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An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models.

Gérard C, Xiao X, Filali M, Coulombe Z, Arsenault M, Couet J, Li J, Drolet MC, Chapdelaine P, Chikh A, Tremblay JP - Mol Ther Methods Clin Dev (2014)

Bottom Line: This mutation leads to a reduced expression of frataxin.We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN).The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche du Centre Hospitalier Universitaire de Québec and Department of Molecular Medecine, Faculty of Medecine, Laval University , Québec, Canada.

ABSTRACT
Friedreich ataxia (FRDA) is a genetic disease due to increased repeats of the GAA trinucleotide in intron 1 of the frataxin gene. This mutation leads to a reduced expression of frataxin. We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN). This AAV was delivered by intraperitoneal (IP) injection to young conditionally knockout mice in which the frataxin gene had been knocked-out in some tissues during embryogenesis by breeding them with mice expressing the Cre recombinase gene under the muscle creatine kinase (MCK) or the neuron-specific enolase (NSE) promoter. In the first part of the study, different doses of virus were tested from 6 × 10(11) v.p. to 6 × 10(9) v.p. in NSE-cre mice and all leading to an increase in life spent of the mice. The higher and the lower dose were also tested in MCK-cre mice. A single administration of the AAV9-hFXN at 6 × 10(11) v.p. more than doubled the life of these mice. In fact the MCK-cre mice treated with the AAV9-hFXN were sacrificed for further molecular investigations at the age of 29 weeks without apparent symptoms. Echography analysis of the heart function clearly indicated that the cardiac systolic function was better preserved in the mice that received 6 × 10(11) v.p. of AAV9-hFXN. The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models. Thus, gene therapy with an AAV9-hFXN is a potential treatment of FRDA.

No MeSH data available.


Related in: MedlinePlus

Detection of the hFXN transgene in various tissues of neuron-specific enolase (NSE)-cre. (a) on the left: The human frataxin (hFXN) transgene was detected by polymerase chain reaction (PCR) in the DNA of muscles (M), L (liver), heart (H), kidney (K), and brain (B) of mice injected with AAV-hFXN at 6 × 1011 v.p. (named AAV-hFXN) and in 6 × 1010 v.p. (named d1/10) and in 3 × 1010 v.p. (named d1/20). a, on the right: The expression of the human frataxin transgene (hFXN) was then detected by RT-PCR in all these tissues. (b) Finally, an ELISA test (Dipstick) was made to detect the human FXN protein in skeletal muscle (M), liver (L), heart (H), kidney (K), and brain (B). The human frataxin protein was estimated in these tissues in NSE-cre mice treated with AAV9-hFXN at 6 × 1011 v.p. (n = 9), at 3 × 1011 v.p. (n = 4), at 6 × 1010 v.p. (n = 3), at 3 × 1010 v.p. (n = 3), at 1.2 × 1010 v.p. (n = 3), at 6 × 109 v.p. (n = 3). The presence of human frataxin was strong with the highest dose of virus and decreased in the tissues with the dilutions except for the heart where the frataxin was still strong at the lowest viral dose.
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fig2: Detection of the hFXN transgene in various tissues of neuron-specific enolase (NSE)-cre. (a) on the left: The human frataxin (hFXN) transgene was detected by polymerase chain reaction (PCR) in the DNA of muscles (M), L (liver), heart (H), kidney (K), and brain (B) of mice injected with AAV-hFXN at 6 × 1011 v.p. (named AAV-hFXN) and in 6 × 1010 v.p. (named d1/10) and in 3 × 1010 v.p. (named d1/20). a, on the right: The expression of the human frataxin transgene (hFXN) was then detected by RT-PCR in all these tissues. (b) Finally, an ELISA test (Dipstick) was made to detect the human FXN protein in skeletal muscle (M), liver (L), heart (H), kidney (K), and brain (B). The human frataxin protein was estimated in these tissues in NSE-cre mice treated with AAV9-hFXN at 6 × 1011 v.p. (n = 9), at 3 × 1011 v.p. (n = 4), at 6 × 1010 v.p. (n = 3), at 3 × 1010 v.p. (n = 3), at 1.2 × 1010 v.p. (n = 3), at 6 × 109 v.p. (n = 3). The presence of human frataxin was strong with the highest dose of virus and decreased in the tissues with the dilutions except for the heart where the frataxin was still strong at the lowest viral dose.

Mentions: An AAV9 coding for the human frataxin (AAV9-hFXN) was injected IP in NSE-cre mice between 5 and 9 days of age. Their organs were collected at the time of the sacrifice. A PCR for the human frataxin was made on genomic DNA to detect the presence of the human frataxin transgene. This transgene was detected in all the investigated tissues (i.e., brain, heart, muscle, kidney, and liver) for 6 × 1011 v.p. injected (Figure 2a on the left). Dilutions 1/10 and 1/20 were also tested in the same conditions. The human frataxin band was observed in muscle, liver, and heart for these dilutions. The intensity of the band decreased with the dilutions, but the strongest band was always detected in the heart. RT-PCR also produced similar results (Figure 2a on the right). For an injection of 6 × 1011 v.p., human frataxin expression was observed in all tissues tested, with the strongest band in the heart and the intensity of the bands were decreasing with the dilutions of the virus.


