Thursday, July 14, 2011

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.

24 comments:

Anonymous said...

will they have to pay a royalty to Benitec for use under their patent?

Dirk Haussecker said...

The technology would seem to fall under Graham. Whether they will actually pay or not is a different question, and may depend on how and when they monetize this candidate if they have not already.

Anonymous said...

Like most patent, Graham patent has loop holes one can circumvent rather than fight head on like Nucleonics. Hopefully, players in RNAi field have the decency to realize the applications for RNAi are vast and beneficial for human health in general; there are a lot of profit to be made for everyone. The strategy to corner a promising technology with patent and lawsuits is just not going to benefit anyone including the patent holders.

Anonymous said...

possibly unlike synthetically delivered RNAi, trying to circumvent licence obligations for endogenous derived RNAi, would be like explaining to your girlfriend's father you only got her half pregnant.

i wonder if the cry for decency would be extended to the drug developers when they take the drug commercial, or would goal posts suddenly be shifted with another breathtaking leap in logic?

benitec holders actually deserve some decency after putting up with unscrupulous, lengthy court and patent challenges, which certainly delayed the clinical programmes in cancer, hcv, hbv, hiv and pain we are now seeing.

Anonymous said...

Dirk,
I am an outsider of this dd-RNAi tech, Would you please explain this to me? My question is how the cell know which DNA is for mRNA transcription, which one is for siRNA transcription, One thing I can think of is the DNA length. And how to avoid the off-target effect?

Dirk Haussecker said...

Hi- DNA makes RNA makes protein. DNA serves as the template for the generation of RNA in a process called transcription. Depending on the structure and sequence of the RNA, the cell determines what to do with the RNA, e.g. as mRNA for protein production (signals are e.g. a 'cap structure', having an 'Open Reading Frame' and a poly-A tail) or as an RNAi trigger (hairpin double-strand structure) for the down-regulation of mRNAs. For ddRNAi triggers, the RNA structure is key.

Anonymous said...

What is the advantage of ddRNAi vs. siRNAi? Why don't we see more development with ddRNAi? Why Alnylam or Tekmira or Merck or Silence seems to focus on siRNAi only? Any comments? Any prediction of the future? more siRNAi or more ddRNAi?

Anonymous said...

Dirk,
Depending on the structure and sequence===========Do you have article discussing it?
The LNP/SNALP is only for dsRNA delivery, right? Any drawback of virus delivery ?

Anonymous said...

Hi Dirk,
Thank you for doing your best to provide the small community with a communication channel and provide the knowledgeable facts as the result of your research efforts. Some of your presented facts might not be received well as we can see from a few boisterous and rather nasty comments, but I believe there is a silent majority who enjoys and benefits from your web site. Please keep up the good work and continue to educate us with broad subjects relate to RNAi. I think you are a true RNAi advocate. Thanks.

Dirk Haussecker said...

ddRNAi versus synthetic siRNA…they are largely complementary approaches and it depends on the application. I like ddRNAi Rx especially for chronic disorders of CNS, ocular, some liver, and stem cell-related disease strategies. I believe that in the immediate future we’ll continue to see more investments by the commercial space into synthetic approaches, although ddRNAi should increasingly be on the radar of companies like Sanofi/Genzyme, Pfizer, GSK as gene therapy shows more and more success in the clinic and orphan diseases and targeted therapies areas still somewhat sheltered as healthcare budgets are cut.

Structure and sequence…I don’t have an article that discusses it in layman’s terms, but maybe this video on RNAi will help: http://www.pbs.org/wgbh/nova/body/rnai.html

Yes, SNALP-delivery has been optimized for the delivery of short synthetic RNAi triggers although its origins are in the delivery of large plasmid DNA.

Anonymous said...

Dirk, good review of the advances in this area. I'm not surprised about the lack of efficacy/marking in the City of Hope trial. The ALD patients you reference actually had about 0.5-0.7 vector copies/CD34 cell at transduction, and then decreased to 0.1-0.2 a few years post transplant. The reason for decline is that the CD34 cell population contains mostly short term progenitors, which are more transducible than long term progenitors, and much more so that HSCs (about 1% of CD34 cells). So the decay in marking you see is from the short term progenitor population dying out.
It's also unfortunate that they transplanted unmodified cells as well, as they will be in competition with the transduced pool. Without looking at the data, it seems to me that they set themselves up for therapeutic failure by banking too much on safety.
-Asail

Anonymous said...

So, with this kind of suprisingly inefficient transduction efficiency, what would Calimmune do diffferently from City of Hope? I believe lentiviral vector has rather efficient transduction efficiency in lab setting, why would it be so indefficient in the clinical setting? How can Calimmune program fit into the California regeneration medicine initiative?

Anonymous said...

Lentiviral transduction efficiency is very much a function of the quality of vector (number of virus particles per infectious), nature of the transfer vector (e.g. size), transduction protocol (cytokines, concentrations of cells and vector), etc. So just because you're using lentivirus, it doesn't mean you're guaranteed to have adequate levels of transduction. Players with the best knowhow in this space will see the best results.
-Asail

Dirk Haussecker said...

