Gene Therapy

Aim: To deliver a healthy gene to Duchenne muscles, to allow normal dystrophin production.
Background: Genes consist of DNA and are located on chromosomes, which are present in the nuclei of all cells. The dystrophin gene contains the genetic code for dystrophin, which can be read by the cell and translated into the dystrophin protein.
Challenge 1: We have a lot of muscle – about 30-40% of our body weight is muscle, and we have
more than 750 different muscles, each consisting of billions and billions of cells. The healthy gene has to be delivered to a significant portion of the cell nuclei of all muscles.
Solution: Fortunately, there is an organism that is quite good at injecting genes into cells: the virus. Thus, the gene therapy field has developed viral vectors, where the viral genes are removed, so there is room for the new gene and the modified viruses are no longer pathogenic.
Challenge 2: Most viruses like to infect dividing cells. Muscle tissue hardly divides and thus is a poor target. In addition, muscle fibers are enveloped by layers of connective tissue, which trap viral particles, so the virus cannot reach the muscle fiber to inject its dystrophin gene.
Solution: There is a virus that is relatively good at infecting muscle cells, the so called AAV virus. This virus can infect human cells but is not pathogenic (it does not cause a disease).
Challenge 3: Unfortunately, AAV is so small that the dystrophin genetic code does not fit (the entire gene is ~500 times to big, the genetic code ~4 times too big).
Solution: Scientists have attempted to create the smallest possible dystrophin, containing only the bare essential domains (micro-dystrophin). The genetic code of this micro-dystrophin is small enough to fit into the AAV vector.
In the Duchenne mouse model (mdx mouse) treatment with microdystrophin containing AAV viruses resulted in an improved muscle quality and function.
Challenge 4: When a dog model (golden retriever muscular dystrophy or GRMD) was treated with AAV-microdystrophin, this resulted in an immune response. Consequently, cells infected with the AAV containing microdystrophin were destroyed by the immune system. From clinical trials in humans with other genes (e.g. to treat hemophilia) we know that AAV also induces an immune response in humans. The immune response will attack all foreign intruders (viruses, bacteria, parasites) and has no way of knowing this time the virus carries a good gene.
Solution: Ways to reduce the immune response are currently under investigation. This can be done by suppressing the immune response with high doses of corticosteroids.
To consider: About 20% of individuals have been infected with a subtype of AAV. These individuals have antibodies against AAV that would preclude them from receiving viral vectors of that specific subtype.
Clinical trials: A first clinical trial where patients received local AAV-microdystrophin injections in the arm muscle was performed in the USA (Mendell, Xiao Xiao and Samulski). Results of this trial have been published. The authors report very poor expression of their microdystrophin version, and the anticipated immune response to AAV.
Challenge: As mentioned muscles make up 30-40% of our body. To have an effect all muscles or muscle groups have to be treated rather than a small area of a single muscle. However, this requires huge amounts of virus (a young boy weighs ~4000 times more than a young mouse).
Solution: After optimizing the manufacturing process, a lot more viral particles can be produced at clinical grade, allowing for trials where the viral particles are injected intravenously and the whole body is treated rather than just small pieces of muscle.
Clinical trials


There are currently 3 clinical trials ongoing with AAV-microdystrophin. In a clinical trial in Colombus, Ohio (coordinated by Sarepta Therapeutics) AAV-microdystrophin is delivered intravenously in young Duchenne patients together with high doses of corticosteroids. Thus far three patients have been treated. Analysis of muscle biopsies 90 days after treatment revealed that for each patient the majority of muscle fibers expressed the micro-dystrophin. The total levels were ~40% of dystrophin levels expressed in healthy muscles. This trial was put on hold in August 2018 due to an impurity that was discovered in the raw materials used for manufacturing.

A trial coordinated by Solid Ventures using a slightly different AAV-microdystrophin system was put on hold after the first patient experienced serious adverse events after infusion of the AAV-microdystorphin. The trial reinitiated in June 2018.
In parallel a trial is ongoing  with a slightly different AAV-microdystrophin system by Pfizer.

To Consider: A recent safety study in dogs and monkeys shows that high doses of AAV vectors can lead to liver and brain problems. Note that the vector type used in this study differs from the ones that will be used for microdystrophin trials.

AAV does not integrate in the DNA. This is good from a safety perspective, but also means that over time the microdystrophin gene may be lost. Studies in dystrophic dogs suggest that most of the delivered gene is lost after 5 years. It is not known whether this is also the case in humans.

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