Viruses, Beware! This time, RNAi Therapeutics Mean Business

Viral infections have long been thought of as an attractive therapeutic area for RNAi Therapeutics.  Unfortunately, with the exception of Tekmira’s Ebola biodefense effort funded by the US Department of Defense, this area has had trouble taking off: Nucleonic’s ddRNAi-based HBV program should never have gone into the clinic (and as expected was soon terminated thereafter), and there is considerable concern that the main mechanism of action of Alnylam’s ALN-RSV01 for respiratory viral infection is due to innate immune stimulation of the unmodified RNAi trigger, not RNAi-mediated gene knockdown.

As RNAi Therapeutics as a whole has turned the corner in 2012, so has antiviral RNAi Therapeutics. 

This assessment is based on two quality programs that have either made it into the clinic recently, Calimmune’s ddRNAi candidate for HIV (LVsh5/C46), or is close to it (Arrowhead’s DPC-delivered anti-HBV candidate ARC520 for which an IND is planned in Q2 2013).  In addition, there is expectation that Tekmira’s Ebola program will be able to take advantage of the significant improvements in SNALP delivery technology, thereby considerably increasing the odds for an FDA approval under the Animal Rule (note the recentapproval of a second drug under this rule).

Suppressing Immune Suppression

Antiviral RNAi Therapeutics have to overcome the important theoretical limitation that even a potent, e.g. 99% knockdown of a viral transcript or particles may not be sufficient as in theory a single infected cell may fuel viral rebound.  It turns out that rather than blindly aiming at knockdown potency, RNAi Therapeutics are likely to be more successful when targeting an important mechanism employed by virtually all viruses: avoiding detection or removal by the immune system.

In the case of HBV, a disease affecting North of 200 million patients worldwide, the virus produces large amounts of the Hepatitis B Surface antigen (HBsAg).  This is thought to suppress, by acting as a decoy, the development of a productive anti-HBsAg immune reponse.   It is thus widely believed in the industry that reducing HBsAg is required to finally generate a drug that can achieve a functional cure, essentially paralleling the recent developments in HCV.  Interferon-based treatment regimens may actually partially work via this mechanism, but cure rates are rather low and come with considerable side effects in the form of severe flu-like symptoms.  Moreover, protein-targeting anti-HBV agents such as small molecule-based polymerase inhibitors do not seem to reduce HBsAg.  This leaves RNAi Therapeutics as the most promising mechanism of action.

A recent article in PLOS Pathogen suggests that the Ebola virus similarly churns out decoy viral proteins so as to subvert the immune system into making antibody duds that do not effectively remove the real viral particles.  It is therefore intriguing that an Ebola drug candidate by Tekmira should not only aim at providing the immune system with more time, but also that it would facilitate it mount a more effective antibody response.

No Escape

Another attraction of the RNAi Therapeutics approach for viral diseases is the fact that such agents may be more successful in prohibiting the virus to mutate around the drug and thereby escape its actions (viral escape).  Consequently, all antiviral RNAi trigger selection strategies focus on sites that are conserved in the various genotypes and quasispecies.  Even if the virus is successful at mutating around conserved sites, it is then relatively simple to include a second (such as in Tekmira’s Ebola program) or third RNAi trigger targeting a conserved site such that the virus would have to mutate around two sites at the same time- a highly unlikely event.

In addition to these general antiviral mechanisms, RNAi Therapeutics may also work through more virus-specific mechanisms.  Calimmune’s ddRNAi-based HIV candidate LVsh5/C46 for example down-regulates the cellular receptor for viral entry, CCR5, such that HIV particles cannot enter cells and integrate into their genomes in the first place.  As an ddRNAi gene therapy approach, LVsh5/C46 further takes advantage of the fact that you can express a therapeutic protein along with the RNAi trigger, thus uniquely combining mechanisms of actions in a single drug.    

Smooth Sailing Ahead

Of course, it is impossible to tell whether an RNAi Therapeutic will actually overcome a virus in each case and receive regulatory approval.  Nevertheless, I believe that the above candidates for Ebola, HBV, and HIV stand a real chance. 

The Ebola program by Tekmira is arguably the most advanced, and it is difficult for me to see how based on the non-human primate data and the lower dosages required for SNALP delivery, which should widen the therapeutic window, approval can be denied under the Animal Rule.

For the HBV and HIV candidates that are being developed along more conventional regulatory pathways, I  am similarly optimistic that they will generate some excitement in the near-term.  This is because viral load is a powerful biomarker, often also an approvable endpoint, and even early clinical studies should be able to generate such outcome data (if Arrowhead could help it, they should go straight into patients with ARC520). 

After orphan diseases involving the liver and oncology, antiviral applications are therefore poised to become the third major support of the RNAi Therapeutics platform.

