual rates varied between 6% and 9% (Nucleonics, Inc. and Alnylam, unpublished data). A number of explanations for the discrepancy between this rate and the 20% knockdown of HBsAg have been postulated.’
‘A 20% knockdown of HBsAg’ to me does not sound like a robust pre-clinical efficacy result in support of clinical studies. I doubt that the FDA would have let the
I know, a non-viral ddRNAi approach sounds great on paper, and would you believe it, not just the VC investors and Nucleonics management were fooled, but also a company like J&J was about to contribute a fair amount to a planned $25M round C capital raise.
NucB1000 phase I results
Altogether only 3 of the planned 15 patients received NucB1000 in the phase I study. This was the first dose cohort which involved 5mg of plasmid DNA, ~0.06mg/kg. As expected, immune stimulation was observed in all 3 patients: a transient fever, accompanied by cytokine elevations, which quickly resolved following antipyretic treatment. This was described by the authors as a mild event and not a reason why patients would have dropped out of the trial- definitely not ‘life-threatening’.
Needless to say, given the nature of NucB1000 and the size of the trial, no signs of antiviral efficacy was seen.
To this day, it is not clear to me why exactly the trial was terminated. Did Nucleonics and their investors attempt a Hail Mary with the first dose cohort before cutting their loss on an increasingly improbable investment, or did they suddenly get cold feet as allegations of scientific misconduct relating to the pre-clinical data were raised? Given that money and lawsuits were involved, I favor the former with the latter serving as justification.
Overall, the Gish et al. paper is a most unusual one. The introduction already states:
'The proprietary nature of these data imposes limitations; however, the authors felt the results merited presentation and discussion as they involve a first-in-human study with potentially important clinical implications.'
I agree that the data was valuable, if not somewhat predictable, and am glad the authors had the courage to publish them. At the same time, the publication can also be read as a justification for why the trial went ahead.
It is also a curious publication because it suggests that Alnylam did not just acquire the Nucleonics IP estate to bolster its synthetic RNAi IP, as Barry Greene stated at the time, but that it was quite a bit more interested in DNA-directed RNAi Therapeutics in general, and NucB1000 in particular than one would have thought. While it cannot be determined conclusively how much NucB1000-related work was actually performed on Alnylam’s dime, the following passages suggest that it was not insignificant:
‘Exhaustive cell culture studies have demonstrated specific down-regulation of all HBV RNAs, including pregenomic RNA as compared with mismatch controls (Nucleonics, Inc.; Alnylam Pharmaceuticals; CS and CP, unpublished data; Recombinant DNA Advisory Committee).’
‘Additionally, significant inhibition of HBV antigen expression and viral replication is also observed, and is consistent with mRNA knockdown (Nucleonics Inc. and Alnylam Pharmaceuticals; CS and CP, unpublished data).’
'When administered intravenously, the nanoparticle been shown in preclinical models to transfect hepatocytes and delivers the eiRNA plasmid to hepatocytes and to the cells of some other tissues, such as the skin (Nucleonics, Inc. and Alnylam Pharmaceuticals, unpublished data).’
Etc, etc, I think you get the idea. But before Benitec supporters get excited about this, I would caution that Alnylam’s situation 3 years ago was much different from what it is today. With a dwindling cash reserve and after Tuschl, Tekmira, and Bass, Alnylam’s appetite for confrontation and lawsuits should have cooled considerably.
This story is yet another example of how concern about appearances (virus = scary) and short-term profit thinking (questionable model systems and lack of scientific rigor) in RNAi Therapeutics caused precious capital to flow into undeserving projects and lawsuits.