University of Pittsburgh
Asklepios BioPharmaceutical, Chapel Hill, N.C.
Delivering minidystrophin genes using an adeno-associated viral vector
Estimated start date for clinical trial
In 1986, Xiao Xiao was a biology instructor at Wuhan University in Hubei Province, China. He had earned a bachelor’s degree in pharmaceutical chemistry at Shanghai University and a master’s in biochemistry at Wuhan, but he wanted to work on gene therapy. The best way to do that, he decided, was to move to the United States.
So, in 1986 he emigrated, and by 1988 was in the Biological Sciences Department at the University of Pittsburgh, working as a teaching assistant under the tutelage of virologist Jude Samulski.
“Since the beginning of graduate school [in the United States], you could say that I’ve been working on gene therapy projects,” Xiao says, “initially on vector development, trying to figure out if adeno-associated viruses can infect dividing and nondividing cells, and where the DNA they’re carrying goes.”
When he started with Samulski, he says, “AAV was still not a very popular virus. Not because it’s a bad virus, but because it’s not a very important virus.
“There’s a joke among virus people, who say AAV means ‘almost a virus.’ It’s called ‘almost a virus’ because it does not cause any disease. If a virus does not cause any disease, people will not study it extensively.”
Xiao stayed at Pittsburgh for four years and then spent a year at Avigen, a California biotechnology company. In 1994, he moved to the University of North Carolina’s Gene Therapy Center, where he and Samulski again worked together. Four years later, Xiao moved back to Pittsburgh, seeking his own niche in the research community.
“As soon as I moved back to the University of Pittsburgh, I started to establish my own lab. I thought a lot about muscle gene transfer, because AAV worked so well in muscle. I looked at the papers, and at the commonly used animal models for the muscular dystrophies, like the mdx mouse for DMD and the hamster model for LGMD.”
Xiao thought first about working with one of the LGMD genes, because of their small size and the relative ease with which they could be inserted into an AAV vector.
“On the other hand,” he says, “that disease is rare, very rare, and Duchenne muscular dystrophy is a very prominent disease and affects a lot of children.”
Enlarging the rod domain
By that time, Xiao had been joined in the lab by colleagues, among them his wife, Juan (pronounced Jwan), also a researcher and a physician. Together, he says, they recognized that although the Duchenne gene, dystrophin, was too large to fit into an AAV, there might be other solutions.
“We read a lot of the papers, and we found out that there are a lot of naturally occurring deletions. You can delete a large chunk of the DNA without compromising the major function of the protein [that’s made from the DNA].”
The team was experimenting with making deletions in the dystrophin gene, when, in 1997, a Japanese group announced that a “minidystrophin” gene missing 23 out of 24 parts of its midsection, known as the rod domain, showed some promise in the mdx mouse. But the group had used another virus to package the gene, and when Xiao’s group tried the experiment using the AAV vector, they didn’t see the same therapeutic effect.
Wanting to pursue the use of the newly developed and apparently safe AAV vectors, Xiao and colleagues started to make their own minidystrophin genes.
“It was apparent that the central rod domain cannot be too short, and that if it is, the protective function of dystrophin will be lost. So we made our own versions of minidystrophins with longer central rod domains,” he explains.
“If we added more central rod domain repeats to the minidystrophin, we knew we would have to get rid of something else to make this dystrophin small enough” to fit inside the AAV vector.
Xiao decided to delete another part of the gene, the C terminus.
“That gave us the capacity to add a few more rod repeats in the middle. We made a series of constructs with different lengths of central rod domains, and we tested them in the mdx mice and saw a very good therapeutic effect with many of them, when they were packaged in an AAV.” By that time, Xiao was being supported by MDA, and, in 2000, he and Bing Wang, a postdoctoral student, published a key paper in Proceedings of the National Academy of Sciences describing the effectiveness of their minigene in the mdx mouse.
“After the good results from my mouse study, many Duchenne parents, as well as the Muscular Dystrophy Association and other scientists, said, ‘What’s your next step?’
“We thought we should somehow translate this from bench to bedside. That’s our long-term goal, to do that. Obviously, there are a lot of hurdles to this.
“To do a clinical trial you have to apply for a grant, because without funding, you cannot do it. And you have to do the preclinical studies, as required by the FDA, and you have to deal with regulatory institutions and committees. You have to produce a clinical-grade virus. Just like with any pharmaceutical clinical trial, you have to have the drug.”
For this endeavor, Xiao once again approached Samulski, whose lab in Chapel Hill had produced a clinical grade AAV vector for a trial in Canavan’s disease.
“He had been working on adeno-associated viruses since graduate school, and so had I. He said he wanted to translate this vector technology into a gene therapy clinical setting and do something with it.”
By this time, the fledgling company that would become Asklepios was in development, and muscular dystrophy gene therapy seemed like a perfect fit for its mission. “We thought our dystrophin technology and his company’s vector development and production technique would be a perfect combination — so that’s how we got started.”