RESUMO
Lafora disease (LD) is a fatal juvenile epilepsy characterized by the accumulation of aberrant glucan aggregates called Lafora bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay has been developed to detect VAL-0417 post-treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found that intracerebroventricular administration provided robust CNS delivery when compared to intrathecal administration. These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.
Assuntos
Encéfalo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Doença de Lafora/tratamento farmacológico , alfa-Amilases Pancreáticas/genética , alfa-Amilases Pancreáticas/farmacocinética , Animais , Fusão Gênica Artificial/métodos , Encéfalo/metabolismo , Modelos Animais de Doenças , Portadores de Fármacos/metabolismo , Ensaio de Imunoadsorção Enzimática , Glucanos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Plasmídeos/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Distribuição Tecidual , Resultado do TratamentoRESUMO
Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.
Assuntos
Encéfalo/metabolismo , Glucosamina/metabolismo , Glicogênio/fisiologia , Processamento de Proteína Pós-Traducional , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glicogênio/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Glicogenólise/genética , Glicosilação , Doença de Lafora/genética , Doença de Lafora/metabolismo , Doença de Lafora/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Abnormal carbohydrate structures known as polyglucosan bodies (PGBs) are associated with neurological disorders, glycogen storage diseases (GSDs), and aging. A hallmark of the GSD Lafora disease (LD), a fatal childhood epilepsy caused by recessive mutations in the EPM2A or EPM2B genes, are cytoplasmic PGBs known as Lafora bodies (LBs). LBs result from aberrant glycogen metabolism and drive disease progression. They are abundant in brain, muscle and heart of LD patients and Epm2a-/- and Epm2b-/- mice. LBs and PGBs are histologically reminiscent of starch, semicrystalline carbohydrates synthesized for glucose storage in plants. In this study, we define LB architecture, tissue-specific differences, and dynamics. We propose a model for how small polyglucosans aggregate to form LBs. LBs are very similar to PGBs of aging and other neurological disorders, and so these studies have direct relevance to the general understanding of PGB structure and formation.