604. TALE-VP64 Targeting the Frataxin Promoter Increase the Expression of That Gene in Friedreich Fibroblasts

Abstract

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.