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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

Detection of 3XFLAG-TALEfrat#8-VP64 and FXN proteins in the liver of 4-month-old YG8R mice (group 1). In all figures, proteins extracted from the liver: control mice (CON) received a saline injection; mice were treated either with a diluted dose of virus (1.2 × 1011 vg, Td) or with a high dose of virus (6.0 × 1011 vg, Tc). In (a) the 3XFLAG-TALEfrat#8-VP64 protein (90 kDa) was detected by western blot using an anti-3XFLAG monoclonal antibody; the expression was barely visible at the low viral dose (Td) but was more strongly expressed at the high viral dose (Tc). In (b), the western blot was made with a monoclonal antibody against human FXN (17 kDa). In the lower horizontal panels of (a and b), the expression of α-tubulin (55 kDa) was similar in the three samples. In (c), FXN expression detected with dipsticks was increased by 1.4-fold with a low dose (Td) and by 1.7-fold with a high dose (Tc) of the virus. A goat anti-mouse antibody (upper line) is included on all dipsticks as internal standard.
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fig5: Detection of 3XFLAG-TALEfrat#8-VP64 and FXN proteins in the liver of 4-month-old YG8R mice (group 1). In all figures, proteins extracted from the liver: control mice (CON) received a saline injection; mice were treated either with a diluted dose of virus (1.2 × 1011 vg, Td) or with a high dose of virus (6.0 × 1011 vg, Tc). In (a) the 3XFLAG-TALEfrat#8-VP64 protein (90 kDa) was detected by western blot using an anti-3XFLAG monoclonal antibody; the expression was barely visible at the low viral dose (Td) but was more strongly expressed at the high viral dose (Tc). In (b), the western blot was made with a monoclonal antibody against human FXN (17 kDa). In the lower horizontal panels of (a and b), the expression of α-tubulin (55 kDa) was similar in the three samples. In (c), FXN expression detected with dipsticks was increased by 1.4-fold with a low dose (Td) and by 1.7-fold with a high dose (Tc) of the virus. A goat anti-mouse antibody (upper line) is included on all dipsticks as internal standard.

Mentions: The 3XFLAG-TALEfrat#8-VP64 protein detected by western blot in the liver of the 4-month-old mice (Figure 5a) is parallel with the increased FXN protein expression determined either by western blot (Figure 5b) or by an enzyme-linked immunosorbent assay method (Figure 5c). These increases were ~1.4- to 1.7-fold, respectively, for diluted (1.2 × 1011 vg Td) and high (6 × 1011 vg Tc) doses. In the liver of the mice treated with a low (Td) or a high dose (Tc), a parallel increase was observed between the 3XFLAG-TALEfrat#8-VP64 protein detected by western blot (Figure 5a), the FXN mRNA measured by qRT-PCR (Figure 4b), the FXN protein estimated by Dipstick (Figure 5c) and the recombinant virus copy number (Figure 4c) observed in the liver of the same mouse in function of the dose tested. This last observation suggests that a minimum of 100 viral copies per 40 ng gDNA (Figure 4c) is necessary in the liver to increase FXN gene expression in vivo.


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)

Detection of 3XFLAG-TALEfrat#8-VP64 and FXN proteins in the liver of 4-month-old YG8R mice (group 1). In all figures, proteins extracted from the liver: control mice (CON) received a saline injection; mice were treated either with a diluted dose of virus (1.2 × 1011 vg, Td) or with a high dose of virus (6.0 × 1011 vg, Tc). In (a) the 3XFLAG-TALEfrat#8-VP64 protein (90 kDa) was detected by western blot using an anti-3XFLAG monoclonal antibody; the expression was barely visible at the low viral dose (Td) but was more strongly expressed at the high viral dose (Tc). In (b), the western blot was made with a monoclonal antibody against human FXN (17 kDa). In the lower horizontal panels of (a and b), the expression of α-tubulin (55 kDa) was similar in the three samples. In (c), FXN expression detected with dipsticks was increased by 1.4-fold with a low dose (Td) and by 1.7-fold with a high dose (Tc) of the virus. A goat anti-mouse antibody (upper line) is included on all dipsticks as internal standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Detection of 3XFLAG-TALEfrat#8-VP64 and FXN proteins in the liver of 4-month-old YG8R mice (group 1). In all figures, proteins extracted from the liver: control mice (CON) received a saline injection; mice were treated either with a diluted dose of virus (1.2 × 1011 vg, Td) or with a high dose of virus (6.0 × 1011 vg, Tc). In (a) the 3XFLAG-TALEfrat#8-VP64 protein (90 kDa) was detected by western blot using an anti-3XFLAG monoclonal antibody; the expression was barely visible at the low viral dose (Td) but was more strongly expressed at the high viral dose (Tc). In (b), the western blot was made with a monoclonal antibody against human FXN (17 kDa). In the lower horizontal panels of (a and b), the expression of α-tubulin (55 kDa) was similar in the three samples. In (c), FXN expression detected with dipsticks was increased by 1.4-fold with a low dose (Td) and by 1.7-fold with a high dose (Tc) of the virus. A goat anti-mouse antibody (upper line) is included on all dipsticks as internal standard.
Mentions: The 3XFLAG-TALEfrat#8-VP64 protein detected by western blot in the liver of the 4-month-old mice (Figure 5a) is parallel with the increased FXN protein expression determined either by western blot (Figure 5b) or by an enzyme-linked immunosorbent assay method (Figure 5c). These increases were ~1.4- to 1.7-fold, respectively, for diluted (1.2 × 1011 vg Td) and high (6 × 1011 vg Tc) doses. In the liver of the mice treated with a low (Td) or a high dose (Tc), a parallel increase was observed between the 3XFLAG-TALEfrat#8-VP64 protein detected by western blot (Figure 5a), the FXN mRNA measured by qRT-PCR (Figure 4b), the FXN protein estimated by Dipstick (Figure 5c) and the recombinant virus copy number (Figure 4c) observed in the liver of the same mouse in function of the dose tested. This last observation suggests that a minimum of 100 viral copies per 40 ng gDNA (Figure 4c) is necessary in the liver to increase FXN gene expression in vivo.

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