Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities

Abstract

Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism. Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism. IntroductionCreatine has an essential role in energy homeostasis, being particularly important in muscle and the brain due to their fluctuating energy demands. Outside of the buffering and transport function of high-energy phosphates, creatine is important for neurite growth cone migration, dendritic and axonal elongation, co-transmission on GABA postsynaptic receptors in the central nervous system (CNS)1Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis.Biochem. J. 1992; 281: 21-40Crossref M. Creatine and of creatine in K. J. D. of receptors a and guanidinoacetate guanidinoacetate deficiency brain on creatine energy for the of a of high-energy of and energy T. M. The creatine kinase system and of to creatine in in the and the of guanidinoacetate (GAA) and guanidinoacetate N-methyltransferase the of creatine GAA M. M. M. imaging in the brain of and to creatine and guanidinoacetate creatine is the and is the cellular creatine a and a Creatine deficiency to to to tissues of at of creatine is in transport result in cerebral creatine deficiency cerebral creatine deficiency include the creatine disorders GAMT Creatine deficiency in guanidinoacetate deficiency, a new inborn of creatine and Creatine deficiency of creatine transport The of this of disorders is the of creatine in the T. of creatine in muscle and brain in an with GAMT and the and can to intellectual disability, and seizures are in Creatine deficiency M. J. M. J. with and in J. Creatine deficiency and the of creatine in the the creatine deficiency disorders of the disorders with a M. M. M. imaging in the brain of and to creatine and guanidinoacetate the creatine deficiency GAMT to result in the guanidinoacetate guanidinoacetate deficiency brain the is to in M. treatment of guanidinoacetate (GAMT) to in M. D. J. to of guanidinoacetate deficiency in of the GAMT with a in J. D. new with guanidinoacetate deficiency and of in the GAMT to in J. of guanidinoacetate deficiency in the of in the GAMT being the have been J. D. new with guanidinoacetate deficiency and of in the GAMT to be the with The of the system to be of early brain M. M. M. imaging in the brain of and to creatine and guanidinoacetate is at Creatine deficiency and are Creatine deficiency D. treatment of guanidinoacetate reduction of guanidinoacetic acid in and ornithine (GAMT) in and for treatment and is an J. M. the of guanidinoacetate have is with marked intellectual and seizures are and to deficiency of creatine is markedly while in the and is to be the of the Creatine deficiency D. treatment of guanidinoacetate reduction of guanidinoacetic acid in and ornithine with the due to the deficiency of creatine and the of guanidinoacetate guanidinoacetate deficiency brain GAMT deficiency, treatment creatine due to the low (GAMT) in and for treatment and Creatine and guanidinoacetate transport at and is creatine has an at to to In creatine is having in in M. M. of J. creatine GAA guanidinoacetate guanidinoacetate deficiency brain Creatine and guanidinoacetate transport at and and while ornithine D. treatment of guanidinoacetate reduction of guanidinoacetic acid in and ornithine can be to J. D. new with guanidinoacetate deficiency and of in the GAMT can plasma and brain levels can (GAMT) in and for treatment and at for seizures and due to the of K. of and guanidinoacetic acid in the brain of we studies a gene therapy approach for GAMT deficiency to the of oral creatine gene to weight normalization of plasma and GAA levels, of brain and plasma and resolution of behavioral to a murine model of the disorder. These findings have implications for the development of a new approach for GAMT in control of GAA and of creatine with a mice in a model and GAMT deficiency in with markedly guanidinoacetic acid and markedly creatine in plasma and J. M. levels, weight homeostasis and in a model of guanidinoacetate N-methyltransferase (GAMT) mice are to human behavioral have been D. D. and of a model for GAMT mice of of of human codon-optimized GAMT a to the for long-term of and blood to and in mice at increased with and for for for and for codon-optimized GAMT in gene GAMT increased with and GAMT with an to in for GAMT with increased to and GAMT also in situ with a codon-optimized human in codon-optimized GAMT demonstrated the of in wild-type GAMT increased with demonstrated human in in GAMT and biochemical mice human codon-optimized GAMT and the in situ is demonstrated in and in the plasma biochemical to plasma creatine and plasma guanidinoacetic acid levels and levels in plasma at plasma creatine of and with in and plasma guanidinoacetic acid in guanidinoacetic in and the marked in GAMT is an