Jorgina SATRÚSTEGUI, PhD
Centro de Biología Molecular Severo Ochoa (CBMSO), Universidad Autónoma de Madrid, Spain

Gene therapy for Citrin deficiency. A proof of principle

CTNL2 is caused by mutations in citrin/SLC25A13/AGC2, the liver isoform of the mitochondrial aspartate-glutamate carrier (AGC). At present treatment of CTNL2 consists of a lifelong dietary control or hepatic transplant. Gene therapy with non-mutated citrin/AGC2 may produce an immune reaction in some patients, especially when the mutation is nonsense and no protein at all is expressed by the patient’s cells.

Our aim is to test the possibility that citrin/AGC2 can be replaced by the isoform of the carrier that is present in brain and skeletal muscle, aralar/AGC1. Because aralar/AGC1 is a protein expressed normally in the patients, it is not expected to trigger an immune response. Both proteins perform the same metabolite exchange between mitochondrial matrix and cytosol and play a role in the malate-aspartate shuttle. However, tissue specific requirements for correct expression and function for AGCs in liver may exist and this would prevent a correct function of AGC1 in liver. The aim of the present project financed by CITRIN foundation and to be carried out in collaboration with Prof. Saheki is to test the feasibility of citrin/AGC2 substitution with aralar/AGC1 by generating a liver specific aralar transgenic mouse in which functional testing may be carried out. Transgenic mouse generation is now underway.

Citrin deficiency is caused by mutations in citrin/SLC25A13/AGC2, the liver isoform of the mitochondrial aspartate-glutamate carrier (AGC).

As components of the malate aspartate shuttle (MAS), AGCs participate in the transfer of redox equivalents of NADH to mitochondria, and allow the mitochondrial export of aspartate to the cytosol. This is critical for the urea cycle and for the synthesis of proteins and nucleotides. Aspartate efflux from liver mitochondria is disrupted in citrin deficient patients due to mutations in citrin/AGC2. As a consequence: i) No aspartate is available in the cytosol for the activity of the urea cycle enzyme argininosuccinate synthetase (ASS), with a decrease in ureogenesis and accumulation of citrulline in plasma. ii) MAS disruption with failure of gluconeogenesis from lactate and low pyruvate levels.

Our hypothesis is that restoring mitochondrial aspartate transport in citrin deficient patients with wild type AGC will overcome the subsequent metabolic defects and become an effective therapy of affected individuals. As wild type citrin may behave as a foreign protein and trigger an immune response in citrin deficient patients who have no protein (nonsense mutations), we plan to test wild type aralar.

As proof of principle, the project financed by CITRIN foundation and carried out in collaboration with Prof. Saheki will allow to test the possibility of transgenic aralar/AGC1 to replace citrin/AGC2 in citrin-knock out mice (Sinasac et al., 2004; Contreras et al., 2010). Aralar/AGC1 and citrin/AGC2 have similar transport properties but aralar/AGC1 has lower activity and slightly different calcium regulation properties (Contreras et al., 2007; Palmieri et al., 2001). Aralar is expressed in many cell types, particularly in liver Kupffer cells (del Arco et al., 2002) and therefore it is unlikely that it would trigger an immune response.

Using a simple transgenic mouse with liver specific expression of aralar enables testing whether citrin can be replaced by aralar in the liver, which is the immediate aim of this project, particularly the effects of aralar/AGC1 in restoring MAS activity in citrin-deficient mouse liver mitochondria. So far, a vector with the coding sequence of aralar/AGC1 under the control of a strong hepatic promoter has been generated, and has been used to confirm the capacity of liver cell lines to successfully express aralar/AGC1 in mitochondria. The generation of the transgenic mice is underway.

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(Updated August 2018)