Enzyme replacement therapy (ERT) is based on the periodic intravenous administration of specific enzymes produced with recombinant DNA technology. ERT consists in restoring a lacking or non functioning enzyme with its native counterpart or with an enzyme able to degrade toxic metabolites or secondary compounds of the reaction catalyzed by the deficient enzyme. But there are many limitations in the use of the ERT, some of them are safety, immunogenicity and delivery. On the light of the above, this thesis will focus on the use of engineered Red Blood Cells (RBCs) in drug delivery and in particular in Enzyme Replacement Therapy and, then, will display two applications of engineered RBCs in the treatment of two metabolic diseases: phenylketonuria (PKU) and Guanidinoacetate-N-methyltransferase (GAMT) deficiencies. PKU is an autosomal recessive metabolic disease characterized by severe intellectual impairment caused by a deficiency of the enzyme phenylalanine hydroxylase that catalyzes the conversion of the amino acid L-phenylalanine (L-Phe) into L-Tyr. In this work, we aimed to demonstrate the ability of rAvPAL (recombinant Anabaena variabilis PAL) -loaded RBCs to act as an effective bioreactor system, in vitro and in vivo, capable of reducing PHE levels. GAMT deficiency is a rare autosomal recessive disorder characterized by low creatine levels resulting in its deficit and causing serious damage, especially in the CNS. In this thesis, we wanted to demonstrate the ability of the engineered red blood cells, loaded with both rGAMT and rMAT2A enzymes, to act as bioreactors to effectively reduce GAA levels and increase Cr. In both cases, we developed the recombinant proteins by cloning, expression and purification of recombinant enzymes to treat these types of inherited metabolic disorders. Then, we optimized the enzymes’ loading procedure in murine erythrocytes to test the strategy in preclinical studies. For PKU, the excellent results obtained in the murine model, which led to and normal values of blood L-Phe concentration, lay the basis for further preclinical studies with repeated administrations. For the GAMT deficiency, the results obtained in the preclinical study are encouraging. Physiological values have not been yet reached, but further studies are ongoing to find the optimal enzyme concentrations and improve the system efficiency.

Erythrocytes loaded with recombinant proteins as enzyme replacement therapy for the treatment of rare diseases

Aliano, Mattia Paolo
2020

Abstract

Enzyme replacement therapy (ERT) is based on the periodic intravenous administration of specific enzymes produced with recombinant DNA technology. ERT consists in restoring a lacking or non functioning enzyme with its native counterpart or with an enzyme able to degrade toxic metabolites or secondary compounds of the reaction catalyzed by the deficient enzyme. But there are many limitations in the use of the ERT, some of them are safety, immunogenicity and delivery. On the light of the above, this thesis will focus on the use of engineered Red Blood Cells (RBCs) in drug delivery and in particular in Enzyme Replacement Therapy and, then, will display two applications of engineered RBCs in the treatment of two metabolic diseases: phenylketonuria (PKU) and Guanidinoacetate-N-methyltransferase (GAMT) deficiencies. PKU is an autosomal recessive metabolic disease characterized by severe intellectual impairment caused by a deficiency of the enzyme phenylalanine hydroxylase that catalyzes the conversion of the amino acid L-phenylalanine (L-Phe) into L-Tyr. In this work, we aimed to demonstrate the ability of rAvPAL (recombinant Anabaena variabilis PAL) -loaded RBCs to act as an effective bioreactor system, in vitro and in vivo, capable of reducing PHE levels. GAMT deficiency is a rare autosomal recessive disorder characterized by low creatine levels resulting in its deficit and causing serious damage, especially in the CNS. In this thesis, we wanted to demonstrate the ability of the engineered red blood cells, loaded with both rGAMT and rMAT2A enzymes, to act as bioreactors to effectively reduce GAA levels and increase Cr. In both cases, we developed the recombinant proteins by cloning, expression and purification of recombinant enzymes to treat these types of inherited metabolic disorders. Then, we optimized the enzymes’ loading procedure in murine erythrocytes to test the strategy in preclinical studies. For PKU, the excellent results obtained in the murine model, which led to and normal values of blood L-Phe concentration, lay the basis for further preclinical studies with repeated administrations. For the GAMT deficiency, the results obtained in the preclinical study are encouraging. Physiological values have not been yet reached, but further studies are ongoing to find the optimal enzyme concentrations and improve the system efficiency.
2020
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2681345
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