Publicação
Phenylalanine hydroxylase (pah) enzymosomes : development of a nanosystem for enzyme substitution/replacement therapies
| Resumo: | Abstract An impaired activity of human phenylalanine hydroxylase (hPAH) results in phenylketonuria (PKU). To prevent the psycho-motor deficits developed by PKU patients, treatment must be initiated as soon as possible after birth. Presently, dietary restriction, BH4 supplementation and enzyme substitution therapy are available. Technological advances in the production of recombinant human proteins boosted the development of enzyme reposition therapies. Although very promising, in the case of PKU their use implies preservation of hPAH activity and 3D structure during the formulation process. Due to the observed conformational instability, formulation of the 200 kDa homotetrameric cytoplasmatic enzyme as an active pharmaceutical ingredient (enzyme reposition therapy; ERT) is a very challenging process. hPAH-enzymosomes may be a desirable formulation to use as an ERT for PKU treatment. In this system the enzyme is covalently attached to the outer surface of liposome conferring protection from degradation, long-circulating properties, and lower immunogenicity. Preliminary data obtained by our group indicated that modification of hPAH lysine residues with N-succinimidyl S-acethylthioacetate (SATA) to allow the covalent reaction with the liposomal system preserve enzymatic activity. In this work, hPAH modification conditions were optimized using different molar ratios of SATA:hPAH. Study of modified hPAH was performed regarding enzymatic activity, thermostability and quaternary structure. As the SATA:hPAH molar ratio increased a decrease in the recovered protein with a slight decrease in enzyme activity concomitant to a change in the oligomeric state was observed. Using the selected ratios, the storage conditions were evaluated, and it was possible to conclude that the enzyme preserved its enzymatic activity and without significant loss in terms of protein content, and therefore, successful functional enzymosomes were obtained. The optimization of the enzymosome preparation process,, which must be thoroughly documented in terms of stability and effectiveness tests to confirm the maintenance of functional features, must be the subject of further research. |
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| Autores principais: | Francisco, Mafalda Vaz |
| Assunto: | Phenylalanine hydroxylase Phenylketonuria Enzyme reposition therapy Enzymosomes |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Abstract An impaired activity of human phenylalanine hydroxylase (hPAH) results in phenylketonuria (PKU). To prevent the psycho-motor deficits developed by PKU patients, treatment must be initiated as soon as possible after birth. Presently, dietary restriction, BH4 supplementation and enzyme substitution therapy are available. Technological advances in the production of recombinant human proteins boosted the development of enzyme reposition therapies. Although very promising, in the case of PKU their use implies preservation of hPAH activity and 3D structure during the formulation process. Due to the observed conformational instability, formulation of the 200 kDa homotetrameric cytoplasmatic enzyme as an active pharmaceutical ingredient (enzyme reposition therapy; ERT) is a very challenging process. hPAH-enzymosomes may be a desirable formulation to use as an ERT for PKU treatment. In this system the enzyme is covalently attached to the outer surface of liposome conferring protection from degradation, long-circulating properties, and lower immunogenicity. Preliminary data obtained by our group indicated that modification of hPAH lysine residues with N-succinimidyl S-acethylthioacetate (SATA) to allow the covalent reaction with the liposomal system preserve enzymatic activity. In this work, hPAH modification conditions were optimized using different molar ratios of SATA:hPAH. Study of modified hPAH was performed regarding enzymatic activity, thermostability and quaternary structure. As the SATA:hPAH molar ratio increased a decrease in the recovered protein with a slight decrease in enzyme activity concomitant to a change in the oligomeric state was observed. Using the selected ratios, the storage conditions were evaluated, and it was possible to conclude that the enzyme preserved its enzymatic activity and without significant loss in terms of protein content, and therefore, successful functional enzymosomes were obtained. The optimization of the enzymosome preparation process,, which must be thoroughly documented in terms of stability and effectiveness tests to confirm the maintenance of functional features, must be the subject of further research. |
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