Development; GAMT; AGAT; Creatine; 3D organotypic brain cell cultures; KI rats; SLC6A8; Guanidinoacetate; In vivo models; Brain
Stöckler Sylvia, Braissant Olivier, Schulze Andreas (2022), Creatine disorders, in Ferrreira CR, van Karnebeek CDM, Blau N, Vianney-Saban C, Dionisi-Vici M (ed.), Springer-Verlag, Berlin, Heidelberg, New York, 1-21.
Duran-Trio Lara, Fernandes-Pires Gabriella, Grosse Jocelyn, Soro-Arnaiz Ines, Roux-Petronelli Clothilde, Binz Pierre-Alain, De Bock Katrien, Cudalbu Cristina, Sandi Carmen, Braissant Olivier (2021), Creatine transporter deficient rat model shows motor dysfunction, cerebellar alterations and muscle creatine deficiency without muscle atrophy, in Journal of Inherited Metabolic Disease
Fernandes-Pires Gabriella, Braissant Olivier (2021), Current and potential new treatment strategies for creatine deficiency syndromes, in Molecular Genetics and Metabolism
Duran-Trio Lara, Fernandes-Pires Gabriella, Simicic Dunja, Grosse Jocelyn, Roux Clothilde, Binz Pierre-Alain, Sandi Carmen, Cudalbu Cristina, Braissant Olivier (2021), A new rat model of creatine transporter deficiency reveals behavioral disorder and altered brain metabolism, in Scientific Reports
, 11(1), 1636.
Diez-Fernandez Carmen, Hertig Damian, Loup Marc, Diserens Gaelle, Henry Hugues, Vermathen Peter, Nuoffer Jean-Marc, HÃ¤berle Johannes, Braissant Olivier (2019), Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three‐dimensional organotypic brain cell cultures, in Journal of Inherited Metabolic Disease
, 42(6), 1077-1087.
Rackayova Veronika, Cudalbu Cristina, Pouwels Petra J.W., Braissant Olivier (2017), Creatine in the central nervous system: From magnetic resonance spectroscopy to creatine deficiencies, in Analytical Biochemistry
, 529, 144-157.
SchulzeAndreas, BraissantOlivier (2017), Creatine deficiency syndromes, in Sarafoglou K, Hoffmann G, Roth K (ed.), McGraw-Hill, New York, 181-190.
Cerebral creatine deficiency syndromes: New in vivo AAV approaches to treat SLC6A8 deficiency.Applicant: PD Dr Olivier Braissant, PhD / Co-applicant : Dr Cristina Cudalbu, PhD1:Summary of the research plan1.1:BackgroundCreatine (Cr) is synthesized by a 2-step pathway involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Cells take up Cr by a specific Cr transporter, SLC6A8. Cr deficiency syndromes (CDS), due to AGAT, GAMT or SLC6A8 deficiencies, are inborn errors of metabolism (IEM) affecting the brain, causing severe neurodevelopmental delays and intellectual disability. CDS are characterized by absence of brain Cr when measured by MRS. AGAT and GAMT deficiencies can be treated with Cr, improving their neurological outcome. SLC6A8 deficiency cannot be treated so far, and patients show an irreversible neurological degradation.1.2:Working hypothesisAttempts to treat SLC6A8 deficiency, the most frequent of CDS, was not successful so far due to the absence of functional Cr transporter SLC6A8 on blood brain barrier (BBB). We recently designed adeno-associated viruses (AAV) that are able to transduce every brain cell type in models of CDS by RNAi. As AAV are more and more accepted as promising tools for human gene therapy including in CNS, we hypothesize that AAV vectors re-establishing a functional Cr transporter on BBB and in CNS would allow restoring brain Cr in SLC6A8-deficient patients and improving their neurological status. We propose here to develop a new AAV-driven in vivo model of treatment for the so far untreatableSLC6A8 deficiency in the rat. 1.3:Specific aims- To develop and produce AAV vectors transducing the functional SLC6A8-eGFP fusion Cr transporter in BBB and brain cells.- To demonstrate the feasibility of SLC6A8 deficiency treatment in vivo, through systemic injection of AAV/SLC6A8-eGFP vectors in a newly developed Slc6a8Y389C/y KI rat, coupled to Cr supplementation to improve CNS Cr restoration.- To demonstrate CNS Cr restoration and improvement of neurological status in AAV/SLC6A8-eGFP-injected Slc6a8Y389C/y KI rats, as well as faster replenishment of their brain Cr upon Cr co-treatment.1.4:Experimental design and methodsAAV1, 2 and 9 vectors (selected for high tropisms for BBB and brain cells) will be developed to transduce the functional SLC6A8-eGFP fusion protein in BBB and surrounding parenchymal brain cells. AAV/SLC6A8-eGFP vectors will first be tested in vitro, by expression in rat 3D organotypic brain cell cultures, as well as by restoring Cr uptake in human SLC6A8-deficient fibroblasts. AAV/SLC6A8-eGFP vectors will then be tested in vivo, first in wild type (WT), second in Slc6a8Y389C/y KI rats. Facilitation (WT) or restoration (Slc6a8Y389C/y) of Cr uptake and synthesis by CNS will be evaluated. Expression of SLC6A8-eGFP at BBB and within brain parenchyma will be characterized, together with the detailed analysis of their CNS involvement (development and cell differentiation, morphology, cell death). SLC6A8 deficiency treatment will be evaluated, combining injection of AAV/SLC6A8-eGFP vectors with Cr co-treatment. Restoration of brain Cr is expected, which will be followed longitudinally in vivo on a long term range (up to 1 year post-AAV injection) by high resolution 9.4T 1H- and 31P-MRS, as well as by LC/MS-MS on brain extracts. The treatments outcome (neurological improvement) will be evaluated through behavioral tests.1.5:Expected value of the projectWe believe that our innovative in vivo AAV-driven approach to restore the functional Cr transporter SLC6A8 (at BBB and in brain parenchyma), coupled to Cr co-treatment, will become available for clinical testing in humans, helping to find new therapeutic strategies for the so far untreatable SLC6A8 deficiency. This approach, by facilitating Cr transfer to CNS, will also become available to improve the treatment of AGAT and GAMT deficiencies, including the potential reduction of brain GAA intoxication found in GAMT deficiency as we have shown recently in 3D organotypic brain cell cultures. Finally, this strategy would then also become applicable to any other IEM showing a specific defect in a transport system affecting CNS development and function.