The gene therapy is likely to become an important tool in the fight against pediatric rare diseases, especially those derived by the alteration of one single gene. This is the case of the Rett Syndrome (RTT), caused by the loss of function of MECP2, a gene involved in the control of the transcription of different genes, whose function is crucial in the neurons. The loss of the activity of the MECP2 leads to the impairment of the motor functions and a reduced life expectancy in children.

An article published by Sarah Sinnett et al. focused on gene therapy in RTT animal models, describing the efforts and the achievements obtained by the insertion of the human MECP2 in the genome of mice deprived of it. The fine tuning (messa a punto) of a good gene therapy experiment is often laborious, because of some drawbacks strictly connected with this technique, as well described by the authors of the study.

AAV9 first generation results and drawbacks

The first obstacle to overcome lies in the choice of an adequate vector for the transport of the exogenous gene into the cell genome. The vector is generally a virus modified in order to be not pathogenic. Moreover, the virus may spread the therapeutic gene through all the body or may show a tropism, i.e. a specificity, for a target tissue. The researchers chose a strain of Adeno Associated Virus (AAV9) with a high tropism for the neuronal cells.

Two groups of mice were treated with the AAV9 carrying the MECP2 gene: a wild-type, healthy group as a control, and a group without the MECP2 that showed RTT-like symptoms as a short life and an abnormal gait. The so-called first generation of AAV9 was engineered to carry the MECP2 into the capsid in order to insert it into the genome of the mice cells. The AAV9 were injected intravenously but in this way, most of the viruses infected the cells of the liver instead of reaching the neurons, due to the presence of the hemato-encephalic barrier that acts as a shield around the brain. As a consequence, the mice had an improvement of the life duration, but the liver showed high levels of markers, such as the alanine aminotransferase (ALT), indicating a high toxicity.

So that, the researchers decided to change the route of administration of the AAV9 and injected the viruses directly into the brain, in the cerebrospinal fluid (CSF). The mice life duration had an increase of 47%, nevertheless, they kept showing motor impairment: this apparent contradiction was caused by the overexpression of the MECP2 in the body of the animals. Both MECP2 overexpression and lack of expression led to the same phenotype, so the problem was that the expression of the MECP2 was not regulated.

AAV9 second generation results and drawbacks

To overcome the lack of genic regulation, a second generation of AAV9 was created. The researchers added some genic control elements close to the MECP2 in order to better modulate its expression in the transduced cells. Such regulatory elements helped to control the MECP2 expression and allowed to obtain good results by injecting a viral dose 10 or 100 times lower than the first generation of AAV9. This was a good achievement because a lower dose of viruses meant a lower expression of MECP2, thus leading to fewer motor problems in the animals as the transgene was not overexpressed as happened with the first generation AAVs.

The main results obtained were the increase of the lifespan, an increase in the body weight, a fall in the toxicity for the liver, and a certain improvement in the motor functions. In any case, in spite of the good results obtained, the second generation of AAVs was not able to permanently reverse the RTT symptoms in mice, and the life duration was still shorter with respect to the wild-type animals.

Final remarks

Despite the encouraging results obtained so far with the administration of the human MECP2 gene in mice with RTT symptoms, many efforts are needed to improve the safety and the efficacy of this gene therapy method. A third generation of viral vectors should be developed to increase the therapeutic benefits injecting viral doses as low as possible. Also, experiments in superior animal models as dogs will be necessary before thinking to reach the clinical phase in humans. In conclusion, gene therapy is another promising therapeutic option against monogenic rare diseases.


Sinnett S.E. et al. Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery. Mol Ther Methods Clin Dev. 2017 Apr 19;5:106-115.

The elaboration of this post has been financed by the project PI15/01082, as a part of the National Plan of I+D+I and co-financed by the ISCIII – General Deputy Direction for Evaluation and Development of Health Research – and the European Regional Development Fund (ERDF).