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New frontiers in drug discovery

Published on 11/09/12 at 04:44pm
The RNAi therapeutic mechnism
The RNAi therapeutic mechnism

Despite the success of small molecule and biologic drugs such as monoclonal antibodies, there remain many areas of unmet medical need, and thus the need for innovative approaches to drug discovery and drug delivery.

A number of new technology platforms are promising to provide new ways through some long-standing obstacles. Among these, RNAi and therapeutic vaccines are among the most high profile, and have generated a number of products which have overcome technical challenges and are well on the road to commercialisation.

Advances in RNAi delivery

In the field of RNA interference, where small oligonucleotides are used as drugs and work by regulating gene function, delivery has long been the major challenge. SMI’s RNAi & Nanotechnology conference held in London recently presented some examples of RNAi delivery that are approaching the moment of truth.

Companies in the field are essentially choosing between one of three broad approaches to the delivery conundrum - ‘naked’ delivery (no delivery vehicle), viral delivery and non-viral (lipid or polymer vehicle) delivery.

Santaris Pharma is pursuing a ‘naked’ delivery strategy. Troels Koch, vice president and chief technical officer at Santaris spoke about its process called gymnosis which could be an elegant solution to the problem. This uses locked nucleic acids (LNAs) in their sequence in place of standard nucleic acids (the process has been developed in collaboration with Cy Stein, Professor, City of Hope Medical Centre).

The appeal of this approach is that it could provide long-lasting gene silencing with fewer toxic effects and more target specific delivery than other RNAi delivery technologies. Most cell lines will take up these ‘naked’ molecules, including hard to transfect primary cells and suspension cell lines. “When LNAs are incorporated into an oligonucleotide, it induces a high affinity for RNA in the molecule,” said Koch.

LNAs also confer nuclease resistance, and therefore stability, to an oligonucleotide. They allow the use of shorter oligonucleotides which have the same, or higher, potency as those made with conventional monomers.

So, typically a 12-16mer with LNA can bind an mRNA target and a 8-15mer will bind to a microRNA (miRNA). “LNA will nearly always give a potency advantage and shorter oligos are often more potent than longer ones,” said Koch.

Santaris has used the liver-specific microRNA miR-122, which is involved in fatty acid and cholesterol metabolism, as a target to get proof of concept for their LNA platform. miR-122 is also a host factor which is essential for Hepatitis C virus (HCV) replication, and is required by all HCV genotypes.

Santaris’ lead compound miravirsen is the first microRNA inhibitor in man, and provides a unique mode of action against the HCV virus. The molecule is a LNA-modified oligonucleotide ( LNA-AntimiR) which binds to miR-122 and is in Phase II clinical trials for treatment of HCV infection.

The compound forms a duplex in a region on the HCV genome where there are two matches for miR-122. “Affinity is important but it is not the only factor that makes this drug such an efficient miR-122 inhibitor,” said Koch. Miravirsen also has high biostability and good pharmacokinetic properties.

HCV affects 170 million patients worldwide and is probably underdiagnosed, as the virus may remain silent for many years. The firstline treatment has for many years been based on PEG inter-feron(INF)/ribavirin(RBV), a combination that is poorly tolerated and only produces a sustained viral response of 40-50 per cent.

The protease inhibitors boceprevir and telaprevir have recently been approved for use with INF/RBV therapy, and that has improved the sustained viral response rates. However, several direct acting agents are presently in clinical trials aiming for providing better tolerated and more efficient HCV therapies. Future HCV therapies are likely to be based on combinations of agents with distinct modes of action.

Two Phase I clinical trials have now been completed and the drug was well tolerated, as well as decreasing cholesterol. It was demonstrated in a Phase IIa trial - conducted in HCV infected individuals - that HCV RNA titres were dose dependently declining over time. In these patients, blood serum levels of liver enzyme levels went down, indicating a marked improvement in liver health.

Six out of nine patients were good responders, and in four out of nine patients, levels of HCV went undetectable. This is a remarkable result considering it was a single agent dosing regimen over just four weeks. “We think this has potential as a pan-genotype HCV drug alone or in combination with others,” Koch said.

“This is a first in class drug and clinical proof of concept for our LNA platform.”

Dynamic polyconjugates

David Lewis, vice president and site head, Arrowhead Research spoke about DPC (dynamic polyconjugates), a targeted polymer-based siRNA delivery platform.

