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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

(a) Detection of AAV9_3XFLAG-TALEfrat#8-VP64 transgene and protein in the muscle, heart and liver of YG8R mice. Muscle (M), liver (L) and heart H) were removed from 4-month-old mice treated with 6 × 1011 vg (left panel), from 9-day-old mice treated with 6 × 1012 vg (right panel) or from control mice injected with saline (middle panel). The expected 446 pb PCR product was strongly detectable in the liver but barely detectable in the muscles and heart of 4-month-old mice. The amplicon was also strongly detected in the three tissues of the 9-day-old mice. (b) Proteins extracted from the liver of 4-month-old (left panel) and from the heart of 9-day-old (right panel) YG8R mice were loaded on a 10% SDS-polyacrylamide gel. In both panels, the left lane contains proteins from a control mouse treated with saline and the right lane contains proteins from mice injected intraperitoneally with AAV9_3XFLAG-TALEfrat#8-VP64. The transgenic protein (90 kDa) was detected by western blot with a monoclonal antibody against the 3XFLAG in tissues of treated mice but not in control tissues.
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fig2: (a) Detection of AAV9_3XFLAG-TALEfrat#8-VP64 transgene and protein in the muscle, heart and liver of YG8R mice. Muscle (M), liver (L) and heart H) were removed from 4-month-old mice treated with 6 × 1011 vg (left panel), from 9-day-old mice treated with 6 × 1012 vg (right panel) or from control mice injected with saline (middle panel). The expected 446 pb PCR product was strongly detectable in the liver but barely detectable in the muscles and heart of 4-month-old mice. The amplicon was also strongly detected in the three tissues of the 9-day-old mice. (b) Proteins extracted from the liver of 4-month-old (left panel) and from the heart of 9-day-old (right panel) YG8R mice were loaded on a 10% SDS-polyacrylamide gel. In both panels, the left lane contains proteins from a control mouse treated with saline and the right lane contains proteins from mice injected intraperitoneally with AAV9_3XFLAG-TALEfrat#8-VP64. The transgenic protein (90 kDa) was detected by western blot with a monoclonal antibody against the 3XFLAG in tissues of treated mice but not in control tissues.

Mentions: A total of 1.2 × 1011 or 6 × 1011 vg AAV9_3XFLAG-TALEfrat#8-VP64 were injected intraperitoneally in 4-month-old YG8R mice and 6 × 1012 vg were injected in 9-day-old YG8R mice. In both groups of mice, the transgene was detected by PCR amplification with specific primers corresponding to the C terminal of the TALE gene producing a 446 pb band (Figure 2a). However, in the 4-month-old mice (group 1), the signal intensity of the PCR band was stronger in the liver and weaker in the heart and the skeletal muscle. In contrast, in the 9-day-old mice (group 2), a strong PCR amplification was observed in the three tissues (muscle, heart and liver). The 3XFLAG-TALEfrat#8-VP64 protein (Mw ~90 kDa) was detected by western blot in the liver (4-month-old mice, group 1) and in the heart (9-day-old mice, group 2) (Figure 2b).


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)

(a) Detection of AAV9_3XFLAG-TALEfrat#8-VP64 transgene and protein in the muscle, heart and liver of YG8R mice. Muscle (M), liver (L) and heart H) were removed from 4-month-old mice treated with 6 × 1011 vg (left panel), from 9-day-old mice treated with 6 × 1012 vg (right panel) or from control mice injected with saline (middle panel). The expected 446 pb PCR product was strongly detectable in the liver but barely detectable in the muscles and heart of 4-month-old mice. The amplicon was also strongly detected in the three tissues of the 9-day-old mice. (b) Proteins extracted from the liver of 4-month-old (left panel) and from the heart of 9-day-old (right panel) YG8R mice were loaded on a 10% SDS-polyacrylamide gel. In both panels, the left lane contains proteins from a control mouse treated with saline and the right lane contains proteins from mice injected intraperitoneally with AAV9_3XFLAG-TALEfrat#8-VP64. The transgenic protein (90 kDa) was detected by western blot with a monoclonal antibody against the 3XFLAG in tissues of treated mice but not in control tissues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: (a) Detection of AAV9_3XFLAG-TALEfrat#8-VP64 transgene and protein in the muscle, heart and liver of YG8R mice. Muscle (M), liver (L) and heart H) were removed from 4-month-old mice treated with 6 × 1011 vg (left panel), from 9-day-old mice treated with 6 × 1012 vg (right panel) or from control mice injected with saline (middle panel). The expected 446 pb PCR product was strongly detectable in the liver but barely detectable in the muscles and heart of 4-month-old mice. The amplicon was also strongly detected in the three tissues of the 9-day-old mice. (b) Proteins extracted from the liver of 4-month-old (left panel) and from the heart of 9-day-old (right panel) YG8R mice were loaded on a 10% SDS-polyacrylamide gel. In both panels, the left lane contains proteins from a control mouse treated with saline and the right lane contains proteins from mice injected intraperitoneally with AAV9_3XFLAG-TALEfrat#8-VP64. The transgenic protein (90 kDa) was detected by western blot with a monoclonal antibody against the 3XFLAG in tissues of treated mice but not in control tissues.
Mentions: A total of 1.2 × 1011 or 6 × 1011 vg AAV9_3XFLAG-TALEfrat#8-VP64 were injected intraperitoneally in 4-month-old YG8R mice and 6 × 1012 vg were injected in 9-day-old YG8R mice. In both groups of mice, the transgene was detected by PCR amplification with specific primers corresponding to the C terminal of the TALE gene producing a 446 pb band (Figure 2a). However, in the 4-month-old mice (group 1), the signal intensity of the PCR band was stronger in the liver and weaker in the heart and the skeletal muscle. In contrast, in the 9-day-old mice (group 2), a strong PCR amplification was observed in the three tissues (muscle, heart and liver). The 3XFLAG-TALEfrat#8-VP64 protein (Mw ~90 kDa) was detected by western blot in the liver (4-month-old mice, group 1) and in the heart (9-day-old mice, group 2) (Figure 2b).

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