The time for genome editing is now

Back from recent CRISPR conferences in Ireland and Denmark, I wanted to share my current thinking about the present and foreseeable future of clinical genome editing.

CRISPR is a clinically mature platform

First of all, genome editing, and in particular CRISPR Therapeutics have arrived big time.  Ignore for a moment the 90%+ declines in the share prices of Intellia, Verve, and Metagenomi, that all, with the exception of perhaps Beam Therapeutics, suggest that the industry will not survive.  Focus instead on the transformational results last week by Verve Therapeutics which will shape the management of chronic metabolic and cardiovascular disease for decades to come, Beam Therapeutics last month announcing that it  for the first time fixed a genetic disease (AATD) by reversing a point mutation, or the three ongoing phase 3 programs by Intellia Therapeutics which promise patients live unintruded by their disease.

So you say that this is only half the truth and many of them will develop cancer?  With hundreds of subjects having been given CRISPR therapeutics, we still have yet to hear about such cases, including for the hematopoietic stem cell applications, probably the most stringent test in that regard given the history of retroviral gene therapy.  This is supported by ample detailed genomic analyses demonstrating that many CRISPR modalities, in particular prime editing, often do not have any detectable off-targeting (!) and therefore are safer than going out in the sun or eating grilled meat.  And don’t get me started with analysts and investors pretending that small molecule drugs, 'dirty drugs' by design, are much safer in that regard when many of them like methotrexate and hydroxyurea are known to cause cancer which barely gets a mention or is routinely accepted as part of the risk-benefit of a drug.  Similarly, the currently more widely embraced antisense and RNAi technologies exhibit considerably more off-targeting, which in theory could result in cancer development with chronic use, than the average CRISPR therapeutic candidate today. 

Prime editing is the ultimate CRISPR manifestation

Prime editing is the foreseeable endgame for genome editing involving edits up to 50 nucleotides.  This is because in addition to being able to knock out genes like (Cas9) nuclease or base editing, it allows the developer to change nucleotides at will and with incredible specificity, and for gain-of-function.  It has therefore surprised me to see that prime editing is still such a niche part in these conferences and barely used in industry and even academia where IP is less of an issue.  This can likely be explained with it requiring a good deal of mental gymnastics when devising a prime editing strategy and subsequent tinkering to optimize efficacy.  Prime editing will likely also be part of strategies for achieving even larger edits, including whole gene replacements that are often desirable.

In some ways this is reminiscent of zinc fingers which make for poor screening and quick molecular biology tools and were quickly forgotten when CRISPR came along with the promise of simple sgRNA design.  However, a panel at the Copenhagen CRISPR meeting by genome editing veterans made it clear that when optimized, zinc fingers can be every bit as powerful as CRISPR and currently have a leg up where delivery is going to be viral for some time to come.  

This is particularly true for the CNS, the fourth major tissue/organ target for genome editing applications after the liver, hematopoietic stem cell, and immune cells.  Not only is it impossible to fit in prime and base editors in single AAV vectors, expressing a CRISPR effector nuclease of bacterial origin for years may be tricky (but not unsolvable) in terms of immunogenicity.  There is also some unease about having nucleases loiter around in cells for years after their job has been done.  Self-targeting/inactivation strategies may here be called for.  Zinc fingers by being small and mimicking human proteins by contrast are ready for clinical use, a fact, as the deal flow by Sangamo Therapeutics illustrates, is increasingly being recognized by the wider industry.

Genome editing versus non-permanent modalities

Another popular argument against CRISPR Therapeutics questions why one should take a ‘risk’ with permanently altering the genome when you have oligonucleotide therapeutics options that need to be administered as infrequently as every quarter, half annually, or even annually.  

While there is some application overlap between genome editing and oligonucleotide therapeutics, especially when it comes to knocking down genes, a closer look shows that the overlap is limited.  Where there is clear genetic evidence that a gene can be disabled for life without safety risks, then genome editing is the endgame, or at least capture a good portion of the market, and transient modalities may serve to de-risk the targets first.  

