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.

A new player in RNAi Therapeutics: Dicerna Pharmaceuticals

IN VIVO Blog (http://invivoblog.blogspot.com/2007/10/dicerna-crashes-rnai-party.html) reported today that a new RNAi Therapeutics company, Dicerna Pharmaceuticals, is about to debut. According to the same source, a $13M Series A financing round is expected to be announced in November. This company is aptly named after an enzyme in the RNAi pathway, as it is founded on the slightly unorthodox way to induce RNAi by providing synthetic Dicer-substrate siRNAs (D-siRNAs) of 26-30bp in length to effect gene silencing.

Dicer is this cool enzyme that digests ('dicerna' in Malay means: 'digested') long double-stranded RNAs into the shorter 21-23bp siRNAs with 3’ overhangs, the structure discovered by Tuschl and colleagues to efficiently induce gene silencing by RNAi in mammalian cells. Once delivered inside the cells, D-siRNAs are then processed by Dicer into 21-23bp effector siRNAs which then are incorporated into the RiSC complex to mediate gene silencing. Tuschl-like 21-23bp siRNAs are currently by far the most widely used method of inducing RNAi in human cells and fairly well understood.

In an elegant Nature Biotech paper in 2005 (Kim et al.: “Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy”), Drs. Kim and Rossi from the City of Hope, in collaboration with Mark Behlke from the nucleic acids synthesis company IDT, found that D-siRNAs can effect remarkably potent RNAi in human cell culture, often more potent than siRNAs of the same sequence. Importantly, in this and follow-up work they worked out some of the basic rules that would make D-siRNAs more practical inducers of RNAi such as better predicting strand incorporation and blocking one end of the dsRNA with non-RNA residues and modifications to force directional Dicer processing.

This Nature Biotech paper was accompanied by a similar paper from the Hannon group in Cold Spring Harbor which found that DNA-directed small hairpin RNAs (shRNAs) with double-stranded RNA stems longer than minimal 19-21base pairs similarly make them often more potent inducers of RNAi. Like Rossi and colleagues, it was speculated that this is due to biochemical coupling of Dicer processing to the RiSC effector complex. In addition to certain advantages in terms of potency, which I feel need further validation on a larger scale, D-siRNAs may in some instances facilitate RNAi delivery where covalent linkage of parts of the RNAi delivery system with D-siRNA is helpful as the active siRNA would be freed from the carrier by Dicer cleavage, although again it remains to be shown that the covalent attachment of e.g. peptides by itself is not inhibitory to Dicer processing.

[Erratum: The 2005 Hannon paper described the use of synthetic, not DNA-directed hairpins, with extended duplex length.]

In addition to these potential biological advantages, certainly a big part of the motivation that went into founding the company from an investors’ perspective is that Dicerna should be sufficiently distinct from the Tuschl siRNAs, a space clearly dominated both in terms of IP and know-how by Alnylam Pharmaceuticals. One can therefore expect that the new chairman and co-founder Douglas Fambrough from Oxford Bioscience Partners will do his best to make Dicerna his second Sirna Therapeutics, which he and his partners sold to Merck last year for a whopping return on their investment.

However, like with Sirna Therapeutics his claims of having freedom-to-operate will likely be clouded by uncertainty as there are a number of areas where Alnylam’s pre-dating IP will significantly overlap with Dicerna’s claims. This is not helped by comments, also cited in the IN VIVO Blog, of new CEO James Jenson stating that Tuschl’s landmark work had been conducted in flies, when Tuschl II –which by the way has issued and is exclusively licensed to Alnylam- is all about RNAi in mammalian cells, all this after laying the groundwork in work described in Tuschl I (also claimed by Sirna and CytRx, but has not issued) through amazing biochemical work in flies: Tuschl the prolific!

Importantly, Tuschl’s work as described in Tuschl II essentially discovered that RNAi operates in mammals and defined the basic rules of synthetic siRNAs. This, in my mind, should go a long way in the patent courts. In its worst case, D-siRNAs could therefore be regarded as simple pro-drugs of siRNAs. This also includes the 3’end overhangs which are thought to be beneficial for D-siRNAs since they are an important recognition element for Dicer.

