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Gene Therapy Corrects Mitochondrial Dysfunction in Hematopoietic Progenitor Cells and Fibroblasts from Coq9R239X Mice.

Barriocanal-Casado E, Cueto-Ureña C, Benabdellah K, Gutiérrez-Guerrero A, Cobo M, Hidalgo-Gutiérrez A, Rodríguez-Sevilla JJ, Martín F, López LC - PLoS ONE (2016)

Bottom Line: A group of target diseases for these therapeutic strategies are mitochondrial encephalopathies due to mutations in nuclear DNA genes.Ectopic over-expression of Coq9 in both cell types restored the CoQ biosynthetic pathway and mitochondrial function, improving the fitness of the transduced cells.These results show the potential of the CCoq9WP lentiviral vector as a tool for gene therapy to treat mitochondrial encephalopathies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Facultad de Medicina, Universidad de Granada, Granada, Spain.

ABSTRACT
Recent clinical trials have shown that in vivo and ex vivo gene therapy strategies can be an option for the treatment of several neurological disorders. Both strategies require efficient and safe vectors to 1) deliver the therapeutic gene directly into the CNS or 2) to genetically modify stem cells that will be used as Trojan horses for the systemic delivery of the therapeutic protein. A group of target diseases for these therapeutic strategies are mitochondrial encephalopathies due to mutations in nuclear DNA genes. In this study, we have developed a lentiviral vector (CCoq9WP) able to overexpress Coq9 mRNA and COQ9 protein in mouse embryonic fibroblasts (MEFs) and hematopoietic progenitor cells (HPCs) from Coq9R239X mice, an animal model of mitochondrial encephalopathy due to primary Coenzyme Q (CoQ) deficiency. Ectopic over-expression of Coq9 in both cell types restored the CoQ biosynthetic pathway and mitochondrial function, improving the fitness of the transduced cells. These results show the potential of the CCoq9WP lentiviral vector as a tool for gene therapy to treat mitochondrial encephalopathies.

No MeSH data available.


Related in: MedlinePlus

Lack of DMQ9 and increase of CoQ9 in Coq9R239X MEFs after transduction with CCoq9WP vector.Levels of CoQ9 (A) and DMQ9/CoQ9 ratio (B). Representative chromatographs of the three different groups (C-E). Q: 1 hit in MEFs, 200 μl, non-concentrated; R: 1 hit in MEFs, 100 μl, 10x-concentrated; S: 1 hit in MEFs, 25 μl, 10x-concentrated; T: 1 hit in MEFs, 5 μl, 10x-concentrated. Data are expressed as mean ± SD. *P < 0.05, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; ***P < 0.001, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; +++P < 0.001, Coq9R239X-CCoq9WP cells versus Coq9R239X cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group).
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pone.0158344.g003: Lack of DMQ9 and increase of CoQ9 in Coq9R239X MEFs after transduction with CCoq9WP vector.Levels of CoQ9 (A) and DMQ9/CoQ9 ratio (B). Representative chromatographs of the three different groups (C-E). Q: 1 hit in MEFs, 200 μl, non-concentrated; R: 1 hit in MEFs, 100 μl, 10x-concentrated; S: 1 hit in MEFs, 25 μl, 10x-concentrated; T: 1 hit in MEFs, 5 μl, 10x-concentrated. Data are expressed as mean ± SD. *P < 0.05, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; ***P < 0.001, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; +++P < 0.001, Coq9R239X-CCoq9WP cells versus Coq9R239X cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group).

Mentions: COQ9 is needed for the stability and functional activity of the hydroxylase COQ7 [3, 4]. Thus, the Coq9R239X mouse model shows a severe reduction in the tissue levels of COQ7 and accumulation of DMQ9, the substrate of the reaction catalyzed by COQ7 (S1 Fig) [3, 4]. This pattern was also observed in MEFs and mHPCs from Coq9R239X mice (Fig 2A and 2B). The overexpression of COQ9 in transduced Coq9R239X MEFs and mHPCs induced an increase in COQ7 levels, which were even higher than the levels observed in control cells (Fig 2A and 2B). As a result, DMQ9 does not accumulate in CCoq9WP LV-transduced Coq9R239X cells (Figs 3B, 3C, 3D, 3E, 4B, 4C, 4D and 4E) and the levels of CoQ9, the final product of the pathway, were normalized (Figs 3A and 4A). In both wild-type and CCoq9WP LV-transduced Coq9R239X mHPCs, we also observe higher levels of COQ7 and CoQ9 over the time (Figs 2B and 4A; S1 and S2 Tables). This was in parallel to the differentiation of mHPCs during culture time (S2 Fig).


Gene Therapy Corrects Mitochondrial Dysfunction in Hematopoietic Progenitor Cells and Fibroblasts from Coq9R239X Mice.

