An interesting paper relating to the delivery of RNAi Therapeutics to the brain via systemic administration was recently published by the Pardridge group at UCLA in the journal Pharmaceutical Research (Xia et al.: “Intravenous siRNA of Brain Cancer with Receptor Targeting and Avidin-Biotin Technology.”).
The Pardridge group has been working on so called Trojan Horses to get pharmaceutical agents across the blood-brain-barrier. This may be achieved by coupling the active drug ingredient to a monoclonal antibody that targets receptors on the vascular endothelium of the brain which can then ferry the entire complex through the endothelial cell layer and into the brain.
In this study, Xia and colleagues combine streptavidin-antibody fusion proteins to biotinylated siRNAs which form tight complexes mediated by the extremely high affinity of streptavidin for biotin. The antibodies recognize the transferrin and insulin receptors which are the most widely studied receptors in this apparently understudied area of research. After demonstrating that it is possible to generate reasonable amounts of pure Trojan Horses, the authors use them for knock down studies in tissue culture with 75-85% knockdown efficacy which was further dependent on the antibody. Importantly, the biotin-streptavidin interaction did not impair knockdown efficacy and both 5’ and 3’ biotinylation of the passenger siRNA strand was tolerated, in support of the flexibility of this technology.
They then move on to an in vivo tumour model where rat glioma cells expressing a reporter gene (luciferase) are implanted into the brains of rats and grown for 5 days. At this point, the animals were intravenously administered with the Trojan Horses incorporating an siRNA against the reporter gene. Remarkably, at a dosage of as little as 270ug/kg siRNA (this does not include the protein component of the formulation though), luciferase levels stabilized and were 4 fold less compared to animals injected with saline control alone, and no obvious toxicities were observed.
One critical control that I would have liked to see is a Trojan Horse with a control siRNA as the assay did not distinguish whether the relative decline in luciferase activity was the result of actual gene knockdown or due to some cytotoxic effect, so that one could rule out that binding of the monoclonal antibody to transferring receptors on the cancer cells itself was the cause for the reduction in luciferase activity. Another nice experiment would have been to target a cancer-related gene and look at survival and other therapeutic measures. In any case, it will be interesting to follow the progress of this technology which appears to be partnered with the Californian biotech company ArmaGen Technologies.
PS 1: In my 19 June 07 Blog: “New Breakthrough in the Systemic Delivery of RNAi for the Brain” I described a related study published in Nature where a rabies peptide facilitated the transfer of an siRNA across the BBB. In regards to that study, Xia et al. make the following cryptic remark: “The RVG peptide is hypothesized to cross the BBB via receptor-mediated transport on the brain capillary endothelial nicotinic acetylcholine receptor. However, activation of brain microvascular AChR causes BBB disruption.” Although temporarily disrupting the BBB may be a way to get drugs into the brain, it seems to raise safety concerns as the BBB serves to keep bad stuff, such as viruses out of the brain for a reason.
PS 2: Last week, Santaris signed a nice collaboration agreement with GSK for the development of LNA-based antisense drugs valued at up to $700M, further highlighting Big Pharma's interest in RNA-based therapies. While I have much to learn about the safety of LNAs, I have little doubt that they can be quite efficient in binding to their targets and are perhaps particularly attractive for antagonising microRNAs since the simple act of sequestering a microRNA would already be effective. Unfortunately for retail investors, the same week it announced another private funding round for M20 Euros. Santaris should find sufficient interest should it decide to go public.
Showing posts with label transferrin. Show all posts
Showing posts with label transferrin. Show all posts
Monday, December 24, 2007
Friday, October 5, 2007
Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 1
The 3rd Annual Meeting of the Oligonucleotide Society (OTS) got under way in the stylish Langenbeck-Virchow Auditorium situated in the centre of a culturally vibrant Berlin. The OTS brings together the many disciplines in the rapidly expanding field of oligonucleotide-based drug development, ranging from aptamers, ribozymes, antisense, immunostimulatory nucleic acids to RNAi. Reports from clinical studies of these treatment modalities and oligonucleotide chemistries were at the centre of today’s lecture sessions.
Brett Monia from ISIS Pharmaceuticals chaired the first session and set the stage by reporting that while there are only four approved oligo-based therapies, more than 50 are currently in clinical development. Many of these are designed for cancer applications, but infectious, metabolic, and inflammatory disease applications are emerging as major new focus areas. This may not only be due to considerable unmet medical needs, but also because these may offer better success rates for clinical development. A few talks on cancer therapies with ASOs (antisense oligos) documented the challenges of designing smooth clinical trials for cancer therapies. One problem is the heterogeneity of cancers and matching the gene target with the right cancer. Early trials on a given compound typically involve small patient numbers with a wide spectrum of cancer manifestations. Although safety trials, almost anecdotal evidence of biological activity in a few patients then often guide the design of later phases of clinical development. Clearly, for cancer, there is a need for the development of more reliable biomarkers as early indicators of therapeutic efficacy and for patient stratification, which may be something that Rosetta Genomics’ microRNA diagnostics may be able to facilitate.
