ENG: Encuentro Ataxias (Ataxia Encounter) 2008 -- Third scientific presentation -- Dr. Javier Díaz Nido, "Gene Therapy in Friedreich's Ataxia: Cellular Models of Olfactory Bulb and Their Repercussions on Patients"

Summary by Mari Luz González Casas

Translation by Marion Clark


To hear the entire presentation in Spanish on mp3, go to the following link: http://download.yousendit.com/19B64078324243AD



Encuentro Ataxias (Ataxia Encounter) 2008

Organized by the

Colectivo Ataxias en Movimiento

(Ataxias on the Move Coalition)

Madrid, Spain

Third scientific presentation:


Dr. Javier Díaz Nido

Severo Ochoa Molecular Biology Center, Autonomous University of Madrid

“Gene Therapy in Friedreich’s Ataxia:  Cellular Models of Olfactory Bulb and Their Repercussions on Patients”

 Dr. Javier Díaz Nido

Friedreich’s ataxia is a disease caused by the deficiency of frataxin in the mitochondria.  It is fundamental to study what occurs in the cells lacking in frataxin so as to better understand the molecular bases of the disease, and in order to look for biomarkers of the disease in order to test all sorts of treatment strategies, from the most traditional pharmacological ones to the latest ones, such as gene therapy.  Cellular models are consistent with this.

            Many studies have been done, but the majority of these have been conducted in very simple experimental models such as yeasts, various invertebrates, or a very accessible model such as the cultures of fibroblasts taken from the skin of FA patients.  These all have been and still are very useful for studying many functions of frataxin.

            Frataxin deficiency does not affect all cells equally.  FA is not exclusively a neurodegenerative disease, because all the cells of every type in the FA patient’s body are deficient in frataxin.  However, it is fundamentally only certain neurons and a few other cell types which show a pathological change.  Therefore, frataxin deficiency does not affect all the cells equally.  For this reason, this research team has focused on the study of frataxin deficiency in the neurons, coming at it from two angles:

a)     Neuronal cellular models

b)     Gene therapy strategies directed at the nervous system

a)   Neuronal cellular models

There are two types:

--Cells differentiated from human neuroblastoma

--Primary cultures from mouse neurons

Each one of these models has its advantages and its disadvantages.

Human neuroblastoma lines:  As neuronal cellular models, these are made by using a strategy in the form of a trick.  Using this strategy the expression of the frataxin gene is diminished, and this is done in cell cultures.  For this, differentiated cells of human neuroblastoma were used.  These cells, which were obtained from tumors, are manipulated in the laboratory, and the frataxin deficiency is induced in them.


--They offer a very homogeneous population of cells, unlike what happens with primary cultures.  This is proving very useful for a whole series of transcriptional studies, biochemical studies, etc., which are better done in very homogeneous cellular populations.

--They are human cells.


--They are cells which come from a tumor and which are differentiated in the laboratory.  They seem to be mature neurons, but it should not be overlooked that this differentiation is not entirely normal.

The importance of all this is that a great deal of data is being obtained:  the neurons only become more susceptible to frataxin deficiency once they reach maturity.  A cell can proliferate and differentiate itself, and only when it really attains maturity will the frataxin deficit cause its degeneration.  How this degeneration takes place has been studied, and a great deal has been learned from this.

Primary cultures of mouse neurons:  The same thing was done in primary cultures of mouse neurons directly extracted from the central nervous system of the mice.   They are very relevant, because we can isolate neurons from the areas most affected by the disease and from the least affected areas, and thus study the consequences of the frataxin deficiency, identify biomarkers, etc.


--With the primary cultures, the cell populations are very heterogeneous

--They are mouse cells, not human cells; and data exist that make us think that the frataxin deficit in mouse cells is not the same as that in humans.  Therefore, it is important to have human cellular models available as well.

Because none of these models is ideal, attempts are being made to develop new models, neuronal models which allow us to obtain human neurons deficient in frataxin.  There are two ways to do this:

1)     Starting from human embryonic stem cells, using lines currently established, neurons can be differentiated, and frataxin deficit can be induced.  This way we would have neurons which were not derived from tumor cells, in which we could study frataxin deficiency.

