An Interview with Dr. Joop Gäken | Biowire Fall 2011


 

Biowire Fall 2011 — Screening — microRNA Target Identification

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In this issue of Biowire, our thought leader is Dr. Joop Gäken, whose work has not only paved the way for new cancer treatment strategies, but has also generated a powerful new tool for the study of microRNAs (miRNAs).

Dr. Joop Gäken
Division of Cancer Studies, King’s College London

“This [3'UTR-enriched cDNA library] allowed individual targets from the vector library to be transduced into cells for screening, enabling identification of target genes that are strongly regulated by the miRNA of interest.”

 

The role of miRNAs in many cancers is well established, with several miRNAs known to function as oncogenes or tumor suppressor genes; however, the clinically relevant targets of these biomolecules are largely unknown. In a career already spanning over 20 years in Hemato-Oncology, Dr. Joop Gäken from the Division of Cancer Studies at King’s College London has investigated a wide range of gene therapy strategies to help patients with leukemia. His group’s work has generated a number of novel technologies, including new therapeutic approaches, and the benefits of these innovations are now being felt outside of the oncology field.

A molecular biologist by training, Dr. Gäken has been based at King’s College London since completing his Ph.D., but his fascination with what makes the world tick started much earlier. Dr. Gäken explained:

Dr. Joop Gäken:
As a young boy I was always interested in how things worked. I was the kind of child that would take apart an alarm clock just to see how it worked. Of course, once the spring had jumped out, I couldn’t usually put them back together again! I also used to visit the forests around my home in the Netherlands a lot with my parents and, as I was fascinated by nature, I decided to pursue a career in biology. The first year of my university degree was very broad — I studied everything from botany to zoology — but in the second year I had my first experiences with molecular biology, and knew this was the path for me. When I finished my undergraduate degree, I continued on to earn my Ph.D. and came to London to complete my studies. I only intended to stay for six months, but 23 years later I’m still here!

Already working in the Hemato-Oncology field, Dr. Gäken became involved in the investigation of gene therapy as a possible treatment for various cancers in the early 1990s:

Dr. Joop Gäken:
Around that time, an important paper was published demonstrating that the introduction and expression of the T-cell co-stimulator (B7-1) in tumor cells resulted in a strong reaction to these cells by the body’s natural immune response mechanisms. Using the expertise in our laboratory at the time, we began to look at ways of further increasing this immune response as a means of helping the body to naturally recognize and destroy cancer cells, specifically looking at acute myeloid leukemia (AML).

The strategy we developed required co-expression of the B7-1 co-stimulator with a T-cell-activating cytokine, such as interleukin-2 (IL-2). IL-2 would then be secreted by the cells, creating an extracellular environment rich in T-cell activators and augmenting the immune response caused by the co-stimulator. However, the inefficiency of retroviral vectors available at the time made this unworkable as a therapeutic strategy, as it would be virtually impossible to create a sufficient number of cells which co-expressed both transgenes. In an effort to overcome this problem we created a fusion protein of B7-1 and IL-2 with a furin endoprotease recognition site in between. This furin site enabled cleavage of the fusion protein in the Golgi and correct expression of B7-1 on the cell membrane and secretion of IL-2.

First published in 2000, this elegant piece of molecular engineering is known as a fusagene technique, and use of this strategy to target cancerous cells in patients has just entered into clinical trials for the first time:

Dr. Joop Gäken:
Although work on this project moved onto a more clinically focused team following our proof of concept work, it is very satisfying that this has now reached the clinical phase for AML patients. Tumor cells are isolated from the patients’ blood and transduced with a lentiviral vector containing the B7-1/IL-2 fusagene. These cells are then irradiated to prevent replication, and injected under the skin of the patient. T-cell activators such as IL-2 are still expressed for at least a week after the cells are irradiated, and so the presence of these cells should induce an immune response which will also target non-treated AML cells in circulation. This strategy avoids the potential risks associated with insertional mutagenesis, as irradiation of the lentiviral infected cells prevents cell division and viral replication, and are therefore used to develop an in vitro “template” for the patient’s own immune system, rather than live viral vectors being introduced into the body.

Following the success of the fusagene work, Dr. Gäken’s attention turned to miRNAs and their role in the pathogenesis of various types of leukemia:

Dr. Joop Gäken:
We work primarily on forms of pre-leukemia called myelodysplastic syndromes (MDS), which are characterized by various chromosomal aberrations, including 5q minus, monosomy 7 and trisomy 8. Due to the abnormal expression patterns associated with these mutations, miRNAs play a significant role in the disease progression, functioning as either oncogenes or tumor suppressor genes, although the target genes are unknown in the majority of cases. Identification of the targets of these miRNAs is therefore an important step in furthering our understanding of the genetic origins of MDS. Currently, identification of miRNA targets is laborious and inefficient, relying on computer algorithms and subsequent validation by in vitro assays, which produce many false positive results. It also has the potential to miss targets, due to the promiscuous nature of miRNA binding, and so we looked at ways of creating a functional assay that could look at the effect of individual miRNAs in a cellular system.

Using the expertise in vector construction that we had developed during the fusagene work, we began by creating a vector with a fusion of positive and negative selectable markers. We then constructed a 3'UTR-enriched cDNA library which was cloned downstream of the selectable marker fusion. This allowed individual targets from the vector library to be transduced into cells for screening, enabling identification of target genes that are strongly regulated by the miRNA of interest.

We quickly realized that this approach had widespread miRNA screening applications extending beyond our own research, as well as for biopharmaceutical companies developing small interfering RNAs (siRNAs) as novel drugs, and so began looking for a commercial partner to help us further develop this technology. Sigma® Life Science’s broad expertise in genomics made it an obvious choice, and the Target ID Library was the result. This transcriptome-wide plasmid library has been constructed from 10 different human tissues and eight human cell lines, providing one of the most comprehensive cDNA libraries available today. We have already used Target ID to create a library of over 200,000 independent clones using MCF7 breast cancer cells, and are currently using this cellular library to identify the targets of several miRNAs known to play a role in 5q minus syndrome, with the aim of elucidating important gene regulation events in vivo.

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