Final Report Summary - MITO BY-PASS (Molecular by-pass therapy for mitochondrial dysfunction)
The C. intestinalis AOX was engineered for conditional expression in Drosophila melanogaster (Fernandez-Ayala et al. 2009). Ubiquitous AOX expression in Drosophila at the level of a typical abundant mRNA appeared to be benign. The protein was stable and correctly targeted to mitochondria, conferring substantial cyanide resistance to mitochondrial substrate oxidation in vitro. As in human cells, it appeared to be enzymatically inert in the absence of a cytochrome pathway inhibitor (Fernandez-Ayala et al. 2009). Consistent with this, it had only a minimal effect on development with a slightly increased weight loss experienced by young adult flies, suggestive of only a minimal drop in the overall efficiency of catabolism. AOX-expressing flies were also fertile, indicative that AOX expression clearly does not compromise any somatic functions required for fertility in this model organism.
In keeping with the free radical theory of aging, we used the AOX to investigate the role of mitochondrial superoxide overproduction in the aging of the flies, and established that mitochondrial superoxide production correlates with but does not directly regulate lifespan in Drosophila. According to this study (Sanz et al., 2010), mtDNA influences longevity in (female) flies, but does so independently of superoxide production which was maintained at a low level by the AOX. More recently, the C. intestinalis AOX gene was successfully expressed in the mouse (El-Khoury et al. 2013). The gene was recoded to maximize its expression and introduced into early mouse embryos by germ line lentiviral transduction. It was placed under the control of the ubiquitously active, chimeric cytomegalovirus/β-actin/β-globin promoter, together with the Woodchuck hepatitis virus post-transcriptional regulatory element, in order to further enhance AOX transgene expression. This was the first demonstration that a functional AOX can be expressed in a mammal and transmitted between generations, conferring significant cyanide resistance to mitochondrial substrate oxidation and tissue respiration as well as the whole organism (El-Khoury et al. 2013). The enzyme was targeted to the mitochondria but did not interfere or compete significantly with the cytochrome pathway. The AOX in this mouse (MitAOX mouse) was enzymatically functional only upon blockade of the latter, when the pool of ubiquinone is expected to become highly reduced. Altogether, the expression of the AOX did not result in any deleterious consequence, while spectacularly increasing the survival of the mice in the presence of a lethal concentration of gaseous cyanide. Thus, the proposed protective mechanism provided by the AOX to organisms naturally harbouring the enzyme was preserved when the oxidase was expressed in a mammal (El-Khoury et al. 2013).
From data that we have accumulated over the past few years, it appears that the predictions made on the potential use of AOX (Dassa et al., 2009a; Rustin and Jacobs, 2009) have been verified. Firstly, the expression of AOX in human cultured cells, in the fly and in the mouse is essentially innocuous. Secondly, it renders human cells, flies and mice resistant to inhibitors of the cytochrome pathway, such as antimycin or cyanide. Thirdly, it prevents superoxide overproduction triggered by an excessive reduction of the RC. As a result, in accordance with our fourth prediction, it offers an efficient way to prevent the consequences of a wide set of genetic or environmentally determined lesions targeting the cytochrome pathway in model systems. Based on this body of data, it is now possible to define additional perspectives, taking advantage of AOX expression. It is tempting to use the MitAOX mouse to investigate the long list of pathological conditions where overproduction of superoxide by mitochondria has been postulated to be instrumental (Fernandez-Checa et al., 2010), especially when already modelled in the mouse.
This includes, for example, ageing, but also many types of physiological insult affecting mitochondrial function in diverse tissues. As an alternative to the tissue restricted AOX-expressing mouse, the use of inhalable or injectable AOX, including its mitochondrial targeting sequence, delivered by a viral or similar vector, can also be considered. Recent progress made in ensuring the safety and longevity of viral vectors (Bouaita et al., 2012) makes this approach particularly attractive, as it might allow the therapeutic use of the AOX gene.
