With the help of a moth and modern molecular manipulation, researchers are looking at more effective ways to fix genetic flaws
Some of the most debilitating diseases are caused by genetic flaws. Scientists have traced muscular dystrophy, lupus and many other chronic diseases, including numerous types of cancer, back to flaws in human DNA. As researchers have gained a more and more detailed understanding of the human genome, the prospect of fixing genetic flaws that can lead to serious diseases in humans has grown more tantalizing.
Until recently, gene-therapy research focused on harnessing the power of viruses to efficiently insert their own DNA into a host cell’s genome. The virus DNA then becomes part of the host organism’s genome, passed down through subsequent generations. Researchers attached beneficial genes to viruses and injected them into target organisms in the hope that viruses would insert the beneficial gene into the genome, replacing the flawed gene. This method, they theorized, could cure some genetic disorders by replacing flawed DNA with normal DNA.
The virus-insertion technique has proved difficult to manage and manipulate. Scientists have been unable to control where precisely on the human genome viruses insert the beneficial genes. Random insertion has made it very hard for scientists to predict the impact of the gene therapy on surrounding DNA or on the test organism. In three human test cases, the viral DNA insertion deactivated key cancer-repression genes, causing leukemia. In another case, the virus itself caused an extremely strong immune response which killed the patient. The gene insertions, for the most part, created only temporary changes in the cell genome. Due to these problems, gene-therapy testing on human patients has largely halted.
A team of scientists, including University of Hawai‘i researcher Stefan Moisyadi, is developing what could prove to be a safer alternative to the viral-insertion technique. Moisyadi and colleagues from the Medical College of Georgia, Texas A&M, the United States Department of Agriculture, and the University of Zurich studied the DNA insertion efficiency of four known transposons. Also called “jumping genes,” transposons spontaneously change their position on the strands of DNA that make up an organism’s genome. Biologist Barbara McClintock identified the first jumping gene in the 1950s when she noticed that two generations of corn with the same DNA had different kernel colors.
McClintock later won a Nobel Prize for this discovery and scientists located transposons in many other organisms. Entomologists have successfully harnessed insect transposons to insert DNA payloads into other insects, but never into mammalian gene lines. Most of the research to date with mammalian cell insertion focused on a fish transposon called “Sleeping Beauty.” Moisyadi and his colleagues tested four types of transposons from different organisms – including Sleeping Beauty and a transposon, known as “piggyBac,” from the paper moth – on living human liver cells.
The researchers found that piggyBac inserted beneficial genes into the target cells’ DNA four to five times more efficiently than Sleeping Beauty and the other two transposons under study. PiggyBac achieved successful insertion roughly 20% of the time. That compares poorly to viruses, which have a higher insertion success rate. However, the researchers feel that by modifying key characteristics of piggyBac they can significantly increase its insertion activity and its overall insertion rate. By using animal or plant transposons in lieu of viruses, researchers also can test and experiment with these therapies far more economically and quickly. That’s because virus research often requires additional safety precautions and use of specially equipped laboratories.
Moisyadi further believes that the transposase enzyme, which directs transposons, can be modified with molecular tagging mechanisms to control the precise location of DNA insertion. Says David Segal, a gene-therapy expert at the University of California at Davis, “The most significant finding, also not obvious, is that the transposon that works the best also appears to have the greatest capacity for attachment to something that can direct it to a safe site within the cell. These results are very exciting, and move the piggy-Bac transposon system to the front of the class for development as a highly efficient and safe method for therapeutic gene delivery.”
Stefan Moisyadi is an assistant researcher in the Department of Anatomy, Biochemistry and Physiology at the University of Hawai‘i’s John A. Burns School of Medicine. He specializes in mammalian transgenesis and has previously worked in genetic research on coffee plants as well as on mammalian gametes under the noted fertility and cloning pioneer Ryozo Yanagamachi.
Image: Sam Kim