When it comes to bringing pathways back into finely tuned balance and/or the disease is not chronic and likely requires only temporary treatment (e.g. some cases of hypertension), transient options are preferable.  Still in other cases such as prion or Huntington’s disease where it is not clear whether it is the overall level or distribution of gene knockdown that is more important for efficacy, the preferred modality has yet to emerge from clinical studies. 

Part of the Wall Street fashion show

Wall Street is a fashion show when it comes to biotech platforms.  A little less than 10 years ago, it predicted that CRISPR would kill off the RNAi Therapeutics industry. Today, Alnylam sports a $30B market cap, about 4x that of the entire CRISPR industry combined (much of it in cash).  Of course, investing in technologies ahead of their time is fatal. The electric vehicle industry is just one example.  As I have laid out above, genome editing, however, is now and here to stay.  Having the potential to save the healthcare system money (e.g. $2M for a one-and-done HAE/ATTR treatment as opposed to $500k annually) or to lead to drastically improved outcomes (as can be predicted from chronic LDLc lowering) got to be embraced by patients, their caregivers, docs, regulators, and insurers alike.  

It's been a rough couple of years investing in development-stage, cutting-edge biotechs and current politics does not seem helpful, but if this is when it is darkest, then generational wealth awaits the survivors with the guts to hold on during a volatile comeback.  One of my two conviction bets that I look up to in this regard is therefore CRISPR therapeutic biotech Verve Therapeutics.  Uniqure, which works in the similarly unloved gene therapy arena and has a promising DNA-directed RNAi candidate for Huntington’s disease, is the other in case you were wondering.  

Voyager Therapeutics Sets Out to Fulfill Promise of AAV-directed RNAi Therapeutics (and More)

Finally!  Long overdue (because so obvious and compelling), with the founding of Voyager Therapeutics, the foundations have now been laid for a strong AAV (adeno-associated virus) platform gene therapy company, including DNA-directed RNAi Therapeutics.   Taking advantage of new AAV shuffling approaches to identify novel serotypes (Gao and Kay labs) to more efficiently and specifically target cell types of interest and the world-class expertise in RNAi trigger design of  two of its scientific co-founders (Kay and Zamore labs), the new company will make use of the best available science to realize the potential of AAV and ddRNAi technology for the many diseases of high unmet medical need in the CNS (e.g. Huntington's disease).

With $45M in start-up funding from Third Rock Ventures (the VC that has started the comparable lentiviral-based gene therapy company which recently went public, bluebird bio), they will deepen that expertise through in-house research and thereby sail by Benitec which sadly has refused to face the fact that in order to be a platform company and stay relevant you need to have such efforts.  If not at a time like this when interest is high, then when??  The latest publication by Benitec and Pfizer (Denise et al. 2014) was ironically the best illustration of that omission by showing that the first-generation ddRNAi expression technology of Benitec generates a plethora of small RNAs which obviously is less than ideal.  By contrast, work in the Kay lab in particular (e.g. Gu et al. 2012) has shown how to generate much cleaner expression cassettes.

Voyager strutting its RNAi expertise and related therapeutic development plans is further confirmation of the dramatic decrease in the value of Benitec's once-prominent IP position in ddRNAi (Graham patent).  With an expiration of ~2018-2020 and Baulcombe the real gate-keeper (although the clock on that one is also rapidly running down), I don’t see why there should be a need to take a license except for maybe the likes of Calimmune which may have something close to commercialization by then should things proceed without a hitch.

The start of a company like Voyager is always very exciting and I trust that the scientific founders have the expertise and wisdom to guide it along the way.  It is also a good time to acknowledge the likes of Dirk Grimm (Heidelberg), Shuo Gu (NCI), and most recently Leszek Lisowski (Salk) which I had the privilege of working next to in the Kay lab when they were critically contributing to the technology underlying Voyager.  The pharmaceutical industry and indeed the investor community is wasting a lot of potential by not tapping into the skills of such driven scientists just because they may not labor in one of the biotech hotbeds.