2)     The second plan consists of obtaining neuronal progenitors from biopsies on patients with FA.  This is a project which has just begun, in collaboration with Dr. Jose Luis Munoz Blanco, neurologist at the La Paz University Hospital in Madrid.  The objective is to obtain neuronal progenitors from the olfactive mucosa.

The olfactive mucosa is one of the few sites which both are relatively easily accessible and contain neuronal progenitors.  In the olfactive mucosa are the olfactory receptor cells, and near them there is a series of progenitors which continually produce new olfactory receptor neurons, which go to replace those which are being lost all through life.  Thus we can isolate neuronal progenitors, culturing them to be able to obtain neurons from them and ultimately to be able to have neurons from FA patients.

This was begun, obviously, with control subjects—voluntary donors—individuals who are undergoing various nasal surgeries, and who give their consent for a small biopsy to be taken.  From this biopsy cells are obtained in which it is possible to grow neurospheres, since they are neuronal progenitor cells.  At the current time they are in the process of being characterized.  Once this technique has been refined, the next step is to obtain biopsies from FA patients.  Starting from that time, those cells will be used, since they provide a cellular model more appropriate for improving our knowledge and seeking potential treatments.

At this point Dr. Diaz Nido gave a brief summary of the situation as regards pharmacological treatments.  Among the pharmacological treatments, the only one to have reached an advanced phase is Idebenone.  It has been demonstrated that Idebenone protects the heart muscle, and some slight neurological benefits have been reported, although this last claim is not beyond dispute.

Cellular models are very important tools used in testing drugs for a limited cost, then going on to test in mice those which turn out to be promising.

b)   Gene therapy

Conceptually this is a very simple treatment.  There is a defective gene which doesn’t produce enough frataxin.  The idea is to implant the “correct” gene in order to raise the quantity of frataxin and thus cure the disease.

In order to place the gene into the cells it needs to be packaged in a vector or vehicle, and this vector must be very efficient so that it delivers the gene to the greatest possible number of cells--this is the only way the body will function well.  The vector must be one which can deliver the gene to all the neurons.  The vectors must be herpes viral vectors, which have all the necessary characteristics.

The first requirement is to have an animal model of FA, a mouse suffering from the disease.  Mice and humans have significant physiological differences, and it has been very difficult to develop a mouse model that consistently reproduces FA.  Some appear to do so, but there are no good models, and for this reason, it was necessary to resort to a trick:  obtaining genetically modified mice.  A sort of marker was placed in the frataxin gene in such a way that using the Cre recombinase enzyme, it was possible to eliminate in a very specific manner a DNA fragment situated between two segments called IoxP.  In the presence of the Cre recombinase enzyme , whatever DNA sequence is situated between the two IoxP sites will be eliminated from the genome.  So this is how the frataxin gene was eliminated to create genetically modified mice, a mouse model made to mimic what happens in FA.

With this model a first trial of gene therapy was made.  The vector was injected with the Cre recombinase enzyme into the brain stem (one of the regions most affected by the disease).  Due to the absence of frataxin, the mouse shows the symptoms characteristic of FA.

This mouse was given a second injection, and another vector, which in this case delivers the frataxin gene, was applied at the same point.  To verify if this mouse is able to recover its functions, it was submitted to a rotarod test.  One group of mice was injected with a vector without the Cre recombinase enzyme; the other group was injected with a vector that did contain this enzyme.  After two weeks it began to be seen that those mice which had received Cre-recombinase developped worse motor coordination.  After 4 weeks, the differences between both groups were evident, and at 6 weeks these differences became even sharper.

For the next part of this study, 4-week-old ataxic mice were selected.  (4-week-old mice are used because this is a point at which the ataxia is detectable, but the mice still are not very badly affected, and it is thought that at this time it’s easier for them to recover lost function.)