An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models.

Gérard C, Xiao X, Filali M, Coulombe Z, Arsenault M, Couet J, Li J, Drolet MC, Chapdelaine P, Chikh A, Tremblay JP - Mol Ther Methods Clin Dev (2014)

Detection of the hFXN transgene in various tissues of neuron-specific enolase (NSE)-cre. (a) on the left: The human frataxin (hFXN) transgene was detected by polymerase chain reaction (PCR) in the DNA of muscles (M), L (liver), heart (H), kidney (K), and brain (B) of mice injected with AAV-hFXN at 6 × 1011 v.p. (named AAV-hFXN) and in 6 × 1010 v.p. (named d1/10) and in 3 × 1010 v.p. (named d1/20). a, on the right: The expression of the human frataxin transgene (hFXN) was then detected by RT-PCR in all these tissues. (b) Finally, an ELISA test (Dipstick) was made to detect the human FXN protein in skeletal muscle (M), liver (L), heart (H), kidney (K), and brain (B). The human frataxin protein was estimated in these tissues in NSE-cre mice treated with AAV9-hFXN at 6 × 1011 v.p. (n = 9), at 3 × 1011 v.p. (n = 4), at 6 × 1010 v.p. (n = 3), at 3 × 1010 v.p. (n = 3), at 1.2 × 1010 v.p. (n = 3), at 6 × 109 v.p. (n = 3). The presence of human frataxin was strong with the highest dose of virus and decreased in the tissues with the dilutions except for the heart where the frataxin was still strong at the lowest viral dose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362356&req=5

fig2: Detection of the hFXN transgene in various tissues of neuron-specific enolase (NSE)-cre. (a) on the left: The human frataxin (hFXN) transgene was detected by polymerase chain reaction (PCR) in the DNA of muscles (M), L (liver), heart (H), kidney (K), and brain (B) of mice injected with AAV-hFXN at 6 × 1011 v.p. (named AAV-hFXN) and in 6 × 1010 v.p. (named d1/10) and in 3 × 1010 v.p. (named d1/20). a, on the right: The expression of the human frataxin transgene (hFXN) was then detected by RT-PCR in all these tissues. (b) Finally, an ELISA test (Dipstick) was made to detect the human FXN protein in skeletal muscle (M), liver (L), heart (H), kidney (K), and brain (B). The human frataxin protein was estimated in these tissues in NSE-cre mice treated with AAV9-hFXN at 6 × 1011 v.p. (n = 9), at 3 × 1011 v.p. (n = 4), at 6 × 1010 v.p. (n = 3), at 3 × 1010 v.p. (n = 3), at 1.2 × 1010 v.p. (n = 3), at 6 × 109 v.p. (n = 3). The presence of human frataxin was strong with the highest dose of virus and decreased in the tissues with the dilutions except for the heart where the frataxin was still strong at the lowest viral dose.
Mentions: An AAV9 coding for the human frataxin (AAV9-hFXN) was injected IP in NSE-cre mice between 5 and 9 days of age. Their organs were collected at the time of the sacrifice. A PCR for the human frataxin was made on genomic DNA to detect the presence of the human frataxin transgene. This transgene was detected in all the investigated tissues (i.e., brain, heart, muscle, kidney, and liver) for 6 × 1011 v.p. injected (Figure 2a on the left). Dilutions 1/10 and 1/20 were also tested in the same conditions. The human frataxin band was observed in muscle, liver, and heart for these dilutions. The intensity of the band decreased with the dilutions, but the strongest band was always detected in the heart. RT-PCR also produced similar results (Figure 2a on the right). For an injection of 6 × 1011 v.p., human frataxin expression was observed in all tissues tested, with the strongest band in the heart and the intensity of the bands were decreasing with the dilutions of the virus.

Bottom Line: This mutation leads to a reduced expression of frataxin.We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN).The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche du Centre Hospitalier Universitaire de Québec and Department of Molecular Medecine, Faculty of Medecine, Laval University , Québec, Canada.

ABSTRACT
Friedreich ataxia (FRDA) is a genetic disease due to increased repeats of the GAA trinucleotide in intron 1 of the frataxin gene. This mutation leads to a reduced expression of frataxin. We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN). This AAV was delivered by intraperitoneal (IP) injection to young conditionally knockout mice in which the frataxin gene had been knocked-out in some tissues during embryogenesis by breeding them with mice expressing the Cre recombinase gene under the muscle creatine kinase (MCK) or the neuron-specific enolase (NSE) promoter. In the first part of the study, different doses of virus were tested from 6 × 10(11) v.p. to 6 × 10(9) v.p. in NSE-cre mice and all leading to an increase in life spent of the mice. The higher and the lower dose were also tested in MCK-cre mice. A single administration of the AAV9-hFXN at 6 × 10(11) v.p. more than doubled the life of these mice. In fact the MCK-cre mice treated with the AAV9-hFXN were sacrificed for further molecular investigations at the age of 29 weeks without apparent symptoms. Echography analysis of the heart function clearly indicated that the cardiac systolic function was better preserved in the mice that received 6 × 10(11) v.p. of AAV9-hFXN. The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models. Thus, gene therapy with an AAV9-hFXN is a potential treatment of FRDA.

No MeSH data available.


Related in: MedlinePlus