improvement in the plasma creatine with the of the plasma creatine to with to plasma creatine levels at the of also in plasma creatine levels to plasma at with in plasma GAA levels declined. markedly a with treated a of to plasma levels of GAA the of the of plasma creatine levels and of plasma this for and in long-term of human codon-optimized GAMT in weight the of and treated of mice with and in in a and mice while the of blood for with having with treated treated mice demonstrated a in mice in weight while mice gained weight the of treated mice also demonstrated weight with mice to the same of the marked improvement in weight and treatment to of mice weight mice that of the mice of to weight in the In a is of and to a while an is of in mice mice and mice with gene therapy of weight and at of with imaging of and mice treated with gene weight and in and mice of the and treated mice mice of a weight at the of the treated to the mice weight at of at mice nearly in their of weight and at to in treated and mice weight and to mice mice mice of demonstrated the in weight with gene mice with at of and the tissues and in the this the is the the is a of the in the treated mice that that of the in imaging of of the and of in mice mice and mice treated with gene therapy are the mice with and treated of weight treated treated and at treated treated and in mice the marked improvement of weight and improvement in mice findings and imaging also in of with gene of weight in mice treated treated and treated treated and also the marked improvement in weight and is a weight and in in and mice and mice of that the weight at to the of in long-term GAMT with control of GAA and of creatine levels in and are in for at of to the in and mice is J. of murine an for for and for treated the pan-hepatic of GAMT the in studies is for the human and with murine is of GAMT is in the murine of in a of with with and and treated mice and mice and in long-term and GAMT of a of for human GAMT for in human GAMT in murine and GAMT in the in and the of for human GAMT and of in and in and for and to GAMT deficiency is markedly plasma creatine with GAA levels. GAA in mice and markedly in in in and in in GAA levels markedly with gene therapy in treated plasma GAA at treated treated and and GAA at treated treated and The in plasma creatine and Creatine in mice is markedly in in and in creatine levels in treated treated treated and treated creatine treated treated and human codon-optimized GAMT in normalization of guanidinoacetic acid and creatine in plasma and of and to plasma levels of GAA and creatine in and with and mice human codon-optimized GAMT a normalization of plasma GAA and creatine of that for the of the and demonstrated resolution of guanidinoacetic and in urinary creatine for the of the in and errors of the are plasma for is GAMT deficiency, urinary GAA is markedly while creatine is These are with gene therapy at treated is to creatine at treated is to time levels of GAA are markedly in with of the of in to in treated in in to in treated in in to in treated in in to in treated in muscle in to in treated in with human codon-optimized GAMT in improvement in levels of GAA while creatine in and levels are markedly in the brain and muscle of mice of GAA with AAV-based gene therapy in the and with marked in the brain and Creatine is nearly in brain and muscle in mice levels are markedly with in the and muscle in treated treated also demonstrated a marked improvement in creatine levels in in to in treated in to in to in treated in to in to in treated in to in to in treated in to muscle in to in treated in to and behavioral studies resolution of with gene the of the to with high-energy we the of to the brain in is a imaging for in cerebral brain in the of and a treatment in GAMT deficiency, we to the brain of this cerebral with we can in brain is to M. of of on in and in the we the the the this is the in mice to the of GAMT normalization of GAA levels, and of creatine in to to is in the of deficiency brain that is with gene of GAMT to of mice with brain this is gene therapy in of in the of and mice with gene therapy the brain is of of the of with oral and gene therapy and on in the brain and and of the brain the of GAMT deficiency and resolution with AAV-based gene therapy brain demonstrated in to the In the the brain to the the brain the the with a the the gene therapy the and the that include the of and a improvement in brain with the of GAMT in with a gene therapy approach and this to therapy with oral creatine oral creatine and with AAV-based gene therapy in control and In we a in brain with with oral creatine with imaging demonstrated a with creatine at of to for in and the murine biochemical are to with the in guanidinoacetate deficiency, a new inborn of creatine is behavioral D. 