604. TALE-VP64 Targeting the Frataxin Promoter Increase the Expression of That Gene in Friedreich Fibroblasts
Notice bibliographique
Résumé
Friedreich's ataxia (FRDA) is due to a reduced frataxin expression due to a trinucleotide repeat. It is 30% in patients with 200 GAA repeats and only 5% in patients with 900 GAA repeats 33. However, carriers of this disease produce about only 50% of the normal level of frataxin but do not develop symptoms. We have engineered 12 genes coding for TALE proteins targeting different nucleotide sequences present in the frataxin promoter. Each expression plasmid contains a TALEFrat gene fused with a transcription activator, VP64 under the EF1α promoter, a 2A peptide and an EGFP. When one of these plasmids was transfected alone in human cells, only green fluorescence was detected by FACS indirectly confirming the expression of the TALEFrat/VP64 protein. To identify which of our 12 different TALEFrat/VP64 proteins were able to better induce the expression of the frataxin gene, we have constructed a reporter plasmid containing the proximal region of the frataxin promoter, followed by a minimal CMV promoter and a mCherry reporter gene (pCR3.1 proximal-promoter-frataxin-miniCMV-mCherry). This reporter plasmid was initially transfected in human cells alone. Very few cells expressed the red fluorescence because the promoter was not effective without transactivation by binding factors. When the reporter plasmid was co-transfected in human cells with one of the pCR3.1-TALEFrat/VP64-2A-EGFP plasmids, a much higher number of cells expressed the red fluorescence because the TALEFrat/VP64 attached to the proximal frataxin promoter and induced the transcription of mCherry. The 3 TALEFrat/VP64, which induced the strongest expression of mCherry, were targeting promoter sequences close to each other. A plasmid coding for TALEFrat#8/VP64 was nucleofected in normal fibroblasts. Using quantitative RT-PCR, we have confirmed in 3 independent experiments that the expression of the frataxin mRNA (relative to GAPDH mRNA) in human cells was doubled or triple by TALEFrat#8/VP64 when results were normalized with cells transfected with EGFP or non-transfected cells. We have also shown that this TALE also increased by 2 folds the frataxin protein in fibroblasts from a FRDA patient. In a recent preliminary result, we have shown that the transfection of TALEFrat#8/VP64 plasmid in YG8R fibroblasts also increases frataxin mRNA (by about 1.4 to 1.9 fold) and protein (by about 1.4 fold). Such increases would be in the therapeutic range (i. e., 50% of normal frataxin level) for many patients. However, for patients that have less than 25% of the normal level of frataxin expression, a further increase of frataxin would be required and this project may permit to obtain higher frataxin increases and thus this would lead to an increased number of FRDA patients who would benefit from our therapy.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,001 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».