1 2 aim

Animal models

In vivo therapy development

The next step are animal experiments, which is called in vivo (‘within the living’). In the case of DMD several animal models exists that also miss the dystrophin protein. It should be kept in mind that even though these models have a genetic mutation similar to patients, there is no animal model that fully represents the human pathology. Each model has its limitations, which should be taken into account when designing experiments and interpreting the results.

Animal models

Murine models

In medical research, also for DMD, mice are most frequently used. They have the advantage that they are relatively small, thereby limiting the required space and costs. Small amounts of compound are needed for treatment. Furthermore, mice breed easily (short gestation time and large litter sizes).

The most well-known mouse model for DMD is the so-called mdx (X chromosome-linked muscular dystrophy) mouse. This mouse carries a spontaneous mutation in the murine Dmd gene, thereby lacking the dystrophin protein. A draw-back of the mdxmouse is that it is not very severely affected compared to DMD patients. This is, amongst others, caused by the fact that mice have a much better regenerating capacity (they efficiently replace the lost muscle cells by new ones). Furthermore, in mice a protein that is similar to dystrophin is able to compensate for the lack of dystrophin. The usefulness of the model depends on the purpose. It is suitable to test whether a compound that aims to correct the mutation, indeed can restore the production of the dystrophin protein. Furthermore, muscle and respiratory function can be assessed, whereby should be taken into account that the muscle function is less impaired than in humans. In addition, multiple muscles can be assessed in detail, e.g. looking at histology or alterations at a molecular level. A big advantage is that in mice the diaphragm can be studied, which is not possible in humans. Notably, while most skeletal muscles are not that severely affected in the mdx mouse, the diaphragm does show severe pathology.

Nowadays, several other murine models have been developed next to the mdxmouse; each of them having its own strength and limitations. Some of them are more severely affected than others and each of them represents different features of the pathology. Also mice carrying specific mutations are helpful to test mutation-specific therapies.

To ensure comparison between different groups and improve translatability of candidate therapies into humans, it is important to use standardized procedures. These are available here.

Canine models

Next to mouse models, canine models are used for DMD research. Several canine models (X-chromosome linked muscular dystrophy with dystrophin deficiency (CXMD)) exist. The most widely used and well-described model is the golden retriever muscular dystrophy (GRMD) dog. Also this dog lacks the dystrophin protein due to a (spontaneous) genetic mutation and the disease course is more comparable to humans. Furthermore they exhibit cardiomyopathy, as is usually much less seen in mouse models. Even though these dogs are on average more severely affected than mice, there is a huge variation between dogs, even within the same litter. Some dogs show severe signs of muscular dystrophy and die young; others only have mild symptoms and have a normal life span.

The secondly most used canine model is the beagle model (canine X-linked muscular dystrophy (CXMDJ)). These dogs have been generated by breeding the GRMD dog with beagles. The advantage is that this model is of much smaller size, reducing the experimental costs. Overall pathology is milder in CXMDdogs compared to GRMD dogs, due to their smaller size. Similar to GRMD dogs, not all CXMDdogs are equally affected.

Recently a new dog model has been identified. Unlike the GRMD and CXMDdogs, this model has a mutation in the same region where most DMD patients also have a mutation.

There are several reasons why dogs are much less used for research. Dogs are far more costly than mice, both due to their size and longer lifespan. The latter also makes experiments more lengthy. Furthermore, the huge variation between individual dogs is a major drawback for experiments. It makes it difficult to draw conclusions. This is also influenced by the costs. Since, dog experiments are very expensive, only small groups are possible.

Other animal models

Next to mice and dogs, several other animal models exist, either naturally occurring or transgenic (i.e. genetically modified). A spontaneous mutation has been described in cats; however, these cats poorly resemble human pathology and are very little used for research.

Other mammalian models have been made in rats, pigs and rabbits; each having its on disease characteristics. In addition, worms, zebrafish and fruit flies carrying mutations in genes analogue to the DMD gene exist.

Considerations on animal research

None of the animal models fully reflects the human pathology. Therefore, results obtained in these models do not necessarily lead to the same results in humans. Nevertheless animal models are indispensable for drug development. They show proof-of-concept of the hypothesis, are useful for optimisation of the therapy (e.g. dosage, treatment regimen, route of administration) and reveal potential toxicity. Before designing an experiment it has to be carefully considered what is the best model for this type of compound/experiment, how the experiment should be set-up and what are the best outcome measures to examine.

Regulatory requirements

Usually regulatory agencies (FDA, EMA or national regulators) require safety testing in at least two (relevant) mammalian species, one rodent and one non-rodent (e.g. non-human primates). Nowadays lots of attention is paid by both researchers and regulators on the implementation of the 3Rs (Replacement, Reduction and Refinement) to reduce the number of animals required and to ensure proper planning of animal experiments to make sure this is done optimally.

How to choose a clinical trial

Check out an overview of all trials