A novel gene delivery platform based on a commensal human anellovirus demonstrates transduction in multiple tissue types

<i>Anelloviridae</i> is a family of non-enveloped viruses with negative-sense, circular, single-stranded DNA genomes that infect vertebrates and are a ubiquitous component of the human virome. The non-pathogenic and commensal nature of anelloviruses makes them compelling candidates as vectors for next-generation genetic medicines. Here we report development of a gene delivery vector system based on an anellovirus of the <i>Betatorquevirus</i> genus. Production is enabled by the development of the self-amplifying <i>trans</i>-complementation of a universal recombinant Anellovector (SATURN) system, which relies on a self-replicating plasmid to provide viral proteins in <i>trans</i> and a Cre-lox-based recombination mechanism to generate single unit-sized circular genomes inside the MOLT-4 production cell line. We demonstrate that the SATURN system can package a vector genome from a single Betatorquevirus with capsids from multiple Betatorquevirus species, supporting the feasibility of establishing a vector platform that takes advantage of anellovirus diversity. The vector demonstrated long-term <i>in vivo</i> function in the mouse eye for 12 months post subretinal administration. Comparable gene expression was achieved to dose-matched adeno-associated virus 9 <i>in vitro</i> and <i>in vivo</i> when transducing HEK293TT cells and the murine brain, respectively. To our knowledge, this is the first report of a functional anellovirus-based gene expression vector.