A new approach to treating genetic disease Deficiency of the creatine transporter SLC6A8 is linked to intellectual disabilities, including a marked impairment of speech acquisition and several other neurological symptoms in infants. Researchers are developing a new approach to treat this genetic disease, as Professor Olivier Braissant and Dr Cristina Cudalbu explain. A type of
naturally occurring organic compound, creatine plays an important role in recycling adenosine triphosphate (ATP) and storing high-energy phosphates within cells in the human body. While it was previously thought that the brain’s creatine needs were met primarily from peripheral sources, recent research by Professor Olivier Braissant has shown otherwise. “A few years ago we discovered in my group that the brain probably needs its own creatine synthesis – it is not sufficient to take creatine from the periphery,” he says. The blood-brain barrier (BBB) – which protects the brain from the periphery – has a very low permeability for creatine, and so the brain itself expresses the two enzymes that enable the synthesis of creatine. “These two enzymes are called AGAT and GAMT, while the brain also expresses the creatine transporter SLC6A8,” explains Professor Braissant. “Creatine can enter the brain through the BBB with the means of the
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creatine transporter – but it is very inefficient. The brain needs its own creatine synthesis to ensure it has enough creatine for its own needs, and the creatine transporter also appears essential to this.”
transporter deficiency in the brain. “We have designed an in vivo model of creatine transporter deficiency. This is a knock-in rat, which harbours a single nucleotide mutation that has been described in patients with
We hope to re-establish this activity of creatine uptake by brain cells. We have done preliminary experiments with a Green Fluorescent Protein (GFP), under the same promoter that we will use for the creatine transporter. Creatine transporter deficiency A deficiency in this transporter is known to cause intellectual disabilities, a topic of great interest to Professor Braissant, a specialist in inborn errors of metabolism (IEM) based at Lausanne University Hospital in Switzerland. As the Principal Investigator of a new SNSFfunded research project, Professor Braissant is investigating the factors behind creatine
creatine transporter deficiency,” he outlines. This model is representative of the genome of patients with creatine transporter deficiency, now researchers are using it to investigate the disease in great depth. “The first part of the project involved characterising this rat model, to see whether it had a phenotype that we could relate to the disease,” says Professor Braissant. “In the second part of the project, we want to
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Golgi-stained rat cortex, showing various neurons with their dendrites and axons with incorporation of AAV viral particles, illustrating the treatment trial of the disease. Original adapted image: Copyright: ©CHUV/Lara Duran-Trio.
design new ways to try to treat the disease. Creatine transporter deficiency has been known for around 20 years now, but an efficient treatment has not yet been developed.” The aim in the project is to design new gene therapy strategies to treat this disease. Researchers are exploring the possibility of using adeno-associated virus (AAV) vectors to transduce the brain of the rat model, and so establish a kind of proof-of-concept for the treatment of creatine transporter deficiency. “We want to transduce the brain, in order to express a new, active creatine transporter, within the brain of our rat,” explains Professor Braissant. In this approach, researchers essentially package the protein sequence of the creatine transporter within the viral particle, with the appropriate promoter in front. “The AAV viral particle takes this genetic material and will transduce – or enter – the brain cells. Then the coding sequence for the normal, active creatine transporter will be expressed within the brain cells,” continues Professor Braissant “We hope by this method to re-establish this activity of creatine uptake by the brain cells. We have done preliminary experiments with a Green Fluorescent Protein (GFP), under the same promoter that we will use for the creatine transporter.” Researchers have found that they are able to transduce the brain quite efficiently with this virus, with a very high proportion of cells
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expressing the transduced gene, so Professor Braissant is hopeful that this approach will prove effective on the in vivo model. Once it has been demonstrated that the creatine transporter is well expressed in the brain of the rat, the next step will be to assess whether this helps to reestablish a sufficient level of creatine. “We will look at the rat treated with the AAV vector to see whether the creatine concentration has been reestablished. It may be that we won’t reach the wild type level, but even if we can only achieve an intermediate re-establishment of the creatine concentration, this can have tremendous effects
Brain 1H-MRS spectra comparing wild type and creatine transporter-deficient rats, showing the strong deficiency of creatine in the brain under creatine transporter deficiency. Copyright: ©CIBM/Dunja Simicic .
on the rat’s brain and their behaviour,” says Professor Braissant. A number of behavioural experiments will also be performed, as Professor Braissant and his colleagues look to assess the impact of the treatment. “We will try to see whether there are any changes in behaviour in the knock-in rat treated with the AAV vector, in comparison to an untreated rat,” he outlines. The precise level of creatine within the brain parenchyma is difficult to establish, as most creatine is found within cells, so there are no clear reference concentration values. “However, we can approach this measure of brain creatine through magnetic resonance spectroscopy,” says Professor Braissant. “We use an MRI machine with a very high magnetic field, at 9.4 tesla, allowing the measure of numerous metabolites in vivo, including creatine,” explains Dr Cristina Cudalbu, a research staff scientist at the Center for Biomedical Imaging of EPFL in Lausanne, who is closely involved in the project. “We measure brain creatine concentrations in healthy animals using proton magnetic resonance spectroscopy (1H MRS), and then compare them with the creatine transporter-deficient ones who show a very strong decrease in brain creatine.” “We thus hope to observe the reestablishment of creatine within the brain of the AAV-transduced creatine transporterdeficient rats” Professor Braissant outlines. Other important aspects of the project’s agenda include investigating the behaviour of these rats, as well as their metabolism, which can be observed through magnetic resonance spectroscopy.