Solid Calimmune DNA-directed RNAi Therapeutics Candidate for HIV Nearing Clinical Development

With the backing of a $20M grant from the California Institute of Regenerative Medicines (CIRM), Calimmune has made progress in advancing a DNA-directed RNAi (ddRNAi) Therapeutics candidate for the treatment of HIV/AIDS towards clinical development in early 2012 (here a recent blurb in the Financial Times). Similar to an HIV candidate developed by City of Hope (CoH) and Benitec before it, the new treatment involves the modification of a patient’s own blood stem cells (hematopoietic stem cells, HSC) with a gene therapy comprising of an expressed small hairpin RNAi trigger. Although Calimmune is not prepared yet to share the details of this program, based on my review of the research conducted by groups associated with Calimmune, the likely candidate has the potential to become one of the most exciting ddRNAi Therapeutics product candidates to enter the clinic yet.

HIV therapy today and motivation for gene-based stem cell therapies

The treatment of HIV has made tremendous progress. Once a certain death sentence, for those with access it has instead largely become a chronic infection that can be kept in check with cocktails of small molecules targeting a variety of stages in the viral life-cycle (highly active antiretroviral therapies or hAART). Nevertheless, the need for taking daily pills for life comes at the cost of side effects, generally reduced quality of life, and the emergence of viral resistances. There is no cure yet for HIVAIDS.

Actually, there might be one example of a cure for HIV. In 2006, an AIDS leukemia patient, aka the Berlin patient, underwent a bone marrow transplant as a treatment for his leukemia. The doctors selected a bone marrow donor whose cells carried defects in the CCR5 gene on both chromosomes. After the transplantation, the patient was not immediately put back on antiretroviral therapy to allow for recovery of his new hematopoietic system. Surprisingly, despite the absence of drug treatment, the virus has not recurred to this day leading more and more experts to talk of the first functional cure of HIV/AIDS.

In hindsight, this result did not come totally as a surprise. CCR5 had been known to be an important entry receptor for the common CCR5-tropic HIV isolates. Epidemiologic evidence gathered in the mid 90s indicated that people with certain CCR5 deletions on both chromosomes were protected from HIV infection, and those with a CCR5 defect on only one chromosome had, on average, delayed disease progression and improved life expectancies. In fact, this research led to the development and recent approval of a class of drugs blocking the CCR5 protein (e.g. Maraviroc by Pfizer).

There remains, however, great interest in developing gene-based stem cell medicines against CCR5 (and other HIV viral and host targets) in the hope of generating HIV medicines with less side effects, reduced chance of viral resistance (one way of HIV resistance to drugs targeting the CCR5 protein is to bind to CCR5 in the presence of drug), and maybe even a cure. The Berlin patient indicates that CCR5 may be an ideal target for such gene-based stem cell therapies.

Two possible mechanisms by which such a strategy may succeed are based on eradication of HIV-permissive cells as they are killed off by the virus while the CCR5-impaired cells persist, or by improving the immune function of CCR5-impaired cells thereby allowing them to fight HIV infection in other places.

City of Hope/Benitec and the first DNA-directed RNAi Therapeutic for HIV

Calimmune’s ddRNAi candidate is not the first one for HIV. The City of Hope, with the financial backing of Benitec, already entered one into clinical development (rHIV-shl-TAR-CCR5RZ), results from which were reported last year in Science Translational Medicine. Recognizing the advantages, if not need, for targeting multiple stages of the HIV life-cycle at once, this candidate was not a pure ddRNAi therapeutics, but a triple RNA therapeutic that in addition to the shRNA RNAi trigger which targeted the viral tat/rev mRNA involved an expressed TAR RNA decoy and an expressed (RNA) ribozyme targeting CCR5. Notably, all three expression cassettes were driven by U6 promoters.

The expression cassettes were placed in a shared lentiviral vector and thus introduced ex vivo, i.e. outside the body, into hematopoietic stem cells isolated from the enrolled AIDS lymphoma patients. Because hematopoietic stem cell transplantation with full bone marrow ablation is associated with risks, but is standard second-line therapy for AIDS-related lymphoma, this patient population was chosen so that the trial participants would simultaneously receive a treatment benefit for their lymphoma while participating in this experimental trial. As an added measure of precaution, the majority of hematopoietic stem cells were left untreated and given together with the modified stem cells to ensure that the immune system would be reconstituted even if something went wrong with the gene therapy.

Four patients were treated per protocol in the phase I trial. Unfortunately, while there was no obvious significant adverse event as a result of the gene therapy, the molecular analyses indicated that rHIV-shl-TAR-CCR5RZ may not be the most promising RNA therapeutics candidate for HIV. Specifically, while the initial transduction efficiency was in line with what would have been expected for lentiviral delivery (~20%, see X-linked adrenoleukodystrophy trial here), the transduced cell population declined rapidly and the ones that persisted were just about detectable- too few to be therapeutically promising.

