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Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice.

Chapdelaine P, Gérard C, Sanchez N, Cherif K, Rousseau J, Ouellet DL, Jauvin D, Tremblay JP - Gene Ther. (2016)

Bottom Line: Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters.The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied.These results corroborate our previous in vitro studies in the FRDA human fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique Humaine, Axe Neurosciences, Centre de Recherche du Centre Hospitalier de Universitaire de Québec-Université Laval, Québec City, QC, Canada.

ABSTRACT
Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases.

No MeSH data available.


Related in: MedlinePlus

(A) Effect of the treatment of AAV9_3XFLAG-TALEfrat#8-VP64 on the FXN mRNA analyzed by Northern blots and qRT-PCR in YG8R mice. The treatments were carried out at different doses of AAV9: T indicates 6 × 1012 vg per mouse, Tc 6 × 1011 vg per mouse and 1.2 × 1011 vg per mouse. The top figures (a1) are total RNA electrophoresed on 1.2% agarose-formaldehyde gel. These RNAs were extracted from three tissues: heart, muscle of group 2 and liver of group 1 YG8R mice. (a2–a4) Northern blot analyses. (a2) Signal hybridization of 18 S rRNA (used as internal standard). (a3 and a4) The hybridization signal of the FXN mRNA exposed for different times, 2 days (a3) and 10 days (a4). (a5) Histograms corresponding to (a4). The Northern blots suggest increased FXN mRNA in mice treated with the recombinant virus. (B) Three horizontal panels summarize the FXN qRT-PCR results. The top panels (b1 and b2) represent histograms showing increased of FXN mRNA in the heart and muscle (group 2, N=3, ave.±s.d. *for P<0.05, **for P<0.01 and *** for P<0.005). The results are expressed either as copy number or as ratios relative to 18 S rRNA, GAPDH, ATP50 and HPRT1. The middle panel (b2) represents FXN mRNA expressed in the muscle (group 2). The lower panel (b3) illustrates FXN mRNA expressed in the liver (group 1), but the histogram bar represents only one mouse for control and one mouse treated with recombinant virus (Tc).
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fig3: (A) Effect of the treatment of AAV9_3XFLAG-TALEfrat#8-VP64 on the FXN mRNA analyzed by Northern blots and qRT-PCR in YG8R mice. The treatments were carried out at different doses of AAV9: T indicates 6 × 1012 vg per mouse, Tc 6 × 1011 vg per mouse and 1.2 × 1011 vg per mouse. The top figures (a1) are total RNA electrophoresed on 1.2% agarose-formaldehyde gel. These RNAs were extracted from three tissues: heart, muscle of group 2 and liver of group 1 YG8R mice. (a2–a4) Northern blot analyses. (a2) Signal hybridization of 18 S rRNA (used as internal standard). (a3 and a4) The hybridization signal of the FXN mRNA exposed for different times, 2 days (a3) and 10 days (a4). (a5) Histograms corresponding to (a4). The Northern blots suggest increased FXN mRNA in mice treated with the recombinant virus. (B) Three horizontal panels summarize the FXN qRT-PCR results. The top panels (b1 and b2) represent histograms showing increased of FXN mRNA in the heart and muscle (group 2, N=3, ave.±s.d. *for P<0.05, **for P<0.01 and *** for P<0.005). The results are expressed either as copy number or as ratios relative to 18 S rRNA, GAPDH, ATP50 and HPRT1. The middle panel (b2) represents FXN mRNA expressed in the muscle (group 2). The lower panel (b3) illustrates FXN mRNA expressed in the liver (group 1), but the histogram bar represents only one mouse for control and one mouse treated with recombinant virus (Tc).

Mentions: Northern blot analysis showed a significant increase of the FXN mRNA in the heart and muscles of the 9-day-old mice and in the liver of the 4-month-old mice (Figure 3A). Real-time reverse transcription-PCR (qRT-PCR) analyses of the same tissues expressed in terms of copy number only or as ratios with internal standards such as 18S rRNA, GAPDH, HPRT1 and ATP50 are presented in Figure 3B. These results confirmed that AAV9_3XFLAG-TALEfrat#8-VP64 was able to stimulate in vivo FXN transcription. The expression ratio of the treated mice relative to the controls was similar for the four internal standards used for the analysis of this experiment.


Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice.