Barriocanal-Casado E, Cueto-Ureña C, Benabdellah K, Gutiérrez-Guerrero A, Cobo M, Hidalgo-Gutiérrez A, Rodríguez-Sevilla JJ, Martín F, López LC - PLoS ONE (2016)

Lack of DMQ9 and increase of CoQ9 in Coq9R239X MEFs after transduction with CCoq9WP vector.Levels of CoQ9 (A) and DMQ9/CoQ9 ratio (B). Representative chromatographs of the three different groups (C-E). Q: 1 hit in MEFs, 200 μl, non-concentrated; R: 1 hit in MEFs, 100 μl, 10x-concentrated; S: 1 hit in MEFs, 25 μl, 10x-concentrated; T: 1 hit in MEFs, 5 μl, 10x-concentrated. Data are expressed as mean ± SD. *P < 0.05, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; ***P < 0.001, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; +++P < 0.001, Coq9R239X-CCoq9WP cells versus Coq9R239X cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920430&req=5

pone.0158344.g003: Lack of DMQ9 and increase of CoQ9 in Coq9R239X MEFs after transduction with CCoq9WP vector.Levels of CoQ9 (A) and DMQ9/CoQ9 ratio (B). Representative chromatographs of the three different groups (C-E). Q: 1 hit in MEFs, 200 μl, non-concentrated; R: 1 hit in MEFs, 100 μl, 10x-concentrated; S: 1 hit in MEFs, 25 μl, 10x-concentrated; T: 1 hit in MEFs, 5 μl, 10x-concentrated. Data are expressed as mean ± SD. *P < 0.05, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; ***P < 0.001, Coq9R239X and Coq9R239X-CCoq9WP cells versus Coq9+/+ cells; +++P < 0.001, Coq9R239X-CCoq9WP cells versus Coq9R239X cells; (one-way ANOVA with a Tukey's post hoc test; n = 4–6 for each group).
Mentions: COQ9 is needed for the stability and functional activity of the hydroxylase COQ7 [3, 4]. Thus, the Coq9R239X mouse model shows a severe reduction in the tissue levels of COQ7 and accumulation of DMQ9, the substrate of the reaction catalyzed by COQ7 (S1 Fig) [3, 4]. This pattern was also observed in MEFs and mHPCs from Coq9R239X mice (Fig 2A and 2B). The overexpression of COQ9 in transduced Coq9R239X MEFs and mHPCs induced an increase in COQ7 levels, which were even higher than the levels observed in control cells (Fig 2A and 2B). As a result, DMQ9 does not accumulate in CCoq9WP LV-transduced Coq9R239X cells (Figs 3B, 3C, 3D, 3E, 4B, 4C, 4D and 4E) and the levels of CoQ9, the final product of the pathway, were normalized (Figs 3A and 4A). In both wild-type and CCoq9WP LV-transduced Coq9R239X mHPCs, we also observe higher levels of COQ7 and CoQ9 over the time (Figs 2B and 4A; S1 and S2 Tables). This was in parallel to the differentiation of mHPCs during culture time (S2 Fig).

Bottom Line: A group of target diseases for these therapeutic strategies are mitochondrial encephalopathies due to mutations in nuclear DNA genes.Ectopic over-expression of Coq9 in both cell types restored the CoQ biosynthetic pathway and mitochondrial function, improving the fitness of the transduced cells.These results show the potential of the CCoq9WP lentiviral vector as a tool for gene therapy to treat mitochondrial encephalopathies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Facultad de Medicina, Universidad de Granada, Granada, Spain.

ABSTRACT
Recent clinical trials have shown that in vivo and ex vivo gene therapy strategies can be an option for the treatment of several neurological disorders. Both strategies require efficient and safe vectors to 1) deliver the therapeutic gene directly into the CNS or 2) to genetically modify stem cells that will be used as Trojan horses for the systemic delivery of the therapeutic protein. A group of target diseases for these therapeutic strategies are mitochondrial encephalopathies due to mutations in nuclear DNA genes. In this study, we have developed a lentiviral vector (CCoq9WP) able to overexpress Coq9 mRNA and COQ9 protein in mouse embryonic fibroblasts (MEFs) and hematopoietic progenitor cells (HPCs) from Coq9R239X mice, an animal model of mitochondrial encephalopathy due to primary Coenzyme Q (CoQ) deficiency. Ectopic over-expression of Coq9 in both cell types restored the CoQ biosynthetic pathway and mitochondrial function, improving the fitness of the transduced cells. These results show the potential of the CCoq9WP lentiviral vector as a tool for gene therapy to treat mitochondrial encephalopathies.

No MeSH data available.


Related in: MedlinePlus