Focusing on diseases like viral infection and metabolic disease where efficacy can be routinely evaluated in early stages of clinical development (viral titers, LDL-cholesterol, glucose) should therefore be lower hanging fruits for the next wave of oligonucleotide-based therapeutics. This transformation in the industry is illustrated by ISIS’ new focus on metabolic disease and its out-licensing of the more challenging ASO applications. Rosanne Crooke recounted the clinical experience with 301012, ISIS’ lead compound for the treatment of hypercholesterolemia. As I wrote earlier, one major concern with targeting ApoB100 is the risk of steatosis of the liver due to inhibition of a protein required for the export of triglycerides and which has indeed been observed by a number of groups targeting ApoB100 by RNAi. Possibly motivated by the existence of individuals with null mutations for PCSK9, the new target of choice for hypercholesterolemia, with no evidence for adverse effects due to lack of the LDL-receptor processing protein, a conference participants asked about the existence of ApoB100 knockout mice. Interestingly, no such mice exist, because ApoB100 would have additional roles during development.
Despite reports of steatosis due to knockdown of ApoB100 with RNAi, Sirna Therapeutics is yet another biotech company interested in targeting it for lowering cholesterol. In a talk about siRNA delivery to the liver, Barry Polisky, CSO of Sirna Therapeutics/Merck also noted that in Sirna’s experience, there are genes that can be knocked down extremely well (e.g. ApoB100) and some that are relatively refractory to knockdown by RNAi. Although anecdotal, given the extensive experience of Sirna Therapeutics with screening for effective siRNAs by tiling them across entire genes, and similar findings by Qiagen and others, it will be interesting to determine what makes a gene a good target for RNAi, particularly whether it involves its normal regulation by microRNAs.
Polisky further focused on the need for siRNA modifications to make RNAi therapeutics a reality. Currently, Sirna’s standard siRNA design seems to involve si(R)NAs that lack essentially all 2’OH groups, except for the three 5’ nucleotides of the guide strand, while the passenger strand has 5’ and 3’ modifications that avoid it to be loaded into RiSC to reduce off-targeting. Other speakers and posters also noted that since most nucleotide modifications had been developed with antisense therapies in mind, new modifications may exist for optimal RNAi efficacy, particularly in vivo. Indeed, new synthesis strategies are being developed that will allow this to be tested, such as the 6-membered carbohydrate substitutions of ribose reported by Piet Herdewijn from the University of Leuven, Belgium.
Polisky further characterized siRNA delivery to the liver as efficient, albeit not optimal. Progress is clearly illustrated by the fact that >30% of siRNA formulated into liposomal nanoparticles now end up in the liver with first-pass kinetics with no evidence for toxicity in the 1-9mg/kg range (mostly rodent studies), whereas >99% of unformulated siRNAs are rapidly filtered out by the kidney.
The liver is also the organ where the first microRNA-based therapies are likely to be targeted. Like Regulus and Rosetta, I was amazed, or maybe not, at how many groups are currently working at abrogating miR-122 function for treating either hypercholesterolemia or HCV infection. LNAs, owned by the privately held Danish company Santaris, appear to be a particularly promising oligonucleotide class for achieving potent microRNA down-regulation in vivo. Similarly, antisense strategies based on LNA appear to rival siRNA potency in a number of settings, and it will be interesting to learn more about the toxicologies of these compounds, which would certainly be facilitated if Santaris decided to enable to field by allowing better access to their technology. It also highlights the need for specialized chemistries for therapeutic targeting of microRNAs which in my mind is something that Regulus ought to consider.
With regard to delivery outside the liver following systemic administration, PEI-formulated siRNAs (see recent PolyPlus-Alnylam deal) are now being used successfully by a number of groups for lung delivery. Other interesting delivery data was presented by a Portuguese group in a poster on RNAi in the brain. By non-covalently adding transferrin protein to cationic liposome-siRNA particles, they were able to efficiently target neuronal cells both in vitro and in vivo which, similar to cancer cells (see Calando’s cancer cell targeting with transferrin), are studded with transferrin receptors.
PS: A nuisance to most scientists in the field, immunostimulation by oligonucleotides is exploited by Dynavax as an adjuvant for vaccines. Poor adjuvants activity lead to insufficient immune responses and the need for multiple booster injections affecting compliance. HBV vaccination is a case in point, and as a consequence around 50% of patients in the US fail to successfully complete vaccination. Very high HBsAg antibody titers after only two injections of recombinant HBsAg with an immunostimulatory oligo from Dynavax as adjuvant, instead of alum, suggests that they may be able to greatly improve on those numbers.
Brett Monia from ISIS Pharmaceuticals chaired the first session and set the stage by reporting that while there are only four approved oligo-based therapies, more than 50 are currently in clinical development. Many of these are designed for cancer applications, but infectious, metabolic, and inflammatory disease applications are emerging as major new focus areas. This may not only be due to considerable unmet medical needs, but also because these may offer better success rates for clinical development. A few talks on cancer therapies with ASOs (antisense oligos) documented the challenges of designing smooth clinical trials for cancer therapies. One problem is the heterogeneity of cancers and matching the gene target with the right cancer. Early trials on a given compound typically involve small patient numbers with a wide spectrum of cancer manifestations. Although safety trials, almost anecdotal evidence of biological activity in a few patients then often guide the design of later phases of clinical development. Clearly, for cancer, there is a need for the development of more reliable biomarkers as early indicators of therapeutic efficacy and for patient stratification, which may be something that Rosetta Genomics’ microRNA diagnostics may be able to facilitate.