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J. 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M. levels, weight homeostasis and in a model of guanidinoacetate N-methyltransferase (GAMT) brain to be in and GAMT creatine the and of the The gene therapy approach in studies an human GAMT a to is the that this in markedly and creatine levels in the is In in mice express GAMT at levels in with the of that has been demonstrated J. of murine an is reduction in to at in the murine is the of a is to this in be the of this T. K. T. and of and in of creatine are also in the and GAA levels are in the and the brain and markedly have GAA levels. the and reduction in GAA is the plasma reduction the with of GAMT this is in the brain and be for to be of tissues is with an a the to express in tissues levels in studies that the at of mice with time the PET-CT to be due to a marked reduction in has been in murine J. M. levels, weight homeostasis and in a model of guanidinoacetate N-methyltransferase (GAMT) the studies that with the of GAMT is improvement in the to and has been J. M. levels, weight homeostasis and in a model of guanidinoacetate N-methyltransferase (GAMT) the reduction in to be to in levels of levels. is is in mice in studies demonstrated brain this nearly with GAMT gene therapy and to with creatine In the improvement in brain be the PET-CT in the brain with K. K. K. K. of on in M. M. in brain and this have the that creatine have and the with the gene therapy and levels in the brain are to be M. of brain creatine deficiency a and J. levels and of of control of J. with the in is that behavioral findings be in the murine biochemical of high plasma and urinary GAA with low creatine are to of with the in guanidinoacetate deficiency, a new inborn of creatine is behavioral in the murine D. D. and of a model for GAMT In that mice abnormalities; J. M. levels, weight homeostasis and in a model of guanidinoacetate N-methyltransferase (GAMT) the that mice an of of D. D. and of a model for GAMT this a In the behavioral of this we Using the a to the for the of mice to the and a for and in In we of a the to the the mice having an abnormality in mice also a to the These in and are with AAV-based gene the of a in in in of the for the mice In mice a on and while mice and treated mice a a of their findings an abnormality in long-term that is with AAV-based gene therapy with resolution of brain GAA in studies to in function mice a reduction in the of the and with the gene therapy conclusion, studies in a gene therapy for GAMT deficiency to the resolution of the of biochemical in and in and in a murine model of the and brain with a gene therapy GAA levels in the a approach include a and a that has an increased to the in the also for a of of AAV-based gene therapy to GAMT deficiency a for the clinical development of a gene therapy and and The of the and the of and of the and and an is the with the codon-optimized is in at the of M. M. J. and of at In a to and and a the guanidinoacetate J. M. levels, weight homeostasis and in a model of guanidinoacetate N-methyltransferase (GAMT) of of that been on the and for The the the These mice at and and of of a and to include of and mice with of mice in for blood collection and at tissues and with Creatine oral in and and for The GAMT GAMT GAMT for for The and the of at with of in for to the with the of and at for with the of a plasma with at for with the of to and at for with development with the of at for The and the at a of control of mice that been with and levels. and of and of and guanidinoacetic in plasma with acid to plasma and in in and for creatine and the plasma with for at and with for with with the of and in and we and for and we and for and and at the of the with the a of acid and acid and for and and for and of muscle in a and brain a with and on at for of with a time for and muscle and for of for and with and at for to the The and at for for of the with of the and of the and at for The a and the at to the in and at for to the at to the in and a for on with at M. D. of creatine in blood and blood for pharmacological studies in of creatine The system of an system with The at a of and a at on a of acid in and of acid in The at at at at at and at The The at the and The and The at and and and for an and in at of for and and for of to of and for and and for The a of and at a of and at a of in and the at the of the to and to the of the to of the and M. K. M. of in mice in and can be gene in to with and with human GAMT and situ in situ the and the to the with at and the for at with to the The with the at high a and in being in with the to the in and with the and with with with the and for the of in the human and of GAMT and for and a for at a of the with and and to the of the and to The the and their The of mice for and of of in for and in for and of the at and with