Genable Finds EUR5M for ddRNAi-Gene Replacement Approach

5 million Euros these days is serious money in RNAi Therapeutics. Last week, Irish biotechnology company Genable Technologies announced that it succeeded in raising such funds for its pre-clinical DNA-directed RNAi Therapeutics-Gene Replacement combo for the treatment of rhodopsin-linked autosomal dominant retinitis pigmentosa (RHO-adRP), an inherited retinal degenerative condition leading to blindness.

To see it flow into DNA-directed RNAi Therapeutics at that makes it particularly noteworthy. It illustrates that ddRNAi is somewhat uncoupled from the current depression in the synthetic RNAi Therapeutics arena due to some technical differences which make it in many ways more similar to traditional gene therapy. Gene therapy, of course, is experiencing a revival, fueled by more and more clinical validation. Incidentally, it which follows a similar boom-bust cycle as RNAi Therapeutics is going through now.

One of the great benefits of ddRNAi Therapeutics is that it can be combined with other DNA-directed modalities, especially the co-expression of protein-encoding genes from the same DNA template. As such, it is particularly amenable to treat autosomal dominant-negative diseases in which the affected gene is important for cellular function thereby precluding approaches that eliminate both the mutated and the healthy copies of a gene. Accordingly, one could use a single DNA molecule from which to direct the suppression of endogenous versions of the affected gene (wildtype and mutant forms) and then replace gene function by providing a version of the gene that is immune to the RNAi.

In Genable’s case, AAV vector capacity limitations meant that a mix of two AAV vectors had to be co-administered by subretinal injection, one carrying the ‘immunized’ rhodopsin gene, the other the shRNA against the endogeneous rhodopsin copies. This apparently did not affect much treatment efficacy. Indeed, it was argued that the co-administration approach would actually increase dosing flexibility by being able to adjust the amount of each component independently. On the other hand, it is possible that the use of vectors with larger capacities, such as the lentiviral system that Oxford Biomedica and Sanofi-Aventis are busy exploring for ocular gene therapy, may have its advantages here. Importantly, it was shown that therapeutic efficacy in the retinitis pigmentosa animal models strictly depended on the presence of both gene replacement and gene knockdown components.

Although the concern with causing harm by knocking down the wild-type allele is often only a theoretical one, as is for example the case with Huntington’s Disease (HD), and depends on the degree of knockdown, other applications for which this approach holds promise are sickle cell anemia, alpha-1 antitrypsin, pachyonychia congenita (PC), amonst many others.

The PC project by TransDerm and collaborators is a good example in the RNAi Therapeutics space for the challenges faced by the alternative approach to treating such autosomal dominant diseases, namely by specifically directing the drug candidate towards the mutated gene/gene product so as to leave the wild-type copy untouched. The problem here is that because the potential mutations can be broadly distributed over a gene, restricting a therapeutic to targeting only one such genetic lesion (or associated gene polymorphisms) can dramatically reduce the number of patients eligible for such a targeted therapeutic. This also explains why the first clinical trial by TransDerm was a placebo-controlled, split-body dose-escalation trial in a single patient (!) as there may not be more than a handful of known patients in the world with the applicable mutation.

The 5 million Euros will be used to bring Genable’s lead candidate, GT038, into clinical development. RHO-adRP seems like a good ddRNAi application as even the correction of only a fraction, e.g. 1/3 to 1/2 of the affected photoreceptors, may be enough to support vision. It is good to finally see a number of ddRNAi Therapeutics candidates based on sound pre-clinical data move into the clinic.

(For another promising late preclinical candidate, see Calimmune's HIV approach)

The Potential for AAV-mediated RNAi Therapeutics

There is good reason to believe that synthetic siRNA-mediated RNAi Therapeutics should emerge as the most commonly used form of RNAi Therapeutics. Nevertheless, DNA-directed RNAi Therapeutics also has a number of potential uses where it should not only be competitive with, but even superior to synthetic RNAi. Unfortunately, the commercial development of DNA-directed RNAi Therapeutics has been somewhat hampered due to litigation and other management issues as well as funding problems that all things “gene therapies” face. In an effort to dispel some of the myths surrounding DNA-directed RNAi Therapeutics and since I’m somewhat familiar with particularly AAV-mediated DNA-directed RNAi, I would like to take the opportunity here to briefly highlight some of the potential applications for this particular technology.