Kreutzer-Limmer is another important cornerstone of Alnylam’s IP strategy, indeed important enough for them to buy the company (Ribopharma AG) that owned it very early on. Kreutzer-Limmer pertains to dsRNA-mediated gene silencing in mammalian cells, including predicted Dicer substrates, and although less well known in the scientific community due to lack of scientific publication, it is actually thought to have been the first demonstration of such gene silencing. Scientifically, my heart is with Tuschl’s detailed work, but Alnylam played it safe by just removing the uncertainty.

In addition, I would not be surprised if there wasn’t a note-book entry or publication that made use of long siRNAs either by design or accident. This would not be unlike early in the shRNA arena where scientists have made use of shRNAs with minimal and relatively long dsRNA stems alike.

Practically, the relatively small field of D-siRNAs will have to achieve what thousands of researchers around the world have done for siRNAs, namely coming up with siRNA design rules that consider all of potency, off-targeting potential, and the induction cytokines, as Rossi’s work has shown that these rules may differ from that of siRNAs. For these and other reason, I expect the complexity of developing D-siRNA therapeutics to be probably increased.

Nevertheless, I am curious to see more data come out that carefully characterize and compare the potencies of siRNAs and D-siRNAs. Comparative gene tiling studies would be an obvious experiment. This reminds me of the finding of hyperfunctional siRNAs, i.e. the odd siRNA that will be active in the low to mid picomolar range, and I could imagine a situation where efforts to find such siRNAs prove difficult for certain genes, while a D-siRNA is hyperfunctional, and vice versa.

I certainly look forward to Dicerna as a new member of the RNAi Therapeutics community. The science is certainly sound and innovative, and should be tested for use as a human therapy, which we all know would not happen without patent protection.

PS: This new development makes me wonder where that leaves Nastech Pharmaceuticals which has built so much of their RNAi program on Dicer substrates and is about to spin out mdRNA as their pure play RNAi Therapeutics subsidiary. It is clear that COH granted them 5 exclusive targets, but I am less sure about the other rights to Dicer substrates they had obtained.

Benitec and City of Hope Start RNA-targeted HIV Phase I Clinical Trial

After some delay, Benitec and their collaborators from the City of Hope finally announced that they obtained regulatory approval to start phase I clinical trials for an RNA-based HIV gene therapy antiviral. The delay was caused by Benitec’s uncertain corporate future and difficulties generating sufficient amounts of clinical-grade lentiviral vectors, but scientifically the risk-(potential) benefit profile of these studies are promising.

This is the 8th RNAi clinical program and the second involving DNA-directed shRNAs. The gene therapy agent is a lentiviral vector expressing 3 different RNA molecules, each designed to interfere with a different aspect of HIV replication, each through a distinct mechanism of action. The plan is to immunise CD34+ hematopoietic progenitor cells and thereby all their progenies, including T-cells, with such vectors ex vivo and then re-administer the cells to the patients.

The RNAi portion is a U6-driven hairpin RNA targeting the tat/rev mRNA by RNAi. A nucleolar-localised TAR decoy RNA, also under the direction of a U6 promoter, is designed to mimic the HIV TAR element, thereby diverting TAR-binding factors by mimicry. Finally, a ribozyme against the CCR5 mRNA, a co-receptor for HIV infection, complements the 3-pronged approach. This approach is inspired by the current HAART HIV treatment paradigm where a cocktail of antiretroviral drugs has proven to be highly effective in suppressing HIV replication with only slow development of drug resistance.

In fact, the RNA-based vector which has been shown in tissue-culture experiments to be considerably active in inhibiting HIV replication may be synergistic with present therapies due to their unique mechanism of action, although non RNA-based anti-CCR5 treatments are currently developed by other companies as well. While Benitec used to pursue a triple RNAi approach for the treatment of HCV (a program now owned by Tacere), the present strategy may be advantageous since it is known that co-transcribed shRNAs or co-transfected siRNAs may compete with each other for cellular RNAi factors.

