In this blog I will summarise the highlights of the corporate update given today by John Maraganore, the CEO of Alnylam Pharmaceuticals, at the Banc of America Healthcare Conference in Las Vegas. Alnylam can be considered the leader in RNAi Therapeutics, and it is their success so far that has opened up the prospect for a whole new class of drugs based on RNAi. As such, Alnylam is proud to note that their work was cited by the Karolinska Institute in the justification for the recent Nobel Prize to Fire and Mello for their discovery of RNAi.
In his presentation, JM detailed the breadth of Alnylam’s clinical and pre-clinical programs encompassing all major therapeutic areas except for bacterial infection. There are more than 20 such programs ongoing at Alnylam, Alnylam partners, or licensees. Moreover, over 50% of current clinical RNAi programs and over 75% of siRNA reagent sales are covered by Alnylam IP. Besides scientific excellence, IP is a key issue for Alnylam’s financial ability to turn RNAi into drugs, and JM made it clear that Alnylam is the gatekeeper in the commercialisation of RNAi therapeutics: Alnylam has access to all the 8 key issued patents relating to RNAi Therapeutics, 7 of them on an exclusive basis. As a consequence, the company has already taken in over $120M in milestone payments and fees, not including the potentially more than 1 billion USD in future milestone payments.
As key business goals, JM cited continued progress on the science, particularly in delivery, moving along the clinical pipeline (for 2007: phase II for RSV, INDs for PCSK9 and pandemic flu; announcement of 2 more pre-clinical programs) and one or more significant alliances by year-end. What JM seemed to value most, however, is their plan to show human proof-of-concept data for an RNAi Therapeutic within the next 12-18 months. This could either come from their phase II RSV experimental infection studies, slated to start by the end of June, or early results from the PCSK9 hypercholesterolemia program. With regard to PCSK9 and possible competition from ISIS’ antisense compound targeting the same target, JM was very diplomatic in his response saying that he is encouraged by developments in the antisense arena, and the future will show what each technology platform can do therapeutically. Unlike ISIS and in spite of considerable outside interest, Alnylam would not have any immediate plans to partner PCSK9 which Alnylam is pursuing with the leading Brown-Goldstein labs at UT Southwestern.
Overall, a solid performance by JM and everything is appears to be on track.
Note: Alnylam dropped today another 4.5% to $15.49 amid heavy trading, despite an overall positive market. At this point, investors just have to be patient. Policies that make it clear that Alnylam is not for sale and saying certain programs are to stay within the company may not be popular in the short-term, but Alnylam is about building a real business, and is not an M&A project.
Thursday, May 31, 2007
Monday, May 28, 2007
RNAi Therapeutics IP: On the Importance of Being Tuschl
A strong patent position is essential for companies to attract investments for the development of RNAi Therapeutics. Although the number of patents and patent applications surrounding RNAi Therapeutics continues to grow, arguably the two or three key early patents are 1) Fire and Mello describing the discovery and use of long double-stranded RNAs for inducing RNAi-mediated gene knockdown in worms and by extension other organisms, and 2) the Tuschl I and II patent series describing the identification of small 21-23nt small RNAs as the mediators of RNAi (Tuschl I) and the use and properties of synthetic siRNA duplexes for RNAi in human cells (Tuschl II).
While Fire and Mello has been granted in the US and can be licensed by almost anyone that wants it, there is much more controversy surrounding Tuschl I and II. It is undisputed that scientifically Tuschl’s studies describing the use of synthetic siRNAs for RNAi in mammalian cells is what opened up the prospect for RNAi to become the next platform for drug development. Tuschl II which is based on the work by Tuschl, Elbashir, and Lendeckel and owned by the Max-Planck Institute in Germany has been exclusively licensed to Alnylam Pharmaceuticals and has already been granted in the US and some other territories (Tuschl is a scientific co-founder of Alnylam). The patent application was provisionally filed in the US on March 30, 2001, and in Europe on December 1, 2000, and describes in great detail the anatomy of effective synthetic siRNAs.