This group of mice was divided in two. The mice of group A were injected with a vector containing an enzyme, beta-galactosidase, which colors the neurons so that they can be more easily located.  The mice of group B were injected with a vector containing the frataxin gene.  In both cases the mice showed some improvement after the injection, but this improvement was much more obvious in those mice that received the frataxin gene.  What caused this improvement in mice with an apparently inactive gene?  The answer to this question is not known, although this phenomenon is not a new one.  Researchers believe that the injection of the viral vector, which at times can have toxic effects, may possibly stimulate the production of neurotrophic factors which cause a transitory therapeutic effect.  Besides not being toxic, the vector is very well tolerated, and this is a good sign.

At 8 weeks of age, the mice which had received frataxin had recovered all their functions to such a point that they could be confused with the healthy mice.  A complete recovery of the phenotype had taken place.

Conclusion:  In a very simple model, gene therapy is possible.  But this model is not representative of the human disease, because in this mouse model there were very few neurons affected.  It is a proof of the concept but with these challenges remaining:

--To achieve a good distribution of the vector in all the affected neurons.  The disease primarily affects the medulla, the dorsal root ganglia, the brain stem, and cerebellum.

--To achieve this result with the smallest possible number of injections, because each injection of a vector is an invasive procedure.

--To adequately regulate the expression of the frataxin gene.  Frataxin should reach an adequate level, neither too little nor too much.

--To respond to all the physiologic stimuli which regulate frataxin, and to do so with lasting results.

The structures  normally used in laboratories are not really genes, but simplified versions called cDNAs (complementary DNA).  With these structures a more persistent expression cannot be achieved.  To achieve a more persistent expression, it is necessary to work with the specific frataxin gene just as it is in the chromosome, and to form a sort of microchromosome ( a small sequence of this chromosome which carries the frataxin gene), package it in a viral vector, and distribute it to the cells.  Dr. Diaz Nido is of the opinion that using this strategy, a more persistent expression of the gene will be achieved.  In fact the results, although preliminary, indicate that this is the case.  Of course, the expression of these genomic structures called “airbags” would be a means of facilitating the persistence of the expression of the gene.

--To demonstrate the effectiveness of the technique in an animal model, a mouse model which really mimics the disease better.  This has been difficult to find, but now a model is available, a unique model in which the same mutation was introduced, developing in the mouse a disease much lighter than in human beings.

At the present time work is being done to optimize the technique in all these aspects.


Wed May 21, 2008 8:52 pm

"Gian Piero Sommaruga \(casa\)" <gippi@...>
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The legacy of Marie Schlau: literature to help cure Friedreich's Ataxia

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Research projects currently being financed by BabelFAmily

Currently, BabelFAmily is financing two promising research projects aimed at finding a cure for Friedreich's Ataxia. Whenever you make a donation to us or purchase a copy of "The legacy of Marie Schlau", this is where all funds raised will be devoted to:

1) Gene Therapy for Friedreich's Ataxia research project:


The project is the result of an initiative of Spanish people affected by this rare disease who are grouped in GENEFA in collaboration with the Spanish Federation of Ataxias and the BabelFAmily. The Friedreich’s Ataxia Research Alliance (FARA), one of the main patients’ associations in the United States now joins the endeavour.

2) Frataxin delivery research project:

The associations of patients and families Babel Family and the Asociación Granadina de la Ataxia de Friedreich (ASOGAF) channel 80,000 euros of their donations (50% from each organisation) into a new 18-month project at the Institute for Research in Biomedicine (IRB Barcelona). The project specifically aims to complete a step necessary in order to move towards a future frataxin replacement therapy for the brain, where the reduction of this protein causes the most damage in patients with Friedreich’s Ataxia.

The study is headed by Ernest Giralt, head of the Peptides and Proteins Lab, who has many years of experience and is a recognised expert in peptide chemistry and new systems of through which to delivery drugs to the brain, such as peptide shuttles—molecules that have the capacity to carry the drug across the barrier that surrounds and protects the brain. Since the lab started its relation with these patients’ associations in 2013*, it has been developing another two projects into Friedrich’s Ataxia.



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