Ultimately, I expect that long-term expression of ideally all 3 RNAs will be important to confer a survival advantage onto the lentivirally transduced CD34+-derived cells. However, even if expression should be silenced eventually, the treatment is likely to give AIDS patients some reprieve. The study population will be 5 AIDS-related leukaemia patients from which CD34+ will be enriched from blood by apheresis, genetically modified, and then returned to the donor patient.

Given that this is a gene therapy with a vector that hasn’t been used in the clinic before, it is expected that this therapy will be used in AIDS/Lymphoma patients who are no more responsive to conventional treatments. Nevertheless, expect to hear results from this promising phase I trial within a year.

RNAi by another Means: Dicer-substrate RNAs

The most widely known and used triggers of RNAi are small interfering RNAs (siRNAs) and DNA-directed hairpin structures. Whereas siRNAs are channelled into the downstream steps of the RNAi pathway, the RiSC complex, hairpin RNAs in humans first have to undergo processing by the endogenous microRNA pathway to yield the active small silencing RNAs. There is, however, a third way to induce RNAi, and these are so called Dicer-substrate RNAs. Their potential utility for gene silencing was recognised following the finding by the Cleary/Hannon [Siolas et al. (2005) Nat. Biotechnol. 23:181] and Rossi [Kim et al. (2005) Nat. Biotechnol. 23: 222] groups that longer RNA duplexes of around 25-27 bp that undergo initial processing by the RNAi enzyme Dicer to yield the active small RNA are in some cases more efficient in gene than the equivalent siRNA. This is thought to be the consequence of Dicer actually forming part of the RiSC loading complex, thereby ensuring efficient hand-off of the small RNA product into the RiSC silencing complex.

There are a number of reasons why Dicer-substrate RNAs have not become a mainstream tool for inducing RNAi yet. Among these were the difficulty of manufacturing Dicer-substrate RNAs that would yield predictable small RNA effectors, non-specific perturbations of gene expression due to cytokine induction by the dsRNA, and the lack of reliable Dicer-substrate RNA design rules. Finally, RNAi inducers upstream of siRNAs may compete with more elements of the microRNA pathway than necessary and the longer length of RNAs will add to the cost and complexity of synthesis.

Some of these challenges, however, are being met mostly as a result of a collaboration between the Rossi lab of the City of Hope, California, and the nucleic acid synthesis company IDT which licensed Dicer-substrate RNAs for use in non-therapeutic applications. Creating dsRNA with one blunt end that contains DNA nucleotides on one strand and 2 nucleotide 3’ overhangs on the other end introduced directionality into Dicer processing. Moreover, it appears that the same modifications that can be introduced into standard siRNAs to avoid cytokine induction also work well for Dicer-substrate RNAs. One weakness that remains, however, is the lack of efficient Dicer-substrate RNA design rules. However, in collaboration with Bio-Rad, IDT is screening and developing sets of validated Dicer-substrate RNAs that have greater than 85% knockdown efficiencies.

It remains to be seen how well accepted Dicer-substrate RNAs will eventually become. With IDT, possibly the world’s largest synthetic nucleic acids supplier for research purposes, behind the technology, Dicer-substrate RNAs should be able to reach most researchers in the field. It will therefore be their hands-on experience, publications showing the benefits of Dicer-substrate RNAs and word-of-mouth that will determine the success of Dicer-substrate RNAs. As siRNAs have shown, a biotechnology that works predictably does not need much advertisement. Some interest meanwhile is demonstrated by the fact that Novartis is apparently testing a small library of Dicer-substrate RNAs for target validation purposes, and Nastech Pharmaceutical Company has obtained an exclusive license from the City of Hope for developing Dicer-substrate RNAs as therapeutics against a handful of gene targets.

With regards to IP issues, I would expect that their therapeutic use would require some kind of licensing agreement from the beneficiaries of the Tuschl I and II patents partly because of the 2nt 3’ overhang structures and the fact that Dicer-substrate RNAs are the immediate precursors of siRNAs. A precedent for this kind of licensing agreement has been set before by Benitec, which in 2005 has taken a license from Alnylam for the “targeted gene silencing mediated by short interfering RNAs (siRNAs) generated from DNA constructs introduced into cells”.