ABSTRACT
Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.
Subject(s)
Capsid Proteins , Capsid , Dependovirus , Genetic Vectors , Liver , Dependovirus/genetics , Animals , Humans , Mice , Genetic Vectors/genetics , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Liver/metabolism , Transduction, Genetic , Gene Transfer Techniques , Machine Learning , Genetic Therapy/methods , Macaca , Hepatocytes/metabolism , HEK293 Cells , Genetic Engineering/methodsABSTRACT
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.
Subject(s)
Brain , DNA Methylation , Dependovirus , Gene Silencing , Histones , Prion Proteins , Animals , Humans , Mice , Brain/metabolism , Dependovirus/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , Histones/metabolism , Prion Diseases/genetics , Prion Diseases/metabolism , Prion Proteins/genetics , Prion Proteins/metabolism , TransgenesABSTRACT
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.
Subject(s)
Antigens, CD , Brain , Capsid , Gene Transfer Techniques , Genetic Vectors , Glucosylceramidase , Receptors, Transferrin , Animals , Humans , Mice , Antigens, CD/metabolism , Antigens, CD/genetics , Blood-Brain Barrier/metabolism , Brain/metabolism , Capsid/metabolism , Capsid Proteins/metabolism , Capsid Proteins/genetics , Dependovirus , Endothelial Cells/metabolism , Gene Knock-In Techniques , Genetic Therapy , Receptors, Transferrin/metabolism , Receptors, Transferrin/genetics , Glucosylceramidase/genetics , Gaucher Disease/genetics , Gaucher Disease/therapy , Parkinson Disease/genetics , Parkinson Disease/therapyABSTRACT
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.