Meanwhile, much of the work in Tuschl I is focussed on the identification of small RNAs as the mediators of RNAi based on the fact that isolated small RNAs derived from processed double-stranded RNAs in Drosophila cell extracts trigger specific gene knockdown and cleavage of a target message at 21-23nt intervals. Interestingly, most of that work does not mention the fact that these 21-23nt small RNAs should be double-stranded. This conclusion could not be derived from the observation of 21-23nt small RNAs on denaturing polyacrylamide gels, but was deduced through the cloning of these small RNAs which is described in Tuschl II, not I. It is therefore very surprising that, out of the blue, Tuschl I demonstrates the use of synthetic siRNAs with preferably 2-nucleotide overhangs for RNAi in mammalian cells. Subsequent claims then focus on the use of such siRNAs for human therapeutic development. This is rather surprising given that the basis for choosing 2-nucleotide synthetic siRNAs is lacking. It therefore appears as if this example had been appended later so as to make the patent more relevant for human therapeutic use. Otherwise, only fruit fly work, albeit important, would have been described. Given the near-identity of this last claim of Tuschl I with work described in Tuschl II, it is difficult to imagine how Tuschl I could be granted in full in the presence of Tuschl II.
Similarly intriguing is the fact that Tuschl I, which by the way has not issued yet, is co-owned by the Whitehead Institute for Biomedical Research in Cambridge, MA, the MIT, the University of Massachussetts, Worcester, and the Max-Planck Institute. At the same time, from the publication record it is clear that synthetic siRNAs were pioneered by Tuschl, Elbashir, and Lendeckel while at the Max-Planck Institute. Interestingly, the UMass has chosen to co-exlusively license their rights to the patent to Sirna Therapeutics (now a Merck subsidiary), CytRX (now RXi), in addition to Alnylam. Clearly, Merck and RXi would benefit most if Tuschl I would be granted eventually and somewhat limit the dominance that Alnylam currently enjoys in the RNAi patent space. Nevertheless, the fact that Tuschl I, filed on the very same December 1, 2000 date as Tuschl II in Europe, has not issued yet and the sudden appearance of synthetic siRNAs and their use in human cells at the very end of that patent application, raises questions about conflicting interests between the involved parties.
Based on publicly available information, the claims of the Tuschl patent series could therefore be divided as follows: Tuschl I getting credit for identifying 21-23nt small RNAs for mediating RNAi in fruit flies and by extension in other organisms, and Tuschl II for characterising effective siRNAs to be double-stranded with preferably 2-nucleotide 3’ overhangs and the ability of synthetic versions thereof to mediate RNAi in mammalian cells. In such a scenario Tuschl II would carry considerably more weight for the development of RNAi Therapeutics which would in turn reinforce Alnylam’s already leading IP position.
I am aware that parts of my interpretation are based on conjecture and criticisms are welcome (email: dirk_haussecker@yahoo.com). For those interested in the patents themselves, please visit
Tuschl I: http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PG01&s1=tuschl.IN.&s2=zamore.IN.&OS=IN/tuschl+AND+IN/zamore&RS=IN/tuschl+AND+IN/zamore
Tuschl II: http://www.google.com/patents?id=BlV6AAAAEBAJ&dq=rna+sequence+specific+mediators+of+rna+interference
While Fire and Mello has been granted in the US and can be licensed by almost anyone that wants it, there is much more controversy surrounding Tuschl I and II. It is undisputed that scientifically Tuschl’s studies describing the use of synthetic siRNAs for RNAi in mammalian cells is what opened up the prospect for RNAi to become the next platform for drug development. Tuschl II which is based on the work by Tuschl, Elbashir, and Lendeckel and owned by the Max-Planck Institute in Germany has been exclusively licensed to Alnylam Pharmaceuticals and has already been granted in the US and some other territories (Tuschl is a scientific co-founder of Alnylam). The patent application was provisionally filed in the US on March 30, 2001, and in Europe on December 1, 2000, and describes in great detail the anatomy of effective synthetic siRNAs.
Meanwhile, much of the work in Tuschl I is focussed on the identification of small RNAs as the mediators of RNAi based on the fact that isolated small RNAs derived from processed double-stranded RNAs in Drosophila cell extracts trigger specific gene knockdown and cleavage of a target message at 21-23nt intervals. Interestingly, most of that work does not mention the fact that these 21-23nt small RNAs should be double-stranded. This conclusion could not be derived from the observation of 21-23nt small RNAs on denaturing polyacrylamide gels, but was deduced through the cloning of these small RNAs which is described in Tuschl II, not I. It is therefore very surprising that, out of the blue, Tuschl I demonstrates the use of synthetic siRNAs with preferably 2-nucleotide overhangs for RNAi in mammalian cells. Subsequent claims then focus on the use of such siRNAs for human therapeutic development. This is rather surprising given that the basis for choosing 2-nucleotide synthetic siRNAs is lacking. It therefore appears as if this example had been appended later so as to make the patent more relevant for human therapeutic use. Otherwise, only fruit fly work, albeit important, would have been described. Given the near-identity of this last claim of Tuschl I with work described in Tuschl II, it is difficult to imagine how Tuschl I could be granted in full in the presence of Tuschl II.