DNA-directed RNAi can either by delivered by non-viral or viral means. For the most part, current systemic non-viral delivery technologies for DNA vectors that need to get into the nucleus for functional activity may not be adequate as a result of their inability to transfect sufficient cell numbers as well as support long-term expression. By contrast, viral vectors, particularly AAV and lentivirus, are capable of very efficiently and stably transducing many cell types. In fact, in vivo potencies are often greater than with most current synthetic RNAi methods with essentially knock-out phenotypes in the liver and eye observed for months and years using self-complementary AAV8 vectors in work reported by the laboratory I work in and collaborators to name just one example.

Before focusing more on AAV with which I am most familiar with (learning by osmosis), lentivirally delivered RNAi has much potential for disease of the CNS, largely for the same reasons as outlined for AAV below, and in combination with cell therapeutics. The latter would involve the ex vivo transduction of lentiviral RNAi constructs for example into stem cells similar to the ongoing phase I HIV-RNAi trial by the City of Hope and sponsored by Benitec, or also to enhance dendritic cell cancer vaccine strategies. Many of these applications take advantage the stable integration of lentiviral vectors into the host genome such that the vector and its expression/knock down will be maintained even in dividing tissues.

By contrast, due to its largely episomal nature, AAV gets rapidly during cell division thus limiting their applicability for cancer therapy or in other situations that involve cell division (regenerating liver, stem cell differentiation etc). Moreover, in certain settings humoral and T-cell mediated immune responses against AAV viral proteins present another challenge for achieving persistent gene silencing (the transduced cell may be recognized by the immune system and be eliminated) and where repeat-administration is desirable (due to neutralizing antibodies generated following the first administration).

For these reasons, AAV RNAi appears most promising for diseases of the eye and CNS as immuno-privileged sites. Although infusion pumps may address some of the challenges of allowing for long-term intracranial gene silencing by synthetic means, due to the ability to mediated sustained gene silencing for 6-12 months if not several years as suggested by canine AAV studies for hemophilia, the prospect of maybe having to subject a patient only once or very few times to an invasive operation makes AAV and lentivirus attractive alternatives for diseases such as Huntington’s Disease and other neurodegenerative disorders.

Not coincidentally, Targeted Genetics and the University of Iowa are currently pursuing an AAV RNAi program (pre-clinical stage) for Huntingon’s Disease that has shown promise. A critical factor for the success of this program should be the design of the shRNA expression cassette, and I personally would feel more comfortable with an H1 promoter-driven instead of a U6 promoter-driven construct that has been the front-runner so far. Another interesting application may be for the treatment of PML viral infection. Biogen Idec and Alnylam have been working on an siRNA-mediated approach, but due to serious nature of JC virus reactivation during PML, rapid onset of gene silencing by self-complementary AAV RNAi and the efficient vector delivery achieved for a number of neuronal cell types, AAV-mediated RNAi warrants consideration for this devastating disease.

Suitable non-CNS applications for AAV ddRNAi candidate may be instances where a single administration may already be therapeutic without the need for sustained gene silencing and repeat administration. HCV infection of the liver may be one such case as it is now possible to essentially transduce every liver cell, at least in mice, and effect long-term silencing after a single administration. AAV-medicated RNAi could therefore be an important component of combination therapies for patients that do not respond to current therapies and could also quite easily be tailored to the different HCV genotypes. Pfizer just recently acquired co-development rights for the pre-clinical stage AAV RNAi program for HCV from the Benitec spin-off Tacere.

AAV gene therapy is relatively new, but it is making rapid progress. Two independent phase I/II AAV gene therapy trial for Leber’s Congenital Amaurosis caused by RPE65 deficiency, a condition that leads to blindness later in life, demonstrated clear improvement in vision and treating children early on promises to even cure the disease. One of the studies was conducted by an academic group in London and was sponsored by Targeted Genetics, the other by a group from the University of Pennsylvania.

