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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 by qRT-PCR in YG8R mice (group 1). (a and b) The qRT-PCR analysis of FXN mRNA from total RNA extracted from the brain and liver of two control mice (saline) and mice treated with (1.2 × 1011 vg, Td) or (6.0 × 1011 vg, Tc), respectively. The FXN mRNA is expressed as copy number per μg total RNA (left panel), ratio FXN/18 S (middle panel) and FXN/GAPDH right panel. (c) Illustrates the copy number (values within parentheses) of the recombinant virus found in the brain, liver, mus cle, heart, kidney of group 1 mice, followed by treatment with two different doses (1.2 × 1011 vg, Td) and (6.0 × 1011 vg, Tc). Note that each histobar represents the value of one mouse.
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fig4: Effect of AAV9_3XFLAG-TALEfrat#8-VP64 treatment on FXN mRNA analyzed by qRT-PCR in YG8R mice (group 1). (a and b) The qRT-PCR analysis of FXN mRNA from total RNA extracted from the brain and liver of two control mice (saline) and mice treated with (1.2 × 1011 vg, Td) or (6.0 × 1011 vg, Tc), respectively. The FXN mRNA is expressed as copy number per μg total RNA (left panel), ratio FXN/18 S (middle panel) and FXN/GAPDH right panel. (c) Illustrates the copy number (values within parentheses) of the recombinant virus found in the brain, liver, mus cle, heart, kidney of group 1 mice, followed by treatment with two different doses (1.2 × 1011 vg, Td) and (6.0 × 1011 vg, Tc). Note that each histobar represents the value of one mouse.

Mentions: As shown in Figure 4, no FXN mRNA upregulation was observed in the brain between the controls and the treated mice (Figure 4a). However, Figure 4b suggests an FXN mRNA upregulation in the liver of the treated mouse at two doses tested. In the liver, there seems to be a relationship between the number of recombinant viral copy (Figure 4c) and the level of FXN mRNA upregulation (Figure 4b) in relation with the doses used: (6 × 1011 vg per mouse, Tc) and (1.2 × 1011 vg per mouse, Td). Figure 4c indicates that the liver and the heart are the main tissues targeted by the AAV9_3XFLAG-TALEfrat#8-VP64 as indicated by the high copy number in these tissues, whereas the muscle, kidney and brain showed very low copy numbers.


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 by qRT-PCR in YG8R mice (group 1). (a and b) The qRT-PCR analysis of FXN mRNA from total RNA extracted from the brain and liver of two control mice (saline) and mice treated with (1.2 × 1011 vg, Td) or (6.0 × 1011 vg, Tc), respectively. The FXN mRNA is expressed as copy number per μg total RNA (left panel), ratio FXN/18 S (middle panel) and FXN/GAPDH right panel. (c) Illustrates the copy number (values within parentheses) of the recombinant virus found in the brain, liver, mus cle, heart, kidney of group 1 mice, followed by treatment with two different doses (1.2 × 1011 vg, Td) and (6.0 × 1011 vg, Tc). Note that each histobar represents the value of one mouse.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Effect of AAV9_3XFLAG-TALEfrat#8-VP64 treatment on FXN mRNA analyzed by qRT-PCR in YG8R mice (group 1). (a and b) The qRT-PCR analysis of FXN mRNA from total RNA extracted from the brain and liver of two control mice (saline) and mice treated with (1.2 × 1011 vg, Td) or (6.0 × 1011 vg, Tc), respectively. The FXN mRNA is expressed as copy number per μg total RNA (left panel), ratio FXN/18 S (middle panel) and FXN/GAPDH right panel. (c) Illustrates the copy number (values within parentheses) of the recombinant virus found in the brain, liver, mus cle, heart, kidney of group 1 mice, followed by treatment with two different doses (1.2 × 1011 vg, Td) and (6.0 × 1011 vg, Tc). Note that each histobar represents the value of one mouse.
Mentions: As shown in Figure 4, no FXN mRNA upregulation was observed in the brain between the controls and the treated mice (Figure 4a). However, Figure 4b suggests an FXN mRNA upregulation in the liver of the treated mouse at two doses tested. In the liver, there seems to be a relationship between the number of recombinant viral copy (Figure 4c) and the level of FXN mRNA upregulation (Figure 4b) in relation with the doses used: (6 × 1011 vg per mouse, Tc) and (1.2 × 1011 vg per mouse, Td). Figure 4c indicates that the liver and the heart are the main tissues targeted by the AAV9_3XFLAG-TALEfrat#8-VP64 as indicated by the high copy number in these tissues, whereas the muscle, kidney and brain showed very low copy numbers.

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