David Evans, Head of Drug Discovery, Cancer Drug Development Laboratory, The Translational Genomics Research Institute (TGen)

Date Posted: Monday, January 03, 2005

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Can you give us some background to the Cancer Drug Development Lab (CDDL)?

We are a division of TGen - a non-profit biomedical research organization focused on improving human health. The CDDL (Gaithersburg, MD) team includes a multidisciplinary group of scientists focused on developing and applying new technologies to accelerate and improve cancer drug discovery and development.

My specific focus is in the discovery of new therapeutics for cancer. We are using both the siRNA and a small molecule screening approach to be able to identify new targets that, when knocked down by the siRNA improve the response to existing therapeutics or are novel targets for therapeutic development and intervention.

You use a chemo-genomics approach. Can you tell us about this?

We have found global RNAi phenotype profiling to be a very powerful approach for finding context-dependent gene targets that represent vulnerable points for therapeutic intervention. One specific cellular context that we have been able to model in vitro is that of therapeutic response. When a cell is exposed to a drug or chemical agent, the cell response is determined by the status of key genes functionally involved in regulating that response. By systematically knocking down individual genes in parallel, we can empirically identify the genes in the genome that are causally involved in controlling or regulating the phenotype following chemical exposure (the chemotype). We call this 'functional chemo-genomics'.

We are applying this approach in pharmacogenomics to discover genes which improve response to common cancer chemotherapy as well as to emerging targeted therapies. The products of this research have three distinct utilities. The first is that these genes give us a better understanding about the molecular mechanisms that regulate drug response. The second is that these genes and/or their products may be candidates as biomarkers that can be used to measure the state of a key response-determining gene and predict patients that will either benefit from a specific drug, or fail to respond to a competing therapeutic regimen.

Third, these are putative drug targets that can be used as a starting point for the development of combination chemotherapies. For example, we have discovered genes that, when knocked down, increase the sensitivity of breast cancer cells to certain types of chemotherapy, like doxorubicin. Small molecule inhibitors targeting these gene products are currently being tested in combinations to evaluate the extent to which they can improve the response of cancer cells to co-treatment with doxorubicin.

What kind of platform do you use?

The technologies and strategies used to discover contextual vulnerabilities with global RNAi profiling include a fully automated platform for cell based screening integrated with a variety of customized technologies, unique expertise, and very extensive genomic and RNAi resources. As a non-profit research organization, all of our technologies, expertise, and discoveries are fully shared with the research community through various publications and presentations at scientific meetings.

We have spent a significant amount of time, effort and expense to obtain the resources and infrastructure necessary for our goals, but, by far the most challenging aspect was building the expertise required to conduct global RNAi profiling - especially on the assay development side.

It was worth the effort because we are now generating very robust and reproducible RNAi data from multiple model cell lines in various assays run in high throughput (currently over 23,000 transfections per day and a capacity with existing infrastructure of over 70,000) - something that was a dream when we started 2-3 years ago.

What would you say are the benefits of using siRNA?

When compared to competing technologies, we find siRNA to be more potent, and more specific for knocking down the expression of individual genes. The biggest benefit is the scalability to high throughput yet still having a functional readout. With siRNA you are functionally knocking out one gene and you can look at the relevance of that gene on phenotypic endpoints in cancer cells e.g. survival. So you get a direct relationship between the gene knockdown and the effect on a cell. It is a very powerful technology. Right now we are using it both for target discovery, and validation of therapeutic potential.

How do you go about managing off-target effects?

First, let me start off by saying that we find that off-target effects are not a major problem if certain precautions are taken in the experimental methods. To minimize the off-target silencing of genes, we have optimized our assays to use low nanomolar concentrations of siRNA, we use careful design of the siRNA, and optimize cell growth and assay conditions such as the amount and type of transfection agent, mode of administration, etc...

Our SOP's for library design and high throughput screening are now optimized to maximize potency and minimize non-specific and off target effects, but once in a while, we still observe gene silencing with one siRNA which we cannot reproduce with other siRNAs against the targeted gene. Although this is rare, it nevertheless highlights the need for stringent validation of critical hits in the screening. We typically like to see at least two siRNA targeting the same gene exhibiting the same phenotype before we begin to rule out off-target effects.

We (and others) are working towards developing better siRNA design that will minimize off target effects even further. For example, our synthetic RNAi libraries produced by Qiagen are designed using advanced bioinformatics tools to minimize potential off target effects and the individual siRNA species are QC’d at Qiagen using LC/MS to validate the purity and sequence. We are also exploring modifications to the siRNA towards strand selection and further reduction of off-target effects.

What is your background? How did you first get interested in this field?

I have always had an interest in the marriage of genomics and chemical (small molecule discovery) approaches. My background is in assay development, high throughput screening automation and drug discovery at various biotech companies (both large and small) where I have built the infrastructure necessary for running high throughput small molecule screening campaigns, and applied genomics approaches (microarray and other gene expression technologies) to the drug discovery process in ways that extract the greatest value in the data generated. So this was an excellent marriage between using high throughput automation and being able to use a very powerful genomics approach (siRNA knock down of individual genes) to understand more about the gene products that could act as points of therapeutic intervention in disease.

I came at this issue from the industrial side, so I am applying a lot of the processes that are typically used in industry in an academic environment. It is rather a unique opportunity to industrialize a process that can revolutionize our understanding of disease mechanisms. We can now evaluate the effect of each gene in the genome in a disease cell model in combination with co-treatment with a known therapeutic in replicate experiments in a week!

What does the future hold for TGen and where do you see it in 5 years time?

In a matter of only two years, TGen has already become a world class research institute and has attracted a multidisciplinary and distinguished faculty of world class scientists and clinicians, but it is only the beginning and we are expanding rapidly. TGen has a number of programs focused on advancing human health through translating genomic research into the clinic.

Our own program is making great progress in the discovery and validation of novel drug targets and development of agents with unique mechanisms of action that we hope to translate into therapeutic opportunities. Our goal is that in 5 years we will have therapeutics either in -or close to- the clinic for many life threatening diseases. TGen has the infrastructure and expertise to be able to take therapeutic moieties all the way through to clinical trials. Towards that end, the stream of research products and new agents coming out of CDDL will be advanced into preclinical and clinical development by a very experienced team that includes Richard Love (COO, formerly with ILEX oncology) and Daniel von Hoff (our VP of Clinical Development).

The unique thing about our approach is that the clinical development and the early basic research for discovery are intimately linked, providing the setting for the development of agents that are personalized for diseases of specific molecular make-up. This is the basis of personalized medicine and it starts with agents targeting defined cellular contexts, and ends with innovative clinical trials where the right patients get the right drugs.

In addition, we are also working with several Biotechnology and Pharmaceutical companies on various projects related to functional pharmacogenomics, target discovery and validation and we hope that these productive collaborations will help improve their efforts to advance emerging drugs by providing the right molecular tools for rapid and successful clinical development. In addition, such partnerships will also provide opportunities to advance new therapeutics arising from TGen’s internal research efforts. We envision TGen as a pioneering institute providing the much needed bridge between basic research in genomics and medical breakthroughs that actually improve and extend the lives of patients.

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