A Purkinje neuron of the rat cerebellum, successfully transduced by an AAV vector driving the expression of the GFP fluorescent protein under the same promoter as the one to be used for the treatment trial of creatine transporter deficiency. Copyright: ©CHUV/Gabriella Fernandes-Pires.
Cerebral creatine deficiency syndromes: New in vivo AAV approaches to treat SLC6A8 deficiency
Project Objectives
To treat SLC6A8 deficiency through a new AAV vector approach in a newly characterized KI rat model of the disease.
Project Funding
SNSF grant: n° 31003A-175778, 533’618 CHF for 4 years.
Project Partners
• Dr Lara Duran-Trio, PhD, postdoc : SLC6A8-deficient rat characterization. • Gabriella Fernandes-Pires, PhD student : AAV-driven treatment of SLC6A8 deficiency. • Dunja Simicic, PhD student : Brain metabolism by high resolution MRS
Contact Details
Professor Olivier Braissant, PhD Associate professor at the Service of Clinical Chemistry Department of Laboratory Medicine and Pathology Lausanne University Hospital (CHUV) & University of Lausanne 1011 – Lausanne, Switzerland T: +41 79 556 72 07 E: Olivier.Braissant@chuv.ch W: www.chuv.ch Doctor Cristina Cudalbu, PhD, Research Staff Scientist & 9.4T MRI Operational Manager CIBM Center for Biomedical Imaging MRI EPFL Section, Animal Imaging and Technology EPFL (Swiss Federal Institute of Technology in Lausanne) 1015 – Lausanne, Switzerland T: +41 21 693 76 85 E: Cristina.Cudalbu@epfl.ch W: www.cibm.ch L.Duran-Trio, G.Fernandes-Pires, J.Grosse, C.Roux, P.-A.Binz, C.Sandi, C.Cudalbu, O.Braissant (2021). Scientific Reports, 11:1636. A new rat model of creatine transporter deficiency reveals behavioral disorder and altered brain metabolism.
Professor Olivier Braissant Dr Cristina Cudalbu
Human treatment This research is part of the long-term goal of developing more effective treatments for cerebral creatine deficiency syndromes. If researchers can demonstrate that this AAV approach leads to the re-establishment of creatine within the brain of the rat, then this could lead to the development of treatment protocols for human patients, while Professor Braissant and his colleagues are also exploring other research avenues. “We are collaborating with other people who are interested in our rat model, while we’re also looking into other strategies, like the use of chaperone molecules,” he outlines. It’s important to treat genetic diseases like cerebral creatine deficiency syndromes as early as possible. “The younger it is done, the better the outcome for the brain. If you can correct the disease quite early, then it’s much better for the brain development,” stresses Professor Braissant. “This has been observed for patients suffering from the two other creatine deficiencies, AGAT and GAMT deficiencies, who still have the normal creatine transporter
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and could be treated pre-symptomatically by creatine supplementation.” There are also other genetic disorders that affect brain development, for example GLUT1 deficiency syndrome, which affects the transport of glucose to the brain. Professor Braissant says the project’s research also holds implications for the treatment of this and other genetic diseases. “GLUT1 deficiency syndrome is a condition that could be treated in the same way as creatine transporter deficiency syndrome – the transporter is the same size as the creatine transporter. There are also many other genetic diseases that affect enzymes within brain cells,” he continues. The primary focus of attention in the project is creatine transporter deficiency however, and Professor Braissant and his colleagues plan to continue investigating the
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disease over the remainder of the project’s funding term and beyond. “We are preparing a few papers on the characterisation of these knock-in rats, and we should have some results on the effectiveness of the AAV transduction some time this year,” he outlines. The project could potentially be extended, which would enable researchers to perform further tests on the AAV treatment as well as the other treatment strategies, such as the chaperone molecules. The hope is that this will lead in future to improved treatment of not only cerebral creatine deficiency, but also other genetic diseases. “If we can develop an efficient protocol to transduce brain cells with our vectors, it will be available not only for the creatine deficiencies, but also for many other genetic diseases which affect brain development,” says Professor Braissant.
Professor Olivier Braissant is a group leader at the Service of Clinical Chemistry of the University Hospital of Lausanne (CHUV). After a PhD (1994) and a postdoc at the University of Lausanne on nuclear receptors, he moved to the CHUV where he developed his research on inborn errors of metabolism affecting brain development. Dr Cristina Cudalbu is a research staff scientist at CIBM Centre for Biomedical Imaging, Switzerland. After a PhD (2006) at the University Lyon 1 and a postdoc at LIFMET/EPFL, she became a group leader developing her own research on new and fast acquisition and quantification techniques for proton and X-nuclei MRS, particularly in CNS.
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