Posted by: Adam Kay | December 29, 2012

Sleeping Beauty awakens. Seminar reflections by UST Biology’s Kerri Carlson and Ryan Augustin

Below are posts from UST Biology’s Kerri Carlson and one of her students, Ryan Augustin. They are reflections on a departmental seminar from this semester given by David Largaespada from the University of Minnesota Cancer Center.

Dr. Carslon’s reflections

Kerri CarlssonIt has always amazed me how scientists are able to harness the power of naturally occurring phenomenon to develop novel tools to enhance our understanding of genetics.   The use of plasmids and restriction enzymes revolutionized the way we study genes through the advent of gene cloning.  An understanding of homologous recombination led to gene knock out technologies in mice.  In Dr. Largespada’s seminar we were presented with another example: the use of a transposon system called Sleeping Beauty to identify novel cancer genes.  (Transposons are DNA elements that are capable of “jumping” from one DNA location to another DNA location in the cell of an organism-and in some cases cause mutations).

Most of us have likely felt the impact of cancer in our lives. Cancer is a genetic disease and historically, forward genetic screens (in which a researcher causes mutations in a mouse, looks for tumor development and then works to identify the gene mutations that led to that tumor) have moved slowly. This is because forward genetic approaches are long, labor-intensive tasks.  As a geneticist who has been involved in these types of studies (although with heart disease not cancer) I can speak from personal experience that this can be painful and frustrating! The Sleeping Beauty System for causing mutations has made the process of identifying cancer genes significantly quicker and more efficient.  In addition, each cancer type is characterized by a unique group of important genes.  The Sleeping Beauty system has allowed researchers to generate genetic profiles for many cancer types (including cancers that have previously been recalcitrant to studies in mouse models). While there is still a lot of labor and cost involved in using this tool, the advantages over past approaches are significant.  After listening and talking to Dr. Largespada, I can’ t help but feel hopeful that his efforts will one day soon lead to new patient specific therapies for cancer.

While not the focus of his talk, Dr. Largespada also touched upon the use of the Sleeping Beauty Transposon system for gene therapy.  Gene therapy involves correcting defective genes that cause genetic diseases.  As a human geneticist I have interacted with many people that suffer from debilitating genetic diseases, and I have been hearing about the potential of gene therapy for many years.  Sadly success in this field has been a long time coming and many initially enthusiastic researchers have been disheartened.   There are many factors that have prevented progress in this field.  For example many gene therapy researchers rely on viruses to deliver genetic material to patient’s cells.  These approaches are hampered by the risks of immune responses from the patients, a size limit in the DNA material that can be delivered, and concerns that the inactive virus will become active and cause disease.  The Sleeping Beauty system, while not without risks of its own, overcomes many of the concerns that exist with viral gene delivery systems.  After the seminar Dr. Largespada mentioned that (although not yet published) the first human gene therapy trial with a Sleeping Beauty system has shown some success!  This intrigued me.   I am looking forward to reading the published results.

Overall, I left Dr. Largespada’s seminar with a sense of amazement over the progress they have made in only a few short years towards understanding the genetics of different cancers, an appreciation for the power of the Sleeping Beauty Transposon System as a genetic tool, and curiosity (and perhaps cautious optimism) to see how Sleeping Beauty will fit into the gene therapy field.

Mr. Augustin’s reflections

Sleeping Beauty is not often linked with mice, genes, and cancer; but Dr. Laragaespada’s seminar revealed a new approach regarding forward genetics that takes advantage of a specific transposable element called Sleeping Beauty (SB). While some of his talk was beyond my level of understanding, many of the techniques and issues that were brought up provided unique insight into the topics discussed in our St. Thomas genetics course—including forward genetic screens using random mutagenesis, the creation of transgenic mice using deletion vectors and homologous recombination, the identification and amplification of mutated genes using PCR, the quantitative study of gene expression using RT-PCR, and the important use of bioinformatics for studying unknown nucleotide or amino acid sequences and their relation to entire genomic systems.

In his talk, Dr. Laragaespada introduced us to the “old” approach of studying mutations linked to cancer—randomly mutating mice DNA in order to induce tumor growth and subsequently working to find the causative mutation—but he pointed out the difficulty in this approach since the genomes of mice and humans are naturally polymorphic, displaying thousands of dissimilar sequences. When specifically studying cancer, the causative mutation may not be easily detected. Ironically, the “new” approach for studying genetic mutations—using whole genome sequences and bioinformatics software—still leads to the same problem. Even though scientists are realistically capable of attaining entire genomic sequences of cancer patients (or test organisms), the specific mutation linked to oncogenesis is difficult to pinpoint.

For these reasons, Dr. Laragaespada’s research team is taking advantage of a technology that has its roots in the naturally occurring genomes of almost every organism—transposons. Transposons are genetic elements that literally “hop” around the genome, cutting and pasting themselves to and from different sequences. Currently, specific roles for these elements are not known; however, some geneticists are manipulating this random “cut and paste” mechanism to study cancer—specifically cancer linked to mutations that can be mimicked through transposon insertion.

Sleeping Beauty is one of these manipulated, synthetic transposon elements that has been useful for three reasons: it has provided an all purpose mutagenesis system in mouse somatic cells, it is able to locate both oncogenes and tumor suppressor genes, and it can work for most types of cancer. The beauty of this system (no pun intended) is that the transposon’s inherent ability to mutate cancer causing genes can now be linked to an actual location within the mouse genome. Using PCR, the junction between the Sleeping Beauty transposon and genomic DNA can be amplified and identified from tumor cells of mice. A question remains, though, as to how this insertional mutation can be linked to cancer formation. Laragaespada’s lab obtains hundreds of thousands of these amplified sequences and looks for repeatable patterns from mice possessing similar cancers. Using this method, his research team can identify 60-70 “cancer genes” for every 100 tumors analyzed—which, in my opinion, is an amazing feat.

Laragaespada has implemented a tissue specific, insertional mutagenic system for studying cancer causing genes in mice; but this feat still begs the question: Is any of this relevant towards the study of human cancer? Even after acknowledging the present benefits of sequencing technology and the important link between epigenetics and cancer, our speaker provided much hope for the relevance of transposon technology towards the study of human cancer. First, sequencing technology cannot detect subtle promoter differences or rare subsets of human cancers that are evident with Sleeping Beauty. Second, by using mice for these studies, correlations may be discovered between mutated genes and the phenotypes of certain tumors. And finally, this transposon system has provided a large number of candidate genes with known mutations causing either a gain or loss function—important implications for the study of cancer formation.

Is Sleeping Beauty a perfect model for studying human cancer? No. Does it provide valuable information and a promising future? Absolutely. By comparing Laragaespada’s mouse model (along with the list of cancer-linked insertion sites) to the accumulating data from human cancers, future research hopes to find recurrent and causative links between mutations and human oncogenesis. From here, functional assays (e.g. knock-out studies, TALEN technology, and other approaches studied in our genetics course) will hopefully narrow down the most important cancer-causing genes, and also help identify therapeutic steps to help treat and manage mankind’s most elusive disease.

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