RESUMO
Phenylketonuria (PKU), an autosomal recessive disorder caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene, results in the accumulation of blood phenylalanine (Phe) to neurotoxic levels. Current dietary and medical treatments are chronic and reduce, rather than normalize, blood Phe levels. Among the most frequently occurring PAH variants in PKU patients is the P281L (c.842C>T) variant. Using a CRISPR prime-edited hepatocyte cell line and a humanized PKU mouse model, we demonstrate efficient in vitro and in vivo correction of the P281L variant with adenine base editing. With the delivery of ABE8.8 mRNA and either of two guide RNAs in vivo using lipid nanoparticles (LNPs) in humanized PKU mice, we observe complete and durable normalization of blood Phe levels within 48 h of treatment, resulting from corrective PAH editing in the liver. These studies nominate a drug candidate for further development as a definitive treatment for a subset of PKU patients.
Assuntos
Fenilalanina Hidroxilase , Fenilcetonúrias , Camundongos , Animais , Fenilcetonúrias/genética , Fenilcetonúrias/terapia , Fenilcetonúrias/metabolismo , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fígado/metabolismo , Hepatócitos/metabolismo , Modelos Animais de DoençasRESUMO
Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.
Assuntos
Expressão Gênica , Íons/química , Lipídeos/química , Nanopartículas/química , RNA Mensageiro/química , RNA Mensageiro/genética , Administração Intravenosa , Animais , Composição de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Injeções Intramusculares , Camundongos , Estrutura Molecular , Nanopartículas/ultraestrutura , RNA Mensageiro/administração & dosagem , RNA Mensageiro/farmacocinética , Análise Espectral , Distribuição Tecidual , TransfecçãoRESUMO
The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after intramuscular injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clinical trials. The review concludes with an analysis of the determinants of the performance of lipid nanoparticles in mRNA vaccines.