It is not clear whether an immune reaction that eliminated transduced liver cells in a hemophilia trial was specific for the AAV 2 serotype used, as most of us will have been exposed to this type of AAV during childhood and may therefore harbor some immune memory for it. A number of strategies have been proposed to minimize the risk of immune recognition in future trials, for example transient immune suppression or the use of alternative serotypes. The search for and development of alternative AAV serotypes is truly exploding and is rapidly yielding new AAV vectors with various tissue tropisms and immune properties.

The less AAV that needs to be administered the better also from an immune point of view. Very promising in that regard is the finding that the self-complementary AAVs which by-pass the rate-limiting second-strand synthesis step during the establishment of gene expression much more efficiently and functionally transduce target cells than conventional single-stranded AAV vectors. While this halves the vector capacity to less than 2kb, a size that is not very practical for expressing many protein-encoding genes, this does not matter at all in the context of small hairpin expression cassettes and appears to be just made for AAV RNAi. Actually, it was this property of self-complementary AAV vectors that was one of the main reasons for me to come to Stanford to conduct post-doctoral research. A patent for this possibly critically enabling technology has been issued to Targeted Genetics.

RNAi Therapeutics Portfolio Review: Increasing Position of Targeted Genetics

The technology is certainly there to be harnessed for therapy, but the development of AAV RNAi Therapeutics is not trivial and is a collaborative effort that requires careful gene target selection, safe and potent hairpin vectors, thoughtful clinical trial designs, and the manufacture of large amounts of high-quality AAV particles. Nevertheless, with the right team and some luck, it should possible to do.

It has both amazed and scared me to learn in a vivid report by RNAiNews that DNA-directed RNAi company Nucleonics whose lead program was a very long-shot (to put it mildly) RNAi program for HBV, was close to raising $25M in a series C round that would have included a venture capital arm from Johnson & Johnson. How that was even a remote possibility given the odds for that particular HBV RNAi program and the uncertain IP of that company is a mystery to me and makes the ~$13M market cap of Targeted Genetics’ look very cheap by comparison.

For this reason and given the promise of AAV-mediated RNAi Therapeutics in general, Targeted Genetics’ AAV gene therapy know-how and IP, including IP directly related to RNAi -especially the one for the double-stranded AAV and apparently another one for the expression of non-coding RNAs- I will add $680 worth of TGEN to the RNAi Therapeutics model portfolio and will pay for this with the sale of some stock in ISIS Pharmaceutcals (-$280), Oxford Biomedica (-$200), Silence Therapeutics (-$100) and Rosetta Genomics (-$100).

Remember, an investment in Targeted Genetics is highly speculative, its balance sheet somewhat ugly which is made worse by current market conditions which make it almost impossible to raise small biotech capital on reasonable terms. This investment thesis therefore is that Targeted Genetics will be able to win the race against the clock by being an attractive partner for other drug companies interested in RNAi Therapeutics with the resulting license fees and development milestones helping the company through the hard times. Maybe Genzyme with its considerable AAV gene therapy efforts and orphan disease management expertise or Biogen Idec with its long-standing ties to Targeted and interest in PML will bite.

Disclosure: The lab that I work in has an interest in AAV-mediated RNAi Therapeutics. The author has also been accumulating shares in Targeted Genetics between $0.58 and $0.72. The stock is not suitable for most due to adverse market conditions and the precarious balance sheet of the company. The thin trading volume of the stock causes volatilities in share price, usually to the downside, and there is a real chance that the stock will be de-listed from the Nasdaq exchange which will make this little company even more opaque to investors. On the other hand, conditions will improve at some point and in an environment where venture capital exits have become increasingly difficult and considering the attractive relative valuation and maturity of the company and technology, Targeted Genetics may represent an interesting, somewhat more liquid piece of RNAi Therapeutics real estate for investors otherwise specializing in private start-up companies.

Update on AAV-based RNAi for Huntington’s Disease: Time to Move on from U6-shRNAs?

Huntington’s disease (HD) is a dominant genetic disease caused by a triplett expansion in one of the two copies of the huntingtin (htt) gene. So far there is no treatment that appropriately addresses this disease. As with other dominant diseases, suppressing the insulting gene copy by RNAi should bring a therapeutic benefit, hopefully even delay its progression. A recent paper from Beverly Davidson’s group in Iowa reports on their latest experience in developing an DNA-directed RNAi Therapeutic for the treatment of Huntington’s disease [HD; Mc Bride et al (2008): PNAS 105:5868).