Focusing on diseases like viral infection and metabolic disease where efficacy can be routinely evaluated in early stages of clinical development (viral titers, LDL-cholesterol, glucose) should therefore be lower hanging fruits for the next wave of oligonucleotide-based therapeutics. This transformation in the industry is illustrated by ISIS’ new focus on metabolic disease and its out-licensing of the more challenging ASO applications. Rosanne Crooke recounted the clinical experience with 301012, ISIS’ lead compound for the treatment of hypercholesterolemia. As I wrote earlier, one major concern with targeting ApoB100 is the risk of steatosis of the liver due to inhibition of a protein required for the export of triglycerides and which has indeed been observed by a number of groups targeting ApoB100 by RNAi. Possibly motivated by the existence of individuals with null mutations for PCSK9, the new target of choice for hypercholesterolemia, with no evidence for adverse effects due to lack of the LDL-receptor processing protein, a conference participants asked about the existence of ApoB100 knockout mice. Interestingly, no such mice exist, because ApoB100 would have additional roles during development.
Despite reports of steatosis due to knockdown of ApoB100 with RNAi, Sirna Therapeutics is yet another biotech company interested in targeting it for lowering cholesterol. In a talk about siRNA delivery to the liver, Barry Polisky, CSO of Sirna Therapeutics/Merck also noted that in Sirna’s experience, there are genes that can be knocked down extremely well (e.g. ApoB100) and some that are relatively refractory to knockdown by RNAi. Although anecdotal, given the extensive experience of Sirna Therapeutics with screening for effective siRNAs by tiling them across entire genes, and similar findings by Qiagen and others, it will be interesting to determine what makes a gene a good target for RNAi, particularly whether it involves its normal regulation by microRNAs.
Polisky further focused on the need for siRNA modifications to make RNAi therapeutics a reality. Currently, Sirna’s standard siRNA design seems to involve si(R)NAs that lack essentially all 2’OH groups, except for the three 5’ nucleotides of the guide strand, while the passenger strand has 5’ and 3’ modifications that avoid it to be loaded into RiSC to reduce off-targeting. Other speakers and posters also noted that since most nucleotide modifications had been developed with antisense therapies in mind, new modifications may exist for optimal RNAi efficacy, particularly in vivo. Indeed, new synthesis strategies are being developed that will allow this to be tested, such as the 6-membered carbohydrate substitutions of ribose reported by Piet Herdewijn from the University of Leuven, Belgium.
Polisky further characterized siRNA delivery to the liver as efficient, albeit not optimal. Progress is clearly illustrated by the fact that >30% of siRNA formulated into liposomal nanoparticles now end up in the liver with first-pass kinetics with no evidence for toxicity in the 1-9mg/kg range (mostly rodent studies), whereas >99% of unformulated siRNAs are rapidly filtered out by the kidney.
The liver is also the organ where the first microRNA-based therapies are likely to be targeted. Like Regulus and Rosetta, I was amazed, or maybe not, at how many groups are currently working at abrogating miR-122 function for treating either hypercholesterolemia or HCV infection. LNAs, owned by the privately held Danish company Santaris, appear to be a particularly promising oligonucleotide class for achieving potent microRNA down-regulation in vivo. Similarly, antisense strategies based on LNA appear to rival siRNA potency in a number of settings, and it will be interesting to learn more about the toxicologies of these compounds, which would certainly be facilitated if Santaris decided to enable to field by allowing better access to their technology. It also highlights the need for specialized chemistries for therapeutic targeting of microRNAs which in my mind is something that Regulus ought to consider.
With regard to delivery outside the liver following systemic administration, PEI-formulated siRNAs (see recent PolyPlus-Alnylam deal) are now being used successfully by a number of groups for lung delivery. Other interesting delivery data was presented by a Portuguese group in a poster on RNAi in the brain. By non-covalently adding transferrin protein to cationic liposome-siRNA particles, they were able to efficiently target neuronal cells both in vitro and in vivo which, similar to cancer cells (see Calando’s cancer cell targeting with transferrin), are studded with transferrin receptors.
PS: A nuisance to most scientists in the field, immunostimulation by oligonucleotides is exploited by Dynavax as an adjuvant for vaccines. Poor adjuvants activity lead to insufficient immune responses and the need for multiple booster injections affecting compliance. HBV vaccination is a case in point, and as a consequence around 50% of patients in the US fail to successfully complete vaccination. Very high HBsAg antibody titers after only two injections of recombinant HBsAg with an immunostimulatory oligo from Dynavax as adjuvant, instead of alum, suggests that they may be able to greatly improve on those numbers.
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.
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