Similarly intriguing is the fact that Tuschl I, which by the way has not issued yet, is co-owned by the Whitehead Institute for Biomedical Research in Cambridge, MA, the MIT, the University of Massachussetts, Worcester, and the Max-Planck Institute. At the same time, from the publication record it is clear that synthetic siRNAs were pioneered by Tuschl, Elbashir, and Lendeckel while at the Max-Planck Institute. Interestingly, the UMass has chosen to co-exlusively license their rights to the patent to Sirna Therapeutics (now a Merck subsidiary), CytRX (now RXi), in addition to Alnylam. Clearly, Merck and RXi would benefit most if Tuschl I would be granted eventually and somewhat limit the dominance that Alnylam currently enjoys in the RNAi patent space. Nevertheless, the fact that Tuschl I, filed on the very same December 1, 2000 date as Tuschl II in Europe, has not issued yet and the sudden appearance of synthetic siRNAs and their use in human cells at the very end of that patent application, raises questions about conflicting interests between the involved parties.
Based on publicly available information, the claims of the Tuschl patent series could therefore be divided as follows: Tuschl I getting credit for identifying 21-23nt small RNAs for mediating RNAi in fruit flies and by extension in other organisms, and Tuschl II for characterising effective siRNAs to be double-stranded with preferably 2-nucleotide 3’ overhangs and the ability of synthetic versions thereof to mediate RNAi in mammalian cells. In such a scenario Tuschl II would carry considerably more weight for the development of RNAi Therapeutics which would in turn reinforce Alnylam’s already leading IP position.
I am aware that parts of my interpretation are based on conjecture and criticisms are welcome (email: dirk_haussecker@yahoo.com). For those interested in the patents themselves, please visit
Tuschl I: http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PG01&s1=tuschl.IN.&s2=zamore.IN.&OS=IN/tuschl+AND+IN/zamore&RS=IN/tuschl+AND+IN/zamore
Tuschl II: http://www.google.com/patents?id=BlV6AAAAEBAJ&dq=rna+sequence+specific+mediators+of+rna+interference
Sunday, May 27, 2007
MicroRNAs as Therapeutic Targets
MicroRNAs are another class of endogenous cellular small RNAs that like siRNA in RNAi down-regulate target genes. Unlike siRNAs in mammalian cells, these mostly recognise their targets by incomplete base-pair complementarity. Consequently, each of the maybe 1000 microRNAs in humans has the potential to regulate a large number of target genes, with some estimates pegging that number upwards of 100. Together, these microRNAs regulate genes involved in almost all aspects of a cell’s life and some of them have been found to be involved in the pathogenesis of many diseases such as cancer and neurodegenerative conditions. It is therefore hoped that the targeting of microRNAs by antisense techniques or their over-expression in the form of siRNA-based microRNA mimics can be exploited for therapeutic purposes.
When the underlying cause or contributing factor (e.g. viral microRNAs) for a disease is a microRNA, then targeting it should stand a good chance in being an effective treatment as long as loss-of-function of that microRNA in the target cells does not have other undesirable side-effects. It appears to be, however, much more complicated when the idea is to target a microRNA as the regulator of a complex phenotype. One example of this approach is targeting microRNA-122, a highly expressed microRNA in the liver, for treating hypercholesterolemia. Down-regulation of this microRNA has been achieved in the liver in rodents and was shown to result in lower cholesterol levels. It needs to be questioned, however, if this approach is viable for chronic treatment given the importance of microRNAs in general for cell viability and the fact that microRNA-122 in particular makes up about 70% of liver microRNAs. It is interesting to note that targeting microRNAs as modulators of complex phenotypes versus RNAi Therapeutics approaches are derived from quite contrary drug development philosophies. The one philosophy (RNAi Therapeutics) holds that the best treatment with the least side-effect focuses on as few causative genes as possible. The other philosophy, however, proposes that single-gene targeting will only be successful in few instances and the best drugs are those that affect multiple targets. As such many of the small molecule inhibitors on the market today are now known to be actually much less specific than intended and their off-target effects contribute to the therapeutic outcome.