Previous studies from the Davidson lab showed that AAV-delivered shRNAs were able to knockdown target genes in mouse brains and subsequently improve the behavioral deficits and pathology of polyglutamine-repeat diseases such as Huntington’s. These promising results led to a collaboration with the AAV gene therapy company Targeted Genetics and Sirna Therapeutics (pre-Merck acquisition) with the goal of advancing an AAV-based RNAi Therapeutic for HD into the clinic.

Their prior work on Huntington’s was based on a transgenic mouse model which expressed a human fragment of the huntingtin gene containing a triplet expansion. However, as the shRNAs in those studies were directed specifically against the human htt gene, but left the mice’s own huntingtin gene copies untargeted, the present studies were designed to evaluate the safety of knocking down all the huntingtin gene copies in a mouse. This is important for the development of an RNAi Therapeutic for HD in man since targeting a small RNA towards the highly structured triplet expansion is unlikely to be effective, and since the mutant htt allele is not associated with a common polymorphism in the htt mRNA which would facilitate a discrimination between mutant and wildtype huntingtin. This issue is also of particular concern since the huntingtin gene is considered to be essential during early mammalian development and little is known about its requirement in the adult brain.

For that reason, McBride and coworkers chose a different mouse model where a triplet expansion had been inserted into one of the existing mouse htt alleles which has the added advantage that it also preserves the natural expression pattern of htt.

Encouragingly, a 50-60% knockdown of mouse htt was observed over a period of 4 months without any obvious target-specific side-effects. Transduction of this particular AAV administered to the striatum, a region in the brain particularly affected in HD, was fairly broad and specific for neurons, the cell type critically affected during HD. Given that not every neuron was transduced and that non-neuronal cells may also express some htt, this 50-60% knockdown implies that very efficient knockdown of htt can be achieved in a subset of neurons and (based on previous studies) should be therapeutic and safe. Longer term studies are ongoing.

The unpleasant surprise, however, was that as previously reported by a number of other groups, some of the tested U6-driven shRNAs were toxic. This is likely caused by the very high levels of small hairpin RNAs from the strong U6 promoter which may jam up the endogenous microRNA gene regulation which is harnessed by RNAi. Whether it is just the level of the hairpins or also the specific biogenesis of the shRNAs, which only resemble microRNA precursors, but actually undergo a slightly different biogenesis involving an unusual set of enzymes and RNA structures, is an open question. In any case, to make a long story short, it is probably advisable that future DNA-directed RNAi Therapeutics programs avoid U6-driven shRNAs altogether and instead use other Pol III-driven systems or Pol II promoters. The latter have the added safety advantage as they can be chosen to be tissue specific so as to minimize side-effects in non-target cells. Since efficient RNAi does not require high small RNA levels, vector transduction and long-term expression should in many applications be more important considerations than mere promoter strength.

Whether it is advisable to go ahead with the particular U6-shRNA that was found to be non-toxic and efficient in knockdown, or instead go back and re-design all of the vectors and repeat 2-3 years of pre-clinical studies, must be a tough decision to make, a situation similar to that faced by other RNAi Therapeutics programs currently in development given the steep learning curve the field is still undergoing.

It is maybe for these reasons that Merck’s subsidiary Sirna Therapeutics decided not to continue with this program and fully hand over the commercial rights to Targeted Genetics. In some way, the AAV-RNAi program always stood out as being an awkward fit for Sirna Therapeutics as it seemed at odds with their siRNA modification focus. Maybe at the time there were not that many advanced RNAi Therapeutics programs to chose from so that Dr. Davidson’s studies seemed like an opportunity to fill the early pipeline. Given the unpredictability of early studies and the number of RNAi Therapeutics programs under evaluation, it is best to let pipeline decisions be driven not by history, but by where the science is progressing swiftest. AAV RNAi for HD still has much promise and the path forward fairly obvious, but as many things in drug development it will be a long and arduous one.

