Limits...
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

Schema of the AAV9_3XFLAG-TALEfrat#8-VP64, detection of the transgenic protein and vector delivery protocol. (a) The plasmid contains the AAV-ITRs and the CAG promoter driving the 3XFLAG-TALEfrat#8-VP64 transgene (3.6 kb). The TALE contains 13 RVDs and 2 nuclear localization signals (NLS). This plasmid was used to produce the AAV serotype 9 vector. (b) The transgenic protein expression in 293 T cells transfected with pAAV9 TALEfrat#8-VP64 was detected by western blot with an anti-FLAG antibody. Total proteins of 10 and 25 μg were loaded in two wells, electrophoresed on a 10% SDS-polyacrylamide gel and electrotransferred on nitrocellulose membrane for the western blot. (c) Two groups of mice were used to realize the present study. Group 1 was composed of four 4-month-old YG8R mice, including two control mice injected intraperitoneally with saline and two mice injected intraperitoneally with 1.2 × 1011 or 6 × 1011 vg of AAV9_3XFLAG-TALEfrat#8-VP64. Group 2 composed of six 9-day-old YG8R mice, including three control mice injected intraperitoneally with saline and three mice injected intraperitoneally with 6 × 1012 vg of AAV9_3XFLAG-TALEfrat#8-VP64. All mice were killed 1 month later.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940929&req=5

fig1: Schema of the AAV9_3XFLAG-TALEfrat#8-VP64, detection of the transgenic protein and vector delivery protocol. (a) The plasmid contains the AAV-ITRs and the CAG promoter driving the 3XFLAG-TALEfrat#8-VP64 transgene (3.6 kb). The TALE contains 13 RVDs and 2 nuclear localization signals (NLS). This plasmid was used to produce the AAV serotype 9 vector. (b) The transgenic protein expression in 293 T cells transfected with pAAV9 TALEfrat#8-VP64 was detected by western blot with an anti-FLAG antibody. Total proteins of 10 and 25 μg were loaded in two wells, electrophoresed on a 10% SDS-polyacrylamide gel and electrotransferred on nitrocellulose membrane for the western blot. (c) Two groups of mice were used to realize the present study. Group 1 was composed of four 4-month-old YG8R mice, including two control mice injected intraperitoneally with saline and two mice injected intraperitoneally with 1.2 × 1011 or 6 × 1011 vg of AAV9_3XFLAG-TALEfrat#8-VP64. Group 2 composed of six 9-day-old YG8R mice, including three control mice injected intraperitoneally with saline and three mice injected intraperitoneally with 6 × 1012 vg of AAV9_3XFLAG-TALEfrat#8-VP64. All mice were killed 1 month later.

Mentions: Figure 1a represents the scheme of the AAV9 containing the 3XFLAG-TALEfrat#8-VP64 under the control of the CAG promoter (CMV early enhancer element, first exon and first intron of β-Actin and the splice acceptor of the rabbit β-Globin gene). The AAV9_3XFLAG-TALEfrat#8-VP64 plasmid was initially successfully tested in 293 T cells to verify by western blot with an anti-FLAG antibody the expression of a protein of expected molecular weight (i.e., ~ 90 kDa) (Figure 1b). The AAV9 vector was injected intraperitoneally with 1.2 × 1011 vg (viral genome) or with 6 × 1011 vg to two 4-month-old mice (group 1; Figure 1c) and with 6 × 1012 vg to three 9-day-old mice (group 2; Figure 1c). Two controls in group 1 and three controls in group 2 were injected with saline. The liver from the four mice in group 1 and the heart and the skeletal muscles from the six mice in group 2 were collected 1 month after the injection. The effect of AAV9_3XFLAG-TALEfrat#8-VP64 on the FXN expression in these tissues was then investigated.


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)

Schema of the AAV9_3XFLAG-TALEfrat#8-VP64, detection of the transgenic protein and vector delivery protocol. (a) The plasmid contains the AAV-ITRs and the CAG promoter driving the 3XFLAG-TALEfrat#8-VP64 transgene (3.6 kb). The TALE contains 13 RVDs and 2 nuclear localization signals (NLS). This plasmid was used to produce the AAV serotype 9 vector. (b) The transgenic protein expression in 293 T cells transfected with pAAV9 TALEfrat#8-VP64 was detected by western blot with an anti-FLAG antibody. Total proteins of 10 and 25 μg were loaded in two wells, electrophoresed on a 10% SDS-polyacrylamide gel and electrotransferred on nitrocellulose membrane for the western blot. (c) Two groups of mice were used to realize the present study. Group 1 was composed of four 4-month-old YG8R mice, including two control mice injected intraperitoneally with saline and two mice injected intraperitoneally with 1.2 × 1011 or 6 × 1011 vg of AAV9_3XFLAG-TALEfrat#8-VP64. Group 2 composed of six 9-day-old YG8R mice, including three control mice injected intraperitoneally with saline and three mice injected intraperitoneally with 6 × 1012 vg of AAV9_3XFLAG-TALEfrat#8-VP64. All mice were killed 1 month later.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Schema of the AAV9_3XFLAG-TALEfrat#8-VP64, detection of the transgenic protein and vector delivery protocol. (a) The plasmid contains the AAV-ITRs and the CAG promoter driving the 3XFLAG-TALEfrat#8-VP64 transgene (3.6 kb). The TALE contains 13 RVDs and 2 nuclear localization signals (NLS). This plasmid was used to produce the AAV serotype 9 vector. (b) The transgenic protein expression in 293 T cells transfected with pAAV9 TALEfrat#8-VP64 was detected by western blot with an anti-FLAG antibody. Total proteins of 10 and 25 μg were loaded in two wells, electrophoresed on a 10% SDS-polyacrylamide gel and electrotransferred on nitrocellulose membrane for the western blot. (c) Two groups of mice were used to realize the present study. Group 1 was composed of four 4-month-old YG8R mice, including two control mice injected intraperitoneally with saline and two mice injected intraperitoneally with 1.2 × 1011 or 6 × 1011 vg of AAV9_3XFLAG-TALEfrat#8-VP64. Group 2 composed of six 9-day-old YG8R mice, including three control mice injected intraperitoneally with saline and three mice injected intraperitoneally with 6 × 1012 vg of AAV9_3XFLAG-TALEfrat#8-VP64. All mice were killed 1 month later.
Mentions: Figure 1a represents the scheme of the AAV9 containing the 3XFLAG-TALEfrat#8-VP64 under the control of the CAG promoter (CMV early enhancer element, first exon and first intron of β-Actin and the splice acceptor of the rabbit β-Globin gene). The AAV9_3XFLAG-TALEfrat#8-VP64 plasmid was initially successfully tested in 293 T cells to verify by western blot with an anti-FLAG antibody the expression of a protein of expected molecular weight (i.e., ~ 90 kDa) (Figure 1b). The AAV9 vector was injected intraperitoneally with 1.2 × 1011 vg (viral genome) or with 6 × 1011 vg to two 4-month-old mice (group 1; Figure 1c) and with 6 × 1012 vg to three 9-day-old mice (group 2; Figure 1c). Two controls in group 1 and three controls in group 2 were injected with saline. The liver from the four mice in group 1 and the heart and the skeletal muscles from the six mice in group 2 were collected 1 month after the injection. The effect of AAV9_3XFLAG-TALEfrat#8-VP64 on the FXN expression in these tissues was then investigated.

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