The company was acquired by Roche in 2008 and then became independent once again in October 2011 (acquiring the Roche IP) when Roche exited the RNAi field. Arrowhead has carried out a number of experiments on DPCs for delivering siRNA into hepatocytes, noting that NAG (N-acetyl galactosamine) is a particularly good ligand for hepatocytes.

Since the first generation of DPCs were developed, the company has made a vast library of them. The blood clotting protein, Factor VII, has proved to be a good target because, on knocking down the gene with siRNA, there is an increase in clotting time which is readily measurable. This kind of drug could be developed as an antithrombotic.

The target gene knockdown with DPC requires liver-tropic siRNA and a hepatocyte-targeted polymer like NAG. Together they give very effective knockdown. “We don't know exactly how they both get into the cells but it works, that’s the important part,” noted Lewis.

The disease target for DPC technology is hepatitis B virus (HBV) infection, which affects 300 million worldwide and causes more than 500,000 deaths per year, because it often leads to hepatocellular carcinoma (HCC). Therefore HBV represents a high unmet medical need.

The current PEGASYS treatment is not very effective and has side effects. But siRNA can target HBV transcripts in hepatocytes and thereby reduce replication. “This is a novel approach to HBV therapeutics,” Lewis commented. A clinical lead formulation is now being identified and manufactured for Phase I trial in Asia beginning in 2013.

Nanoparticle delivery

Michael Keller, siRNA project leader at Novartis Pharma, spoke about work the company has done on chitosan/siRNA nanoparticles. Chitosan, a linear polysaccharide, is the second most abundant biopolymer on earth and already has a number of commercial applications including water filtration, drug delivery (insulin) and medical dressings.

There are various sources of chitosan available for siRNA work, many of them derived from crustacean shells.

The Novartis team uses a chitosan from Kitozyme, sourced from Agaricus bisporus (common mushroom). They have created various polyplexes between this chitosan and siRNA, capitalising on a strong interaction between the two. These have then been evaluated in various cell types. Selection work also showed that a low N:P ratio in the chitosan was important in determining its activity while exhibiting minimal toxicity.

“There is faster uptake of the smaller chitosans, which is one of the reasons why they are more active,” Keller noted. Preliminary in vivo work involving oral delivery in mice showed that siRNA accumulates outside the stomach and is delivered systemically.

“Therefore there might be potential interest in oral administration targeting the liver,” said Keller. These preliminary experiments also revealed that the siRNA-chitosan polyplexes can downregulate endogenous genes in the GI tract.


One of the key players in the RNAi field is Alnylam. Since it was founded in 2002 the company has been on a rollercoaster ride, strong rises in its share prices being followed by frightening plunges as investors react to the changing fortunes of the firm and the wider cutting-edge field.

The latest news has been positive - Alnylam received a milestone payment in July from GSK as part of an ongoing collaboration based on Alnylam’s VAXiRNA technology to develop a cell-culture based influenza vaccine. VAXiRNA uses RNAi to silence genes that limit or prevent production of vaccine antigens, and may help GSK in its bid to manufacture an influenza vaccine in cells to meet potential demand from pandemic influenza. The milestone will help support Alnylam’s ‘5x15’ initiative, which has the aim of developing RNAi therapies for five unmet medical needs by 2015.

The vital importance of the drug delivery technology has been underlined by a bitter dispute between Alynlam and a partner company. The company is currently being sued by Tekmira, which claims Alnylam has misappropriated Tekmira’s delivery technology, alleging that Alnylam has attempted to copy Tekmira’s methods and even sought to patent some of the techniques.

Therapeutic vaccines

A therapeutic vaccine differs from a preventive vaccine in that it aims to treat a disease in progress rather than stopping it from occurring.

A number of platform technologies are being applied to the generation of therapeutic vaccines, chiefly in the field of oncology. Cancer cells are tagged with specific antigens and the immune system should be capable of destroying them using similar mechanisms to those used against invading pathogens.

But established tumours have developed ways of evading the immune response. A therapeutic vaccine aims to restore the immune response against cancer and has the added bonus of having fewer toxic side effects than conventional cancer treatments.

The very first such therapeutic cancer vaccine is Dendreon’s Provenge, a treatment for hormone refactory prostate cancer which was launched in 2010. The treatment works by by extracting dendritic cells from a patient, loading them with the antigens, and reintroducing them to the patient’s body to trigger an immune response.