Nature Publishes Reassuring Study by Alnylam on the in vivo Delivery of siRNAs and their Effect on the Endogenous microRNA Pathway

Given that therapeutic RNAi takes advantage of an endogenous biological pathway, the introduction of very high levels of small RNAs certainly has the potential to interfere with related small RNA pathways, such as microRNA function.

Indeed, competition with the microRNA pathway in vivo which in some cases caused the death of mice, were first reported in a study by Grimm and colleagues in the journal Nature last year. Ironically, rather than a demonstrating a failure of the viral delivery system used in that study, it was the extreme efficiency with which small RNAs could be expressed with the double-stranded AAV vectors that allowed the competition to be observed. In what is an often overlooked aspect of these studies, compared to non-viral delivery methods, silencing efficiencies of over 95% can be easily achieved with these vectors at doses that have no adverse effects on either the microRNA pathway or the viability of mice.

In an ideal world, the Grimm et al. studies would have been embraced as an opportunity to study the dose-limiting steps of therapeutic RNAi to inform future RNAi therapeutics strategies. Instead, as the Press lives from feeding the public the simple messages, rather than reporting complicated truths, it decided to label the studies as yet another example of the dangers of gene therapy, and - somewhat understandably- caused some companies involved in developing RNAi Therapeutics to distance themselves from DNA-directed RNAi for political reasons.

As the trusted leader of RNAi Therapeutics, Alnylam was therefore given the platform to reassure the RNAi community this week in Nature that, unlike AAV-RNAi, liposomally delivered siRNAs had no obvious adverse effects on the endogenous microRNA pathway (John et al., 2007). The study further highlighted that liposomal siRNA delivery has advanced to a point where around 80% gene silencing in hepatocytes can routinely be achieved following systemic administration of therapeutically viable doses of siRNAs, including their repeat administration.

Although I welcome Nature’s decision to document progress in the important area of RNAi therapeutics, and understand Alnylam’s desire to publish in the highest profile journals, I would like to take this opportunity to address a few misconceptions about the studies. One important misconception is that delivering RNAi with AAV per se is more toxic. To make this point, a direct comparison of the intrahepatic levels of small RNA levels following both routes of administration would have been necessary. Given the >99% transduction efficiency of double-stranded AAV in mice and the consequently extremely high gene silencing efficiencies, it is quite likely that double-strand AAV vectors are currently the most potent delivery system to the liver in terms of small RNA delivery and gene knockdown.

I would therefore not be surprised at all to see similar competition with microRNA function following administration of very high siRNA dosages. This is supported by numerous studies that have shown competition for gene silencing when very high levels of two or more siRNAs were introduced simultaneously into tissue culture cells. However, given the ability of hundreds of microRNAs to function in a given cell at any time, such observations represent only extreme cases and suggest a wide therapeutic index. Unfortunately, the relatively small range of doses used in the John et al. studies (2mg/kg to 5mg/kg) did allow for a careful evaluation of related competition in vivo and concomitant dose-limiting toxicities.

I guess the purpose of this Blog really is my plea to the field of RNAi Therapeutics to keep learning from each other, instead of letting the Press and uninformed “analysts” play on the fears of investors, through their indiscriminate use of buzzwords, thereby polarising and separating what really belongs together. In this spirit, I would like to stress that this study is yet another proof-point of the viability of RNAi for therapy leading up to the possibly first proof-of-concept gene silencing results in Man to be revealed in the coming months- once again by Alnylam.


PS: Although a combination of liposomal delivery methods were used in these studies, the details were not disclosed. Apparently, another study on lipidoid-delivered siRNAs, a technology developed by the Langer and Anderson groups at the MIT, has been submitted to Nature Biotech and is about to be published. Lipidoids differ slightly from the Tekmira-owned SNALP technology, and looks likely to be the technology used for Alnylam’s first clinical systemic RNAi program (liver cancer or hypercholesterolemia), for which an IND is expected by the end of 2007. More than knockdown efficiency, we should be looking for the toxicity profile as I regard this to be the big unknown that will determine the success of this program, particularly if it turns out to be for hypercholesterolemia.