Four investing purposes, four companies that stand to benefit from the development of microRNA-based therapeutics come to mind: Alnylam (ticker: Alny), ISIS (ticker: Isis), Rosetta Genomics (ticker: Rosg), and Asuragen (privately held). Although many more microRNAs exist, Alnylam’s co-founder Tuschl identified the first 120 microRNAs in humans, and these are therefore likely to be amongst the most important in human biology. Alnylam partnered these microRNAs with ISIS Pharmaceuticals which owns many of the fundamental antisense patents required for the targeting of microRNAs. In addition to developing similar microRNA-based therapeutics, Rosetta and Asuragen (a privately-held company that was spun off from Ambion when it was acquired by ABI) are two companies that both expect to generate early revenues through the development of microRNA-based diagnostics, e.g. for the classification of cancers. In addition to their own discovered and characterised microRNAs, they will therefore have to pursue a prudent licensing policy to secure their freedom to operate.
While it is already obvious that microRNAs are extremely important for human gene regulation, much more work remains to be done to confidently identify suitable targets. Fortunately, much of this work is already being undertaken due to the immense interest in microRNAs in biology. In the meantime, let-7 in cancer and microRNA-122 for the treatment of HCV and hypercholesterolemia may lead the way in showing the promise of microRNA-based therapeutics.
When the underlying cause or contributing factor (e.g. viral microRNAs) for a disease is a microRNA, then targeting it should stand a good chance in being an effective treatment as long as loss-of-function of that microRNA in the target cells does not have other undesirable side-effects. It appears to be, however, much more complicated when the idea is to target a microRNA as the regulator of a complex phenotype. One example of this approach is targeting microRNA-122, a highly expressed microRNA in the liver, for treating hypercholesterolemia. Down-regulation of this microRNA has been achieved in the liver in rodents and was shown to result in lower cholesterol levels. It needs to be questioned, however, if this approach is viable for chronic treatment given the importance of microRNAs in general for cell viability and the fact that microRNA-122 in particular makes up about 70% of liver microRNAs. It is interesting to note that targeting microRNAs as modulators of complex phenotypes versus RNAi Therapeutics approaches are derived from quite contrary drug development philosophies. The one philosophy (RNAi Therapeutics) holds that the best treatment with the least side-effect focuses on as few causative genes as possible. The other philosophy, however, proposes that single-gene targeting will only be successful in few instances and the best drugs are those that affect multiple targets. As such many of the small molecule inhibitors on the market today are now known to be actually much less specific than intended and their off-target effects contribute to the therapeutic outcome.
Four investing purposes, four companies that stand to benefit from the development of microRNA-based therapeutics come to mind: Alnylam (ticker: Alny), ISIS (ticker: Isis), Rosetta Genomics (ticker: Rosg), and Asuragen (privately held). Although many more microRNAs exist, Alnylam’s co-founder Tuschl identified the first 120 microRNAs in humans, and these are therefore likely to be amongst the most important in human biology. Alnylam partnered these microRNAs with ISIS Pharmaceuticals which owns many of the fundamental antisense patents required for the targeting of microRNAs. In addition to developing similar microRNA-based therapeutics, Rosetta and Asuragen (a privately-held company that was spun off from Ambion when it was acquired by ABI) are two companies that both expect to generate early revenues through the development of microRNA-based diagnostics, e.g. for the classification of cancers. In addition to their own discovered and characterised microRNAs, they will therefore have to pursue a prudent licensing policy to secure their freedom to operate.
While it is already obvious that microRNAs are extremely important for human gene regulation, much more work remains to be done to confidently identify suitable targets. Fortunately, much of this work is already being undertaken due to the immense interest in microRNAs in biology. In the meantime, let-7 in cancer and microRNA-122 for the treatment of HCV and hypercholesterolemia may lead the way in showing the promise of microRNA-based therapeutics.
Thursday, May 24, 2007
RNAi-related Phenomena and their Relevance for RNAi Therapeutics
Following the discovery of small RNAs as part of RNAi and a growing range of other classes of small RNAs, it soon became clear that RNAi is just one part of a network of small RNA-directed cellular processes. MicroRNAs which are involved in a wide range of post-transcriptional gene regulatory processes are probably best known, but small RNAs have also been found to play roles from chromatin silencing to chromosome rearrangement. The existence and importance of these functions can vary greatly between different organisms. Nevertheless within a given organisms these functions are interconnected by partly making use of overlapping sets of proteins or proteins from the same gene families.