and with in for for of the in a at for The with the the to of the with the and with with and with imaging imaging the and the and tissues in the in the the of in of the brain the brain D. M. In of the brain behavioral mice and of the mice to muscle and and a on a for the and the with at of at of the and the in the the to the that be to the The the and that the the and the to the the of the The and the the the to at a the of the The the of the and to the with the The with the The to the in a the on the of of and mice in the the and in the K. M. J. of and in the in The of a with the and the The in the of the with and for and a for the mice in of on the of the a for the of the The of low with and in the to the of of with an of and The of the the same for mice the the of the to the the the the for being to their and mice a to and long-term the of the the and mice to for of the and mice are to the to the to of of and mice in the the and in the In for the approach to the approach the the of the and the of and to the approach the a the and the of the to the approach the the and in a to the of the with the of the and with for with The IntroductionCreatine has an essential role in energy homeostasis, being particularly important in muscle and the brain due to their fluctuating energy demands. Outside of the buffering and transport function of high-energy phosphates, creatine is important for neurite growth cone migration, dendritic and axonal elongation, co-transmission on GABA postsynaptic receptors in the central nervous system (CNS)1Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis.Biochem. J. 1992; 281: 21-40Crossref M. Creatine and of creatine in K. J. D. of receptors a and guanidinoacetate guanidinoacetate deficiency brain on creatine energy for the of a of high-energy of and energy T. M. The creatine kinase system and of to creatine in in the and the of guanidinoacetate (GAA) and guanidinoacetate N-methyltransferase the of creatine GAA M. M. M. imaging in the brain of and to creatine and guanidinoacetate creatine is the and is the cellular creatine a and a Creatine deficiency to to to tissues of at of creatine is in transport result in cerebral creatine deficiency cerebral creatine deficiency include the creatine disorders GAMT Creatine deficiency in guanidinoacetate deficiency, a new inborn of creatine and Creatine deficiency of creatine transport The of this of disorders is the of creatine in the T. of creatine in muscle and brain in an with GAMT and the and can to intellectual disability, and seizures are in Creatine deficiency M. J. M. J. with and in J. Creatine deficiency and the of creatine in the the creatine deficiency disorders of the disorders with a M. M. M. imaging in the brain of and to creatine and guanidinoacetate the creatine deficiency GAMT to result in the guanidinoacetate guanidinoacetate deficiency brain the is to in M. treatment of guanidinoacetate (GAMT) to in M. D. J. to of guanidinoacetate deficiency in of the GAMT with a in J. D. new with guanidinoacetate deficiency and of in the GAMT to in J. of guanidinoacetate deficiency in the of in the GAMT being the have been J. D. new with guanidinoacetate deficiency and of in the GAMT to be the with The of the system to be of early brain M. M. M. imaging in the brain of and to creatine and guanidinoacetate is at Creatine deficiency and are Creatine deficiency D. treatment of guanidinoacetate reduction of guanidinoacetic acid in and ornithine (GAMT) in and for treatment and is an J. M. the of guanidinoacetate have is with marked intellectual and seizures are and to deficiency of creatine is markedly while in the and is to be the of the Creatine deficiency D. treatment of guanidinoacetate reduction of guanidinoacetic acid in and ornithine with the due to the deficiency of creatine and the of guanidinoacetate guanidinoacetate deficiency brain GAMT deficiency, treatment creatine due to the low (GAMT) in and for treatment and Creatine and guanidinoacetate transport at and is creatine has an at to to In creatine is having in in M. M. of J. creatine GAA guanidinoacetate guanidinoacetate deficiency brain Creatine and guanidinoacetate transport at and and while ornithine D. treatment of guanidinoacetate reduction of guanidinoacetic acid in and ornithine can be to J. D. new with guanidinoacetate deficiency and of in the GAMT can plasma and brain levels can (GAMT) in and for treatment and at for seizures and due to the of K. of and guanidinoacetic acid in the brain of we studies a gene therapy approach for GAMT deficiency to the of oral creatine gene to weight normalization of plasma and GAA levels, of brain and plasma and resolution of behavioral to a murine model of the disorder. These findings have implications for the development of a new approach for GAMT

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