The findings with Drosophila already suggest that AOX expression could be of benefit in a wide spectrum of OXPHOS disorders. However, even in the mouse, a number of questions remain to be answered before it is possible to conclude that AOX expression is a feasible therapeutic strategy. In particular, as the AOX gene we used so far derives from C. intestinalis which lives in highly oxygenated (intertidal zone) and cold water (<20°C), we have to consider the possibility that different temperature and oxygen tension in mammalian organs might affect the functionality of the AOX, and thus its capacity to counteract RC deficiencies, although the first data with regard to the brain are highly encouraging (El-Khoury et al., 2013).
In keeping with the free radical theory of aging, we used the AOX to investigate the role of mitochondrial superoxide overproduction in the aging of the flies, and established that mitochondrial superoxide production correlates with but does not directly regulate lifespan in Drosophila. According to this study (Sanz et al., 2010), mtDNA influences longevity in (female) flies, but does so independently of superoxide production which was maintained at a low level by the AOX. More recently, the C. intestinalis AOX gene was successfully expressed in the mouse (El-Khoury et al. 2013). The gene was recoded to maximize its expression and introduced into early mouse embryos by germ line lentiviral transduction. It was placed under the control of the ubiquitously active, chimeric cytomegalovirus/β-actin/β-globin promoter, together with the Woodchuck hepatitis virus post-transcriptional regulatory element, in order to further enhance AOX transgene expression. This was the first demonstration that a functional AOX can be expressed in a mammal and transmitted between generations, conferring significant cyanide resistance to mitochondrial substrate oxidation and tissue respiration as well as the whole organism (El-Khoury et al. 2013). The enzyme was targeted to the mitochondria but did not interfere or compete significantly with the cytochrome pathway. The AOX in this mouse (MitAOX mouse) was enzymatically functional only upon blockade of the latter, when the pool of ubiquinone is expected to become highly reduced. Altogether, the expression of the AOX did not result in any deleterious consequence, while spectacularly increasing the survival of the mice in the presence of a lethal concentration of gaseous cyanide. Thus, the proposed protective mechanism provided by the AOX to organisms naturally harbouring the enzyme was preserved when the oxidase was expressed in a mammal (El-Khoury et al. 2013).
From data that we have accumulated over the past few years, it appears that the predictions made on the potential use of AOX (Dassa et al., 2009a; Rustin and Jacobs, 2009) have been verified. Firstly, the expression of AOX in human cultured cells, in the fly and in the mouse is essentially innocuous. Secondly, it renders human cells, flies and mice resistant to inhibitors of the cytochrome pathway, such as antimycin or cyanide. Thirdly, it prevents superoxide overproduction triggered by an excessive reduction of the RC. As a result, in accordance with our fourth prediction, it offers an efficient way to prevent the consequences of a wide set of genetic or environmentally determined lesions targeting the cytochrome pathway in model systems. Based on this body of data, it is now possible to define additional perspectives, taking advantage of AOX expression. It is tempting to use the MitAOX mouse to investigate the long list of pathological conditions where overproduction of superoxide by mitochondria has been postulated to be instrumental (Fernandez-Checa et al., 2010), especially when already modelled in the mouse.
This includes, for example, ageing, but also many types of physiological insult affecting mitochondrial function in diverse tissues. As an alternative to the tissue restricted AOX-expressing mouse, the use of inhalable or injectable AOX, including its mitochondrial targeting sequence, delivered by a viral or similar vector, can also be considered. Recent progress made in ensuring the safety and longevity of viral vectors (Bouaita et al., 2012) makes this approach particularly attractive, as it might allow the therapeutic use of the AOX gene.
The findings with Drosophila already suggest that AOX expression could be of benefit in a wide spectrum of OXPHOS disorders. However, even in the mouse, a number of questions remain to be answered before it is possible to conclude that AOX expression is a feasible therapeutic strategy. In particular, as the AOX gene we used so far derives from C. intestinalis which lives in highly oxygenated (intertidal zone) and cold water (<20°C), we have to consider the possibility that different temperature and oxygen tension in mammalian organs might affect the functionality of the AOX, and thus its capacity to counteract RC deficiencies, although the first data with regard to the brain are highly encouraging (El-Khoury et al., 2013).