One concern in the development of RNAi Therapeutics for human use would therefore be that small RNAs may engage unwanted small RNA-directed processes. In humans, the microRNA pathway and RNAi-related chromatin silencing are probably the main concerns. The microRNA pathway is actually the main reason for off-targeting in RNAi experiments, and I have outlined potential strategies about how to selectively engage RNAi and not the microRNA pathway in earlier posts. Silencing chromatin through the introduction of small RNAs in human cells, however, is much more controversial. Even if we assume that this pathway does exist, then according to published papers you would probably need more than one small RNA targeting the same gene. Moreover, one would likely have to introduce small RNAs targeted to the promoter region and not the transcribed gene as siRNAs do in RNAi. A possibly related interesting phenomena that was reported recently is RNA activation, or RNAa. In these examples small RNAs targeted to certain promoters actually upregulated gene activity. It is clear, however, that not any small RNA will have this effect, and again siRNAs targeted towards the transcribed genes are not likely to result in promoter activation.
Alone from the fact that there are 4 Argonaute genes and additional related Piwi genes, and new classes of small RNAs being discovered on almost a monthly basis, one can expect the RNAi-related universe to expand ever more. However, largely based on microarray studies, it seems that exogenous small RNAs almost exclusively engage the RNAi machinery and the related microRNA pathway. It is therefore a focus on the microRNA pathway that is most likely to ultimately improve the specificity of RNAi Therapeutics.
One concern in the development of RNAi Therapeutics for human use would therefore be that small RNAs may engage unwanted small RNA-directed processes. In humans, the microRNA pathway and RNAi-related chromatin silencing are probably the main concerns. The microRNA pathway is actually the main reason for off-targeting in RNAi experiments, and I have outlined potential strategies about how to selectively engage RNAi and not the microRNA pathway in earlier posts. Silencing chromatin through the introduction of small RNAs in human cells, however, is much more controversial. Even if we assume that this pathway does exist, then according to published papers you would probably need more than one small RNA targeting the same gene. Moreover, one would likely have to introduce small RNAs targeted to the promoter region and not the transcribed gene as siRNAs do in RNAi. A possibly related interesting phenomena that was reported recently is RNA activation, or RNAa. In these examples small RNAs targeted to certain promoters actually upregulated gene activity. It is clear, however, that not any small RNA will have this effect, and again siRNAs targeted towards the transcribed genes are not likely to result in promoter activation.
Alone from the fact that there are 4 Argonaute genes and additional related Piwi genes, and new classes of small RNAs being discovered on almost a monthly basis, one can expect the RNAi-related universe to expand ever more. However, largely based on microarray studies, it seems that exogenous small RNAs almost exclusively engage the RNAi machinery and the related microRNA pathway. It is therefore a focus on the microRNA pathway that is most likely to ultimately improve the specificity of RNAi Therapeutics.
Tuesday, May 22, 2007
Promising Cancer RNAi Therapeutics Study Published in Cancer Research
Due to the relative ease of delivering RNAi effector molecules to the eye, lung, skin, and liver, it is expected that RNAi-based therapies for these organs will be the first to successfully navigate their way through the clinical trial maze. Nevertheless, a considerable amount of the early development efforts of RNAi Therapeutics is spent on cancer. I do not think that I need to remind anybody here about the need for more and better cancer therapeutics, but RNAi in particular offers the potential to greatly benefit cancer care. Delivery, however, represents a major obstacle for the development of cancer RNAi Therapeutics. This is even more so when faced with metastases which emphasises the need to find systemic RNAi delivery solutions to reach disseminated cancer cells throughout the body.
I was therefore very encouraged on reading a study published by Pirollo and colleagues from the Lombardi Comprehensive Cancer Center at Georgetown University Medical Center. In a Priority Report in the reputable journal Cancer Research (Cancer Res. 2007: 67 (7)), they report the development of a novel siRNA delivery system made up of nano-sized liposomes that harbour effector siRNAs and antibodies directed against the transferrin receptor. Transferrin receptors are known to be overexpressed on tumour cells and targeting them emerges as a promising strategy for cancer RNAi Therapeutics (Note: Calando, a subsidiary of Arrowhead Research, uses a similar approach in their first clinical RNAi cancer program). Consequently, the authors could show through imaging and nucleic acid detection techniques that accordingly formulated siRNAs were specifically enriched in the tumours of mouse models. The siRNA itself targets HER-2, a gene known to play a role in the survival of cancer cells. Although the siRNA in isolation already exhibited antiproliferative effects in tissue culture and the living animal, the most impressive data was that it could greatly sensitise cancer cells to the commonly used chemotherapeutic agent gemcitabine at low mg/kg doses. In addition to a dramatic reduction in tumour growth, the mice continued to gain weight and generally looked healthy. This study therefore shows the promise of using siRNAs in combination with chemotherapeutic agents, allowing for treatment regimens that are both more efficacious in addition to reducing debilitating side-effects.
