Scientists at Stanford University School of Medicine managed to administer effective gene therapy in mice without triggering an autoimmune reaction. The research, led by Dr. Peggy Ho, Ph.D., was published in the Proceedings of the National Academy of Sciences .
In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is “foreign,” and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.
Gene therapy and autoimmunity
Gene therapy may be extremely useful to replace defects in the genome of a patient. A defective gene encodes for a faulty protein, which may lead to crippling or even lethal diseases. By replacing broken genes with working ones through gene therapy, cells can be instructed to produce functional proteins instead of faulty ones, thereby eliminating the very root of genetic diseases.
However, the new, working protein produced by a newly introduced gene is foreign to the patient’s body; his or her cells have never produced it before, and the immune system is bound to recognize it as a threat and treat it as such, mounting a dangerous immune reaction that might even result in death. For this reason, gene therapy has always been a bit of a double-edged sword.
Finding ways to administer gene therapy and eliminate or mitigate autoimmune reactions is thus a central problem, and Stanford researchers think that they’ve found a way to do it, at least in mice.
The researchers carried out their experiment on a murine model of Duchenne muscular dystrophy—a progressive, crippling pathology that affects all voluntary muscles and, eventually, cardiac and breathing muscles as well. In humans, it occurs primarily in males and exceedingly rarely in females. The rate of incidence of the disease is about 1 in 5,000 males at birth, most of whom will be unable to walk by the time they reach the middle of their teenage years; in a best-case scenario, affected individuals can expect to live to their 30s.
The reason why the researchers chose this disease is that it originates from a single faulty gene. This gene is responsible for the production of a protein called dystrophin; if the protein is lacking or dysfunctional, it will lead to the disease. As curing the disease requires fixing only one gene, it is a relatively simple target.
The researchers’ patients were fifteen 6-week-old mice who had been bioengineered to lack functional dystrophin. In order to deliver a working copy of the dystrophin-encoding gene to the mice, the researchers employed viral vectors—that is, they modified viruses to render them harmless while preserving their cell-penetrating ability, and they equipped them with the gene they wanted to splice into the mice’s genetic code. This gene wasn’t exactly the one encoding for dystrophin, which would be simply too large to fit into the chosen viral vector; instead, they created a smaller version of it that produces microdystrophin, a suitable substitute for dystrophin. The viral vector, designed by one of the study’s authors, would then go on to infecting the mice’s cells and insert the replacement gene into their DNA.
However, this approach may actually worsen immune reactions; the recipient’s immune system would react not only to microdystrophin but also to the viral vector itself. To circumvent this problem, the authors of the study made use of plasmids—small DNA molecules that are most commonly circular, double-stranded, and found in bacteria. These molecules are generally transferred directly from one bacterium to another—a process known as horizontal gene transfer—sometimes facilitating the transmission of antibiotic resistance.
The immune systems of the mice would mount a response against some of the sequences in the DNA contained in the plasmid as well, but in this case, the scientists had replaced those sequences with others capable of dampening the immune reaction to both microdystrophin and the viral vector.
After the mice received the microdystrophin gene through a viral vector, they were divided into three groups, which, for 32 weeks, received a weekly intramuscular injection of a placebo, a placebo plus the plasmid without the dampening sequences, or the immunity-dampening plasmide. At the end of this treatment, the latter group had much stronger muscles and higher levels of microdystrophin than the other groups as well as little immune response to microdystrophin and few inflammatory signals between immune cells.
While an experiment in mice is not sufficient to absolutely determine whether the same approach would work in people, the researchers are optimistic that this method might translate; the same approach has been used to induce immune tolerance to myelin protein and proinsulin in people with multiple sclerosis and Type 1 diabetes, respectively, so there’s reason to believe that it might work for human gene therapy as well.
 Ho, P. P., Lahey, L. J., Mourkioti, F., Kraft, P. E., Filareto, A., Brandt, M., … Steinman, L. (2018). Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy. Proceedings of the National Academy of Sciences, 201808648.