However, the drug’s limited benefits above and beyond other treatments, its very high cost and difficulty of production have all contributed it being a commercial disappointment. Nevertheless, numerous other theraepeutic vaccine candidates are now in clinical trials.


Among the most promising therapeutic vaccine candidates is ImmunoCellular Therapeutics’ ICT-107 for glioblastoma multiforme (GBM), an aggressive brain tumour.

In a Phase I clinical study of ICT-107 in GBM, 16 newly diagnosed patients who received the vaccine in addition to standard of care treatment of surgery, radiation and chemotherapy demonstrated a two-year overall survival of 80% and a three-year overall survival of 55%, a significant advance on the current 26% two-year overall survival and 16% three-year overall survival based on the historical standard of care treatment alone.

Updated data from the trial shows overall survival (OS) of 50% after four years, and 38% of the trial patients are progression free (PFS) for 48-66 months; suggesting the treatment could be a major breakthrough.

Vaxil BioTherapeutics is developing therapeutic vaccines for cancer and infection based on its technology VaxHit.

This identifies the part of a target antigen which will give a strong immune response. ImMucin, the company’s lead therapeutic vaccine product contains a peptide sequence from the MUCI antigen, which is expressed by over 90% of cancers including lung, breast, colorectal, prostate, kidney and pancreatic.

The vaccine works by activating CD4 and CD8 T-cells which then destroy cells expressing the MUC1 biomarker.

Vaxil began a Phase I/II trial of ImMucin in multiple myeloma and an interim analysis on seven patients suggested a good safety profile, a strong immune response and even a complete response in three of the patients.

Cervical, liver cancer

Advaxis’ ADXS-HPV vaccine against cervical cancer was selected as Best Therapeutic Vaccine in the 5th Annual Vaccine Industry Excellence Awards (sponsored by Novartis).

Preliminary data from a Phase II trial of ADXS-HPV, carried out in India with women with recurrent cervical cancer, showed survival at six, nine and 12 months as 64%, 46% and 29% (compared with 0-22% 12-month survival with conventional therapy).

The vaccine targets cells expressing HPV (human papillomavirus) E7 - a gene which transforms cells infected with the virus into a malignant state. It directs antigen-presenting cells to generate a powerful immune response against the HPV infected cells.

Another cervical cancer vaccine is Inovio’s VGX-3100, a DNA vaccine now in Phase II. This is similar to a gene therapy, inserting a piece of DNA which codes for an antigenic protein found in HPV strains into patient’s cells. This causes production of the antigenic protein which triggers the appropriate immune response against the target cancer cells.

Jennerex has recently treated the first patient with liver cancer in their Phase II trial of JX-594. This vaccine is particularly interesting because it works by three different mechanisms. Not only does it cause the lysis of cancer cells, it also shuts down the blood supply to the tumour and, finally, it stimulates an immune response against cancer cells.

JX-594 is the lead product from the Jennerex proprietary SOLVE (Selective Oncolytic Vaccinia Engineering) platform.   It uses pox viruses potential to be engineered to target and destroy solid tumours both systemically and locally.

The pox virus strain used in JX-594 naturally targets cancer cells because of genetic abnormalities in these cells and has been genetically engineered to enhance this response. Liver cancer is a high unmet clinical need because it is the fifth most common cancer, and the third leading cause of cancer death, worldwide, with over 600,000 new cases diagnosed each year.

Chimeric antigen receptors

Meanwhile, Novartis and the University of Pennsylvania recently announced a new collaboration to develop new immunotherapies.

The pharma firm and the academics are to develop chimeric antigen receptor (CAR) immunotherapies and will also set up a new R&D facility, the Center for Advanced Cellular Therapies (CACT), on the Penn campus.

CAR technology takes T cells from a patient’s blood, re-codes them to identify cells expressing proteins on tumours and re-introduces them to bind to - and destroy - those targeted cancer cells.

It is certainly early days but two patients with advanced chronic lymphocytic leukemia (CLL) were in remission for more than a year after a pilot study with Penn’s first CAR investigational therapy, CART-19.

Therapeutic vaccines and immunotherapies, like RNA interference, are sure to yield more drugs which will help advance treatment, but the cutting-edge nature of both fields means that the pace of that progress - and the cost to R&D companies - is unclear, and will remain an unpredicatable path.

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