Chapdelaine P, Gérard C, Sanchez N, Cherif K, Rousseau J, Ouellet DL, Jauvin D, Tremblay JP - Gene Ther. (2016)

(A) Effect of the treatment of AAV9_3XFLAG-TALEfrat#8-VP64 on the FXN mRNA analyzed by Northern blots and qRT-PCR in YG8R mice. The treatments were carried out at different doses of AAV9: T indicates 6 × 1012 vg per mouse, Tc 6 × 1011 vg per mouse and 1.2 × 1011 vg per mouse. The top figures (a1) are total RNA electrophoresed on 1.2% agarose-formaldehyde gel. These RNAs were extracted from three tissues: heart, muscle of group 2 and liver of group 1 YG8R mice. (a2–a4) Northern blot analyses. (a2) Signal hybridization of 18 S rRNA (used as internal standard). (a3 and a4) The hybridization signal of the FXN mRNA exposed for different times, 2 days (a3) and 10 days (a4). (a5) Histograms corresponding to (a4). The Northern blots suggest increased FXN mRNA in mice treated with the recombinant virus. (B) Three horizontal panels summarize the FXN qRT-PCR results. The top panels (b1 and b2) represent histograms showing increased of FXN mRNA in the heart and muscle (group 2, N=3, ave.±s.d. *for P<0.05, **for P<0.01 and *** for P<0.005). The results are expressed either as copy number or as ratios relative to 18 S rRNA, GAPDH, ATP50 and HPRT1. The middle panel (b2) represents FXN mRNA expressed in the muscle (group 2). The lower panel (b3) illustrates FXN mRNA expressed in the liver (group 1), but the histogram bar represents only one mouse for control and one mouse treated with recombinant virus (Tc).
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Related In: Results  -  Collection

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Show All Figures
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fig3: (A) Effect of the treatment of AAV9_3XFLAG-TALEfrat#8-VP64 on the FXN mRNA analyzed by Northern blots and qRT-PCR in YG8R mice. The treatments were carried out at different doses of AAV9: T indicates 6 × 1012 vg per mouse, Tc 6 × 1011 vg per mouse and 1.2 × 1011 vg per mouse. The top figures (a1) are total RNA electrophoresed on 1.2% agarose-formaldehyde gel. These RNAs were extracted from three tissues: heart, muscle of group 2 and liver of group 1 YG8R mice. (a2–a4) Northern blot analyses. (a2) Signal hybridization of 18 S rRNA (used as internal standard). (a3 and a4) The hybridization signal of the FXN mRNA exposed for different times, 2 days (a3) and 10 days (a4). (a5) Histograms corresponding to (a4). The Northern blots suggest increased FXN mRNA in mice treated with the recombinant virus. (B) Three horizontal panels summarize the FXN qRT-PCR results. The top panels (b1 and b2) represent histograms showing increased of FXN mRNA in the heart and muscle (group 2, N=3, ave.±s.d. *for P<0.05, **for P<0.01 and *** for P<0.005). The results are expressed either as copy number or as ratios relative to 18 S rRNA, GAPDH, ATP50 and HPRT1. The middle panel (b2) represents FXN mRNA expressed in the muscle (group 2). The lower panel (b3) illustrates FXN mRNA expressed in the liver (group 1), but the histogram bar represents only one mouse for control and one mouse treated with recombinant virus (Tc).
Mentions: Northern blot analysis showed a significant increase of the FXN mRNA in the heart and muscles of the 9-day-old mice and in the liver of the 4-month-old mice (Figure 3A). Real-time reverse transcription-PCR (qRT-PCR) analyses of the same tissues expressed in terms of copy number only or as ratios with internal standards such as 18S rRNA, GAPDH, HPRT1 and ATP50 are presented in Figure 3B. These results confirmed that AAV9_3XFLAG-TALEfrat#8-VP64 was able to stimulate in vivo FXN transcription. The expression ratio of the treated mice relative to the controls was similar for the four internal standards used for the analysis of this experiment.

Bottom Line: Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters.The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied.These results corroborate our previous in vitro studies in the FRDA human fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique Humaine, Axe Neurosciences, Centre de Recherche du Centre Hospitalier de Universitaire de Québec-Université Laval, Québec City, QC, Canada.

ABSTRACT
Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases.

No MeSH data available.


Related in: MedlinePlus