RESUMO
Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid-onset dementia and death. However, elimination of endogenous PrP halts prion disease progression. In this study, we describe Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates endogenous DNA methyltransferases, thereby reducing transgene size and cytotoxicity. When delivered to the mouse brain by systemic injection of adeno-associated virus (AAV), Prnp-targeted CHARM ablates PrP expression across the brain. Furthermore, we have temporally limited editor expression by implementing a kinetically tuned self-silencing approach. CHARM potentially represents a broadly applicable strategy to suppress pathogenic proteins, including those implicated in other neurodegenerative diseases.
Assuntos
Encéfalo , Metilação de DNA , Dependovirus , Inativação Gênica , Histonas , Proteínas Priônicas , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Dependovirus/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Histonas/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , TransgenesRESUMO
Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an adeno-associated virus (AAV) capsid, BI-hTFR1, that binds human transferrin receptor (TfR1), a protein expressed on the blood-brain barrier. BI-hTFR1 was actively transported across human brain endothelial cells and, relative to AAV9, provided 40 to 50 times greater reporter expression in the CNS of human TFRC knockin mice. The enhanced tropism was CNS-specific and absent in wild-type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared with AAV9. These findings establish BI-hTFR1 as a potential vector for human CNS gene therapy.
Assuntos
Antígenos CD , Encéfalo , Capsídeo , Técnicas de Transferência de Genes , Vetores Genéticos , Glucosilceramidase , Receptores da Transferrina , Animais , Humanos , Camundongos , Antígenos CD/metabolismo , Antígenos CD/genética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Dependovirus , Células Endoteliais/metabolismo , Técnicas de Introdução de Genes , Terapia Genética , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genética , Glucosilceramidase/genética , Doença de Gaucher/genética , Doença de Gaucher/terapia , Doença de Parkinson/genética , Doença de Parkinson/terapiaRESUMO
Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an AAV capsid, BI-hTFR1, that binds human Transferrin Receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. These findings establish BI-hTFR1 as a promising vector for human CNS gene therapy.