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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

Effect of AAV9_3XFLAG-TALEfrat#8-VP64 treatment on FXN mRNA analyzed in five tissues of YG8R mice (group 2) by qRT-PCR in relationship to the recombinant viral copy number found in these tissues. In (a), the qRT-PCR analysis of FXN mRNA from total RNA extracted from heart, muscle, brain, kidney and liver of three control mice (saline) and three mice treated with 6 × 1012 vg. The FXN mRNA is expressed in copy number per μg total RNA (ave.±s.d.). Significant increases (**P<0.01) were observed in the heart and muscle. In (b), the AAV copy numbers per 40 ng of gDNA in the heart and muscle following injection of 6.0 × 1012 vg per mouse are illustrated. Each histobar represents an individual mouse. The copy numbers found in the heart and muscle in (b) parallel the upregulation of FXN mRNA observed in (a) in the same tissues.
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fig6: Effect of AAV9_3XFLAG-TALEfrat#8-VP64 treatment on FXN mRNA analyzed in five tissues of YG8R mice (group 2) by qRT-PCR in relationship to the recombinant viral copy number found in these tissues. In (a), the qRT-PCR analysis of FXN mRNA from total RNA extracted from heart, muscle, brain, kidney and liver of three control mice (saline) and three mice treated with 6 × 1012 vg. The FXN mRNA is expressed in copy number per μg total RNA (ave.±s.d.). Significant increases (**P<0.01) were observed in the heart and muscle. In (b), the AAV copy numbers per 40 ng of gDNA in the heart and muscle following injection of 6.0 × 1012 vg per mouse are illustrated. Each histobar represents an individual mouse. The copy numbers found in the heart and muscle in (b) parallel the upregulation of FXN mRNA observed in (a) in the same tissues.

Mentions: The brain of 9-day-old mice treated with a high dose (6 × 1012 vg per mouse) of recombinant virus showed no significant FXN mRNA upregulation relative to the control mice injected with saline (Figure 6a). However, the heart and muscle (Figure 6a) showed a significant upregulation of FXN mRNA following the AAV9_3XFLAG-TALEfrat#8-VP64 treatment. No upregulation of the FXN mRNA was observed in the liver and kidney (Figure 6a) even if more than 1000 copies of the recombinant virus per 40 ng of gDNA (Figure 6b) were detected in the liver of the young mice treated with the higher viral dose. This last observation is quite different from the upregulation of FXN mRNA observed in the liver of the 4-month-old mice treated at lower doses of recombinant virus (Figures 4b and c).


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)

Effect of AAV9_3XFLAG-TALEfrat#8-VP64 treatment on FXN mRNA analyzed in five tissues of YG8R mice (group 2) by qRT-PCR in relationship to the recombinant viral copy number found in these tissues. In (a), the qRT-PCR analysis of FXN mRNA from total RNA extracted from heart, muscle, brain, kidney and liver of three control mice (saline) and three mice treated with 6 × 1012 vg. The FXN mRNA is expressed in copy number per μg total RNA (ave.±s.d.). Significant increases (**P<0.01) were observed in the heart and muscle. In (b), the AAV copy numbers per 40 ng of gDNA in the heart and muscle following injection of 6.0 × 1012 vg per mouse are illustrated. Each histobar represents an individual mouse. The copy numbers found in the heart and muscle in (b) parallel the upregulation of FXN mRNA observed in (a) in the same tissues.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940929&req=5

fig6: Effect of AAV9_3XFLAG-TALEfrat#8-VP64 treatment on FXN mRNA analyzed in five tissues of YG8R mice (group 2) by qRT-PCR in relationship to the recombinant viral copy number found in these tissues. In (a), the qRT-PCR analysis of FXN mRNA from total RNA extracted from heart, muscle, brain, kidney and liver of three control mice (saline) and three mice treated with 6 × 1012 vg. The FXN mRNA is expressed in copy number per μg total RNA (ave.±s.d.). Significant increases (**P<0.01) were observed in the heart and muscle. In (b), the AAV copy numbers per 40 ng of gDNA in the heart and muscle following injection of 6.0 × 1012 vg per mouse are illustrated. Each histobar represents an individual mouse. The copy numbers found in the heart and muscle in (b) parallel the upregulation of FXN mRNA observed in (a) in the same tissues.
Mentions: The brain of 9-day-old mice treated with a high dose (6 × 1012 vg per mouse) of recombinant virus showed no significant FXN mRNA upregulation relative to the control mice injected with saline (Figure 6a). However, the heart and muscle (Figure 6a) showed a significant upregulation of FXN mRNA following the AAV9_3XFLAG-TALEfrat#8-VP64 treatment. No upregulation of the FXN mRNA was observed in the liver and kidney (Figure 6a) even if more than 1000 copies of the recombinant virus per 40 ng of gDNA (Figure 6b) were detected in the liver of the young mice treated with the higher viral dose. This last observation is quite different from the upregulation of FXN mRNA observed in the liver of the 4-month-old mice treated at lower doses of recombinant virus (Figures 4b and c).

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