Reference:
9_Renieri
Abstract:
Despite the intense scientific effort devoted by researchers around the world to study the molecular mechanisms at the basis of Rett, the obvious limitations due to the fact that the primary defect of the brain disease concerns have prevented until now the development of a good model on cells that represent the primary targets of the pathology: neurons. This has greatly complicated the definition and the study of the processes altered by the genetic defect at the cellular level. This has also made it more difficult to program on a large scale in vitro studies aimed at identifying and testing potential drugs. A revolutionary new procedure, called genetic reprogramming, has recently made it possible to obtain pluripotent stem cells, known as iPS (induced Pluripotent Stem) cells directly from adult human fibroblasts. IPS cells have potential comparable to embryonic stem cells and primitive, as the latter, can be grown in vitro for a long time, give rise to an unlimited number of cells and differentiate into any mature cell type, including neurons, astrocytes, cardiomyocytes, muscle fibers and osteocytes (Takahashi K, 2007; Yu J, 2007; Park IH, 2008 WE Lowry, 2008). IPS cells provide an important opportunity to create an innovative and unique in vitro model of genetic diseases (Park IH, 2008; Dimos JT, 2008 AD Ebert, 2008). Particularly for neurological disorders, it is possible to obtain iPS cells from the patient and differentiate into mature neurons generating the latter, thus, human cells “sick.” This protocol allows for the first time to generate human neurons “sick” in large quantities and accessible to any type of study. The derivation of these cells from patients promises to accelerate discoveries on causal mechanisms and develops a human cell model particularly suitable for pharmacological screening for the identification of therapeutic molecules. In order to create a cell model that allows to study the effects of molecular alterations in load MECP2, CDKL5 or FOXG1 directly on human neuronal cells, we propose to use the technology of genetic reprogramming. Since 1998, the Medical Genetics of Siena has collected a large series of Rett patients, both classic and variants, and created a biobank of DNA samples and cell lines. Among the patients included in the biobank, 196 have a mutation in the MECP2, CDKL5 9 and 2 in the newly identified gene FOXG1. This biobank will be of great help since it will allow to quickly recruit families willing to participate in the study of reprogramming and already have valuable information from clinical and molecular point of view. This project will allow us to start from skin fibroblasts from patients with mutations in MECP2, CDKL5 and FOXG1 and healthy controls in order to obtain the immortal cells that can be maintained and induced to differentiate into neurons. Fibroblasts can be easily obtained by skin biopsy, a well-standardized medical procedure that involves minimal risk and stress for patients. The levy will be made in fact by the standard technique of punch-biopsy under local anesthesia at the level of the region of the forearm or thigh. The reprogramming of these cells into pluripotent stem cells will allow us to have a virtually unlimited source of cells that will be induced to differentiate into neurons. In this way it will be possible to study the development and neuronal function directly on human neurons that have specific mutations found in patients. A comparison of cells obtained from healthy controls with those isolated from patients will allow us to assess whether alterations are present in Rett cell differentiation. In addition, these cells will allow us to determine if there are any changes in the more general process of maturation of neurons. Using microarray technology will be possible to study the effects of mutations in MECP2, CDKL5, FOXG1 on gene expression. This resource will also be crucial for the study of possible therapeutic approaches: the availability of human neurons originating from the cells of patients, and therefore the mutations and the genetic background of the patients, will assess accurately and direct both the effectiveness and the possible toxic effects of potential drug treatments. This will allow the analysis of a much larger number of potential drugs in a much shorter time compared to the use of mouse models, allowing you to select a limited number of promising drugs to be tested on more complex models (mouse models) and possibly enter into clinical studies.
PROJECT DETAILS
beginning: 2008.
end: 2010.
Country of research: Italy
Counry of funding source: Italy
Funding organization: AIRETT
Financing: PRIVATE FUNDERS – 50 000 €