Behind the success of these studies were 2 small tricks applied to the basic concept of nano-immunoliposomally formulated siRNAs. One was the addition of a pH-sensitive
peptide. This significantly enhanced cellular siRNA delivery, possibly by facilitating endosomal release of the siRNA cargo. The other trick was the modification of the siRNA, in which the non-targeting strand of the blunt-ended “siRNA” was greatly modified. This suggests that the systematic modification of basic siRNAs can be considerably advantageous and may yield even better siRNAs in the future.
Let us hope that other laboratories will find this immunoliposome technology equally promising so that it will eventually be tested in clinical trials.
I was therefore very encouraged on reading a study published by Pirollo and colleagues from the Lombardi Comprehensive Cancer Center at Georgetown University Medical Center. In a Priority Report in the reputable journal Cancer Research (Cancer Res. 2007: 67 (7)), they report the development of a novel siRNA delivery system made up of nano-sized liposomes that harbour effector siRNAs and antibodies directed against the transferrin receptor. Transferrin receptors are known to be overexpressed on tumour cells and targeting them emerges as a promising strategy for cancer RNAi Therapeutics (Note: Calando, a subsidiary of Arrowhead Research, uses a similar approach in their first clinical RNAi cancer program). Consequently, the authors could show through imaging and nucleic acid detection techniques that accordingly formulated siRNAs were specifically enriched in the tumours of mouse models. The siRNA itself targets HER-2, a gene known to play a role in the survival of cancer cells. Although the siRNA in isolation already exhibited antiproliferative effects in tissue culture and the living animal, the most impressive data was that it could greatly sensitise cancer cells to the commonly used chemotherapeutic agent gemcitabine at low mg/kg doses. In addition to a dramatic reduction in tumour growth, the mice continued to gain weight and generally looked healthy. This study therefore shows the promise of using siRNAs in combination with chemotherapeutic agents, allowing for treatment regimens that are both more efficacious in addition to reducing debilitating side-effects.
Behind the success of these studies were 2 small tricks applied to the basic concept of nano-immunoliposomally formulated siRNAs. One was the addition of a pH-sensitive
peptide. This significantly enhanced cellular siRNA delivery, possibly by facilitating endosomal release of the siRNA cargo. The other trick was the modification of the siRNA, in which the non-targeting strand of the blunt-ended “siRNA” was greatly modified. This suggests that the systematic modification of basic siRNAs can be considerably advantageous and may yield even better siRNAs in the future.
Let us hope that other laboratories will find this immunoliposome technology equally promising so that it will eventually be tested in clinical trials.
Saturday, May 19, 2007
Journal Club: eIF6 is Part of the Translational Repressor Complex, but not Minimal Human RiSC- Implications for Therapeutic RNAi
This week, the journal Nature published a study by the Shiekhattar group online investigating the molecular identity of the microRNA-mediated translational repressor complex. This is important for RNAi Therapeutics since it is still unclear whether the microRNA complex is distinct from the RNAi effector complex in humans. MicroRNAs mediate gene silencing by translational repression via 7-8nt base-pair complementarity to the target gene mostly in the 3’ UTR of gene transcripts, whereas siRNAs knockdown genes with much larger complementarity by instructing their specific cleavage and destruction. It is thought that the microRNA pathway is largely responsible for so-called off-targeting effects in RNAi experiments given their less stringent base-pairing requirements. A better molecular understanding of what constitutes the microRNA translational repressor complex versus the RNAi mRNA destruction complex should lead the way in identifying strategies to specifically harness the RNAi pathway, thus minimising off-targeting and therefore unpredicted side-effects in the clinical setting.
