ABSTRACT
Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.
Subject(s)
Capsid/metabolism , Dependovirus/metabolism , Directed Molecular Evolution , Gene Transfer Techniques , Muscle, Skeletal/metabolism , Amino Acid Sequence , Animals , Capsid/chemistry , Cells, Cultured , Disease Models, Animal , HEK293 Cells , Humans , Integrins/metabolism , Macaca fascicularis , Mice, Inbred BALB C , Mice, Inbred C57BL , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophy, Duchenne/therapy , Myopathies, Structural, Congenital/pathology , Myopathies, Structural, Congenital/therapy , Protein Multimerization , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/therapeutic use , RNA, Guide, Kinetoplastida/metabolism , Recombination, Genetic/genetics , Species Specificity , TransgenesABSTRACT
BACKGROUND: Adeno-associated virus (AAV) vectors are a promising vehicle for noninvasive gene delivery to the central nervous system via intravenous infusion. However, naturally occurring serotypes have a limited ability to transduce the brain, and translating engineered capsids from mice to nonhuman primates has proved challenging. METHODS: In this study, we use an mRNA-based directed-evolution strategy in multiple strains of mice as well as a de novo selection in cynomolgus macaques to identify families of engineered vectors with increased potency in the brain and decreased tropism for the liver. FINDINGS: We compare the transgene expression capabilities of several engineered vectors and show that while some of our novel macaque-derived variants significantly outperform AAV9 in transducing the macaque brain following systemic administration, mouse-derived variants-both those identified in this study and those reported by other groups-universally do not. CONCLUSIONS: Together, the results of this work introduce a class of primate-derived engineered AAV capsids with increased therapeutic potential and highlight the critical need for using appropriate animal models to both identify and evaluate novel AAVs intended for delivery to the human central nervous system. FUNDING: This work was funded primarily through an anonymous philanthropic gift to the P.C.S. lab at the Broad Institute of MIT and Harvard and by a grant from the Howard Hughes Medical Institute to P.C.S.