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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

Dipstick analysis of the FXN protein in the heart and muscle of 9-day-old YG8R mice. (a) Eight Dipsticks are illustrated. On the top of each Dipstick, a positive control goat anti-mouse antibody line is included as internal standard to ensure that the same amount of proteins has been used for each assay. The four Dipsticks on the left side are dosing muscle FXN protein and four Dipsticks on the right side are dosing heart FXN protein. The illustrated Dipsticks are dosing proteins from two control (CON) mice that received a saline injection and two treated (T) 9-day-old YG8R mice (i.e., group 2) that received 1.6 × 1012 vg AAV9_3XFLAG-TALEfrat#8-VP64. (b) A concentration curve was made to semiquantify the Dipstick results. The FXN protein was increased 1.6-fold in the treated (T, N=3, ave.±s.d.) heart and 2.1-fold in the treated muscles relative to control (CON, N=3, ave.±s.d.). However, these changes did not reach a significant level.
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fig7: Dipstick analysis of the FXN protein in the heart and muscle of 9-day-old YG8R mice. (a) Eight Dipsticks are illustrated. On the top of each Dipstick, a positive control goat anti-mouse antibody line is included as internal standard to ensure that the same amount of proteins has been used for each assay. The four Dipsticks on the left side are dosing muscle FXN protein and four Dipsticks on the right side are dosing heart FXN protein. The illustrated Dipsticks are dosing proteins from two control (CON) mice that received a saline injection and two treated (T) 9-day-old YG8R mice (i.e., group 2) that received 1.6 × 1012 vg AAV9_3XFLAG-TALEfrat#8-VP64. (b) A concentration curve was made to semiquantify the Dipstick results. The FXN protein was increased 1.6-fold in the treated (T, N=3, ave.±s.d.) heart and 2.1-fold in the treated muscles relative to control (CON, N=3, ave.±s.d.). However, these changes did not reach a significant level.

Mentions: FXN protein increases of, respectively, 1.6- and 2.1-fold were detected by Dipstick analysis in the heart and in the skeletal muscle of 9-day-old mice (group 2) treated with 6 × 1012 vg AAV9_3XFLAGTALEfrat#8-VP64 (Figures 7a and b). However, these increases did not reach a significant level.


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)

Dipstick analysis of the FXN protein in the heart and muscle of 9-day-old YG8R mice. (a) Eight Dipsticks are illustrated. On the top of each Dipstick, a positive control goat anti-mouse antibody line is included as internal standard to ensure that the same amount of proteins has been used for each assay. The four Dipsticks on the left side are dosing muscle FXN protein and four Dipsticks on the right side are dosing heart FXN protein. The illustrated Dipsticks are dosing proteins from two control (CON) mice that received a saline injection and two treated (T) 9-day-old YG8R mice (i.e., group 2) that received 1.6 × 1012 vg AAV9_3XFLAG-TALEfrat#8-VP64. (b) A concentration curve was made to semiquantify the Dipstick results. The FXN protein was increased 1.6-fold in the treated (T, N=3, ave.±s.d.) heart and 2.1-fold in the treated muscles relative to control (CON, N=3, ave.±s.d.). However, these changes did not reach a significant level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940929&req=5

fig7: Dipstick analysis of the FXN protein in the heart and muscle of 9-day-old YG8R mice. (a) Eight Dipsticks are illustrated. On the top of each Dipstick, a positive control goat anti-mouse antibody line is included as internal standard to ensure that the same amount of proteins has been used for each assay. The four Dipsticks on the left side are dosing muscle FXN protein and four Dipsticks on the right side are dosing heart FXN protein. The illustrated Dipsticks are dosing proteins from two control (CON) mice that received a saline injection and two treated (T) 9-day-old YG8R mice (i.e., group 2) that received 1.6 × 1012 vg AAV9_3XFLAG-TALEfrat#8-VP64. (b) A concentration curve was made to semiquantify the Dipstick results. The FXN protein was increased 1.6-fold in the treated (T, N=3, ave.±s.d.) heart and 2.1-fold in the treated muscles relative to control (CON, N=3, ave.±s.d.). However, these changes did not reach a significant level.
Mentions: FXN protein increases of, respectively, 1.6- and 2.1-fold were detected by Dipstick analysis in the heart and in the skeletal muscle of 9-day-old mice (group 2) treated with 6 × 1012 vg AAV9_3XFLAGTALEfrat#8-VP64 (Figures 7a and b). However, these increases did not reach a significant level.

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