By performing co-immunoprecipitation experiments with a protein known to be involved in siRNA-guided gene silencing, Chendrimada and colleagues identified another complex containing this protein that was distinct from the one mediating siRNA-guided mRNA cleavage. One of the factors found was eIF6 which is known to be a component of the 60S large ribosomal particle and to prevent the association of 60S with 40S to constitute the active translation complex. The authors then go on to show that by inhibiting the activity of eIF6 (using siRNAs), microRNA-mediated translational inhibition was abrogated. Since eIF6 is not required for siRNA-guided cleavage in vitro, this suggests that the microRNA repressor complex is distinct from the RNAi machinery. It would have been nice, however, to test whether following the inhibition of eIF6, RNAi activity was left intact as would be predicted in such a model. Similarly, it would have been interesting to test whether eIF6 depletion would have abolished microRNA-mediated translational repression in an in vitro system similar to the one used for the identification of the RNAi minimal RiSC.
While this study leaves some of these questions unanswered, it is an excellent demonstration that through a more thorough understanding of how microRNAs and siRNAs effect gene silencing, it should be possible to further increase the specificity of RNAi, a feature already considered to be one of the strengths of this technology. The ultimate goal would be to design siRNAs that cannot be used by the microRNA repressor complex without compromising RNAi activity. It is noteworthy that Dharmacon scientists have demonstrated that through the introduction of a simple modification in the siRNA, microRNA effects can be reduced by 5-10 fold while leaving siRNA activity intact. It is exciting to see how clever experimentation in many labs is gradually breaking down the barriers towards the safe and widespread application of RNAi Therapeutics.
By performing co-immunoprecipitation experiments with a protein known to be involved in siRNA-guided gene silencing, Chendrimada and colleagues identified another complex containing this protein that was distinct from the one mediating siRNA-guided mRNA cleavage. One of the factors found was eIF6 which is known to be a component of the 60S large ribosomal particle and to prevent the association of 60S with 40S to constitute the active translation complex. The authors then go on to show that by inhibiting the activity of eIF6 (using siRNAs), microRNA-mediated translational inhibition was abrogated. Since eIF6 is not required for siRNA-guided cleavage in vitro, this suggests that the microRNA repressor complex is distinct from the RNAi machinery. It would have been nice, however, to test whether following the inhibition of eIF6, RNAi activity was left intact as would be predicted in such a model. Similarly, it would have been interesting to test whether eIF6 depletion would have abolished microRNA-mediated translational repression in an in vitro system similar to the one used for the identification of the RNAi minimal RiSC.
While this study leaves some of these questions unanswered, it is an excellent demonstration that through a more thorough understanding of how microRNAs and siRNAs effect gene silencing, it should be possible to further increase the specificity of RNAi, a feature already considered to be one of the strengths of this technology. The ultimate goal would be to design siRNAs that cannot be used by the microRNA repressor complex without compromising RNAi activity. It is noteworthy that Dharmacon scientists have demonstrated that through the introduction of a simple modification in the siRNA, microRNA effects can be reduced by 5-10 fold while leaving siRNA activity intact. It is exciting to see how clever experimentation in many labs is gradually breaking down the barriers towards the safe and widespread application of RNAi Therapeutics.
Friday, May 18, 2007
A Framework for Progressing the Delivery of RNAi Therapeutics
The delivery of RNAi to the desired target organ is perceived by many to be the main hurdle before RNAi Therapeutics can have a universal impact on treating diseases. Arguably today already, it probably is possible to treat dozens of diseases for the eye and lung alone with current siRNA delivery methods. Furthermore, it is possible to efficiently knock down genes in the liver of primates, either with siRNAs formulations or viral gene therapy vectors. This is because the liver is the first pass organ for most drugs and highly vascularised thereby facilitating drug uptake.