Correct me if I'm wrong, but the 2-year cell marking in the ALD trial was about 20-100 fold higher than in the CoH study. The CoH is quite experienced in HSC transplantation and lentiviral transduction, so I doubt that this and the co-infusion of unmodified stem cells account for the large difference.

The CoH study was well run and a lot of valuable information gained. I believe, however, that ddRNAi design has made sufficiently significant progress that the newer designs should be given a try.

Anonymous said...

OK now I read the paper. They don't look at toxicity from the transgene in vivo (e.g. engraftment in mice, serial transplant), although they see no effect in vitro. So while toxicity may be leading to negative selection against transduced cells, there's no data to back that up. Their transduction efficiency is quite bad. At 4 weeks in vitro, they're down to 1% marking. The ~20% marking earlier on, in my opinion, could be artificially high because of DNA carry over in the manufactured the purified vector. This is observed more so with some purification protocols than others; they don't list their purification details (did they use TFF, SEC?). Another reason why you might see poor marking long term is from incomplete myeloablation. The study they site does not use standard ablation therapy (e.g. busulfan 16 mg/kg), so I don't think they had complete ablation and have not sufficiently cleared the HSC niche. there are a lot of factors that go into determining successful marking, and it is by no means easy to get them right. You may be right that their transgene is detrimental to HSCs, but unfortunately there is not enough data in their paper to know for sure.
-Asail

Anonymous said...

CoH results are from their pilot safety study, therapeutic effect was not on their radar. Very intentionally they used very low modified stem cell count because that's what they advised the FDA in the IND. This is a gene therapy study after all - powerful stuff but makes FDA nervous.
Safety result achieved, CoH's second study planned for later this year to use much higher modified stem cell count to get a therapeutic handle.

Anonymous said...

Dirk,
say US government is cutting the health budget so big pharma can't put more money into RNAi development is an excuse. The financial world is typical example that if there is a business make one dollar become two, then every cent in the market would do that business.

It means there is some obstacle, limit, diffuculty of ddRNAi, which makes the big pharma reluctant to more resource into it. And, it looks the readers of this blog are interested in this topic. Would you please post more article about it?

Dirk Haussecker said...

ddRNAi is part of the blog so I will post more on it.

About investments by for-profits in ddRNAi Rx. There definitely is increased interest in gene therapies and ddRNAi should benefit from that. And guess why? It's not just the advance in the science, but for a large part driven by a reimbursement climate that more and more favors medicines that address the root causes of diseases of high unmet medical need. Still, irrational social biases and scaremongering by incumbents continue to impede the even faster adoption.

Anonymous said...

Dirk,
However, I don't see the what is the diffuculties. There should be one, I think the instrest is still there. Otherwise, Nobel prize would not be given to Menlo and Fire. Would you please tell readers the obstacle met???

Anonymous said...

No matter how hopeful we may think RNAi is in shifting therapeutic paradigm; the investment world sees no proof of concept, plenty of law suits, no means to deliver RNAi agents and big pharma's pull out. As first-in-class therapeutic agents, FDA's learning curve is typically slow. We need at least one good and solid clinical result to reinvigorate the field. Hopefully, those "front-runners" do not screw up.

Anonymous said...

My opinion is the market needs something imminent,say,within one month or so, However, the SNALP/LNP or dd-RNAi needs maybe at least one year period of time. For sure, it is hard to find money. Even in big pharma, all money got interest cost, it looks like SNALP/LNP may have faster developing speed, although dd-RNAi got brighter future, no money last long without return.

I am not a scientist. I don't know why dd-RNAi needs such a long time to develope. However, it looks like Dirk doesn't know either.

Anonymous said...

If you've done a bit of research into Benitec and ddRNAi you'll be aware within less than a year of receiving their seminal ddRNAi patent (Graham), in 2004 Benitec were taken back through the USPTO following a third party observation by Nucleonics. The re-examination soon rejected the Nucleonics observation but in the process the examiner believed they had found other grounds for rejection, that being anti-sense prior art. The USPTO being so slow, it took until late last year for the case to get to the board of appeals. The appeal judges summarily dismissed the exmainer's arguements and reinstated all of benitec's claims.

This 6 years of IP uncertainty had a very negative impact on ddRNAi investment. That's one hurdle that slowed benitec and ddRNAi down.

(Nucleonics went bust and its founders and management began legal action against each other for dodgy behaviour. Interestingly Alnylam bought up Nucleonics peripheral ddRNAi IP in the fire sale)

Another hurdle is ddRNAi is gene therapy - it works via the nucleus. Gene therapy had a couple of spectacular failures earlier on and is only now starting to gain a more credible reputation.

That said, ddRNAi is now beginning to enter the clinic. Dirk's covered many of these programmes in his earlier blog entries. Which you should probably check first.

eg: Pfizer have just finished the lab work in the ddRNAi programme for HCV and next step is phase I trials in the clinic. They have the exclusive non asian commercialisation rights to Benitec / CSIRO ddRNAi IP for this target via Tacere.

Anonymous said...

The patent delay actually did not stop the advance of science. I think the major part is still the delay of science.

At Wall Street, the promising future can always find money easily. It looks the future of RNAi is longer than what market needs. It makes Sirna/Silence stop. But, I wonder what is the diffuculty ?

Anonymous said...

The pfizer ddrnai HCV deal is worth $145M....

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