If this candidate were to be further developed, an important goal would be to increase the fraction of stem cells that are modified. This could either be by improving the transduction efficiency, by only providing stem cells that were treated with lentivirus instead of providing the untreated stem cells as a backup, or by using a protocol that chemically selects for the modified stem cells after their re-infusion. Still, I am skeptical that this would solve the problem as in light of other lentiviral and retroviral clinical experiences the observed decline in transduced cells seemed to be specific to rHIV-shl-TAR-CCR5RZ. It is therefore possible that some inherent toxicity of the expression cassette itself, possibly due to the use of U6 promoters, accounted for the poor long-term persistence of modified stem cells.

The Calimmune approach: A non-toxic, H1-driven shRNA targeting CCR5

The reason why I feel that Calimmune’s approach may have better prospects is that it has fully accounted for the U6-related shRNA toxicities and selected an H1 promoter-based RNAi expression cassette that was shown to be both safe/stable and, equally important, highly efficient in CCR5 knockdown in human and rhesus HSC-derived cells. Also, I like the fact that it is an RNAi trigger, and not a ribozyme, that is targeting CCR5, as I believe this to be the more efficient knockdown modality.

While Calimmune has yet to fully disclose their eventual clinical candidate, the one reservation that I have about the putative candidate at this time is that they may have failed to take advantage of the combinatorial potential of RNAi Therapeutics. With combinatorial potential I do not necessarily mean here combining ddRNAi with other RNA (like CoH/Benitec) or protein expression modalities- in fact, it may be scientifically 'cleaner' to use just RNAi for now- but targeting at least two HIV-related genes instead of one to minimize the emergence of viral resistance.

The panels on the left depict what in my mind have been the most impressive dataset from the development program. It shows the results from a rhesus monkey model in which the ddRNAi trigger was introduced into blood stem cells from two monkeys (RQ3570 and RQ5427 for those with good eyes) which (panel A) led to solid, long-term (!) 6-20% cell marking in the various cell lineages of the blood. Moreover, when the cells were sorted into those that were transduced (black bars, panel B) versus those that were not (grey bars, panel B) and the CCR5 levels measured in the respective cell populations, the CCR5 was found to be down-regulated by 80-90% in the transduced cells. And since your experiment is only as good as your negative controls, data from a control animal that received a lentivirus without the RNAi trigger (2RC003) show no differences in CCR5 levels between the two cell populations.

While I have yet to see the obligatory HIV in vivo challenge studies with this putative candidate, based on CCR5 genetics, a candidate with such transduction levels and knockdown potencies should stand a good chance at improving CD4+ T-cell counts for enhanced immune system vigor and delaying or maybe even eradicating HIV over time.

It is debatable to what degree a full CCR5 knockout compared to a highly potent CCR5 knockdown would bring additional benefits. Sangamo Biosciences for example has made tremendous progress in increasing the efficiency of gene disruption using their Zinc Finger Nuclease technology. Not surprisingly, this company also has a CCR5 hematopoietic stem cell candidate in the early pipeline. In a 2010 Nature Biotechnology paper, Sangamo reported an estimated frequency of 5-7% homozygous CCR5 gene disruption in human hematopoietic stem cells, and another 10% heterozygous gene disruptions.

Simplistically, taking upper estimates, ddRNAi may provide for 90% CCR5 knockdown in 20% of cells whereas ZFN technology may delete CCR5 altogether in 7% of cells and knockdown CCR5 by half in another 10%. Because these numbers are close and a clean knockout in some cells may make up for the slightly decreased overall knockdown levels, I would be even more excited to see Calimmune's current lead candidate paired with at least another shRNAi trigger, thereby exploiting said combinatorial potential of ddRNAi Therapeutics which ZFNs cannot provide as easily.

Benitec license?

Benitec, of course, will follow Calimmune’s developments with great interest as the company has rights to critical ddRNAi trigger patents. Curiously, both companies are based in Australia, but have significant roots also in the US South-West, so it should be possible to come to an amicable agreement.

License or not, it will be good for the entire field of RNAi Therapeutics, and ddRNAi Therapeutics in particular, for this trial to get underway in 2012 as it should attract significant general interest to a what looks like a solid RNAi Therapeutics candidate.

Acknowledgement: The idea for this blog came from a reader that alerted me to this interesting RNAi Therapeutics candidate that had flown below my radar, and maybe also to placate another reader that complained that the Tekmira-Alnylam feud was taking up too much space and there were other interesting things happening, especially in ddRNAi Therapeutics. So if you know of exciting RNAi Therapeutics developments that you believe I may be missing, please let me know by email (first name dot last name at gmail dot com). In most cases, I won’t be able to write about it immediately, but it won’t be forgotten either.

Update: On March 5, 2012, Calimmune acquired a global, non-exclusive license from Benitec to use ddRNAi in HIV/AIDS.