The liver, however, demonstrates already some of the challenges we are facing. In the case of viral vectors, immunogenicity issues need to be addressed to make sure that immune responses against the vector will not abolish long-term gene knockdown. This is being addressed with efforts such as identifying novel serotypes or modifying pre-exising ones such as in the AAV field, but any of these solutions have yet to undergo testing in humans. For non-viral siRNA delivery, the main challenge at the moment is to get highly efficient gene knockdown without causing liver damage. It should be noted that limited liver damage is associated with a number of drugs on the market, however the degree of the damage needs to be tightly monitored. Needless to say, as with any drug, there is always a risk-benefit to consider. Alnylam published last year a study in Nature in which they show highly potent gene knockdown in primate liver. That in itself is a major achievement as only 2 years ago systemic delivery in humans was thought to be far on the horizon, and now it may happen much sooner. However, it was reported in the same study that there was a dose-dependent elevation of liver enzymes associated with the liposomally delivered siRNAs, in some cases quite high. Having established effective gene knockdown in primate liver, I would now expect the teams at Alnylam, Tekmira, Protiva, and Merck/Sirna to work on modifications of the liposomal delivery strategy that marries efficacy with little to no delivery-related side-effects
Beyond the liver, the reticuloendothelial system, kidney, intestines, and cancer, are probably next on the list of organs being targeted by systemic delivery. I expect more varied approaches to be taken to reach these organs, especially the use of targeting strategies such as adding cell-type specific targeting agents such as antibodies, peptides, or RNA-aptamers to a core delivery vehicle. This is an area where I see innovation particularly coming from the academic arena due to their freedom to explore. As a corporate strategy, however, I would be cautious in moving poorly characterised delivery strategies into the clinic, and put emphasis on further understanding and optimising promising technologies that exist today, such as the lipid-based technologies mentioned above. With scientists and funders working on all these fronts, it should be possible to gradually widen the scope for RNAi Therapeutics in a well-managed manner. From a commercial perspective, there are enough targets around now to focus on for the credible players in the field. I should add, however, that for orphan diseases or terminal diseases, more innovative strategies should definitely be considered for the clinic today.
Finally, I would like to appeal to all those involved in legal proceedings about who owns certain IPs in the delivery area. Please stop and realise that this is not helping anybody here, except of course for certain lawyers. It should be possible to set aside bruised egos and reach agreements with which everybody can live with that contributed to the development of a promising technology such as the liposomal delivery of siRNAs to mention just one example. Challenges such as liver toxicities associated with some of the liposomal vehicles are best solved when the scientists that co-developed and understand it best have access to.
The liver, however, demonstrates already some of the challenges we are facing. In the case of viral vectors, immunogenicity issues need to be addressed to make sure that immune responses against the vector will not abolish long-term gene knockdown. This is being addressed with efforts such as identifying novel serotypes or modifying pre-exising ones such as in the AAV field, but any of these solutions have yet to undergo testing in humans. For non-viral siRNA delivery, the main challenge at the moment is to get highly efficient gene knockdown without causing liver damage. It should be noted that limited liver damage is associated with a number of drugs on the market, however the degree of the damage needs to be tightly monitored. Needless to say, as with any drug, there is always a risk-benefit to consider. Alnylam published last year a study in Nature in which they show highly potent gene knockdown in primate liver. That in itself is a major achievement as only 2 years ago systemic delivery in humans was thought to be far on the horizon, and now it may happen much sooner. However, it was reported in the same study that there was a dose-dependent elevation of liver enzymes associated with the liposomally delivered siRNAs, in some cases quite high. Having established effective gene knockdown in primate liver, I would now expect the teams at Alnylam, Tekmira, Protiva, and Merck/Sirna to work on modifications of the liposomal delivery strategy that marries efficacy with little to no delivery-related side-effects
Beyond the liver, the reticuloendothelial system, kidney, intestines, and cancer, are probably next on the list of organs being targeted by systemic delivery. I expect more varied approaches to be taken to reach these organs, especially the use of targeting strategies such as adding cell-type specific targeting agents such as antibodies, peptides, or RNA-aptamers to a core delivery vehicle. This is an area where I see innovation particularly coming from the academic arena due to their freedom to explore. As a corporate strategy, however, I would be cautious in moving poorly characterised delivery strategies into the clinic, and put emphasis on further understanding and optimising promising technologies that exist today, such as the lipid-based technologies mentioned above. With scientists and funders working on all these fronts, it should be possible to gradually widen the scope for RNAi Therapeutics in a well-managed manner. From a commercial perspective, there are enough targets around now to focus on for the credible players in the field. I should add, however, that for orphan diseases or terminal diseases, more innovative strategies should definitely be considered for the clinic today.
Finally, I would like to appeal to all those involved in legal proceedings about who owns certain IPs in the delivery area. Please stop and realise that this is not helping anybody here, except of course for certain lawyers. It should be possible to set aside bruised egos and reach agreements with which everybody can live with that contributed to the development of a promising technology such as the liposomal delivery of siRNAs to mention just one example. Challenges such as liver toxicities associated with some of the liposomal vehicles are best solved when the scientists that co-developed and understand it best have access to.
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