Huntington’s disease, a rare genetic disorder characterized by diminishing brain function, has become an increasingly popular target for RNAi-based therapies. Sirna Therapeutics has been working in conjunction with the University of Iowa’s Beverly Davidson to develop a treatment for Huntington’s disease, while CytRx has said that it is considering starting up its own program for the disease.
Now Gustavo Tiscornia, of the Salk Institute for Biological Sciences in San Diego, is getting in on the game.
Tiscornia works in the lab of Inder Verma, which has worked with retroviral vectors for years and ultimately developed lentiviral vectors capable of transducing non-dividing cells in vivo and in vitro, he told RNAi News.
“The Verma lab has been specializing in the development of lentiviral vectors for almost a decade,” he said. “When the [Thijn] Brummelkamp paper came out in Science [in 2002] describing the use of Pol III promoters to drive hairpin [RNAs], it was a pretty obvious idea to mount this system on a [lentiviral vector] to combine the advantages of it as a system for delivering genes into cells with the specificity of RNAi.”
After joining the Verma lab, Tiscornia began working on this project in vitro and in vivo. The first animal experiments involved transducing eggs from GFP-positive transgenic mice with lentivirus-expressing siRNA specific for GFP. According to Tiscornia, the results of the work — which were published in the Proceedings of the National Academy of Sciences in February 2003 — showed that gene knockdown could be achieved through the creation of a “double-transgenic” animal.
“The way we did that … was by taking early embryos and infecting them with lentiviral vectors,” he said. “You take those embryos that you have made double transgenic, and you implant them into the uterus of a female, and eventually you get pups that are transgenics for whatever the virus was carrying.”
Given the success of the lentiviral approach in knocking down about a dozen genes in vitro, including the oncogene p53 and the NF-kappaB transcription factor subunit p65, the in vivo work with GFP-positive mice, and the Verma lab’s overarching interest in gene therapy, Tiscornia said that he and his colleagues began searching for new applications of the technology.
“I had done my PhD on myotonic dystrophy, and so I was familiar with a group of diseases that are not due to loss of function but rather gain of function,” he said. These disorders, of which Huntington’s disease is one, are caused by the expansion of triplet repeat DNA sequences, Tiscornia said. Specifically, Huntington’s disease is caused by the expansion of a CAG repeat in exon 1 of the gene huntingtin.
“The traditional approach in gene therapy is to find a disease caused by a protein being mutated, and then providing that protein through a lentiviral vector — finding something that is not there and then giving it back,” Tiscornia explained. “In these diseases, the problem is the opposite … so RNAi was, on paper, perfectly suited for this.”
Tiscornia’s project, the first of its kind in the Verma lab, is looking to establish a proof of concept that siRNAs targeting huntingtin and delivered using lentiviral vectors can slow or halt the progression of Huntington’s disease in a transgenic mouse model of the disease.
Tiscornia said that one part of the project involves a mouse model with what he termed a “three-gene situation: The endogenous locus, the normal mouse huntingtin protein, and a transgene, which in the case of the mouse model we’re using is [for] human huntingtin.” This mouse model is transduced with a lentiviral vector carrying an siRNA cassette targeting the human transgene, and the results are analyzed, he said.
Assuming that this effort is successful, Tiscornia said he will then look to achieve similar gene knockdown using a different delivery approach. “Obviously, one cannot propose to do whole-organism-level transgenesis in order to reach enough number of cells,” he said, so the next likely route is direct injection of the RNAi agent into the part of the brain primarily affected by Huntington’s disease: the striatum.
Even if both approaches are able to impact the progression of Huntington’s disease in the mouse model, Tiscornia noted that his work remains quite a long way from the clinic. “Injecting viruses into a human brain is, I think at this point, rather out of the question,” he said. “We’re just trying to see if we can get it to work in mice.
“We’re testing the limits of the technology,” he added. “Once we know what those limits are, then we can think about what to do later.”
Tiscornia said that his project is, theoretically, a three-year effort, with preliminary results expected in about a year. “We have to develop the viruses, which we’re already well on our way to doing; we need to make the transgenic animals, which we’ve already made; but now we need to characterize the results, both on the molecular level and behaviorally,” he said. “You always know where an experiment begins, and you never know where it ends.”