Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning

Therapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals. [Display omitted] In this article, Nonnenmacher and colleagues establish TRACER, a cell-specific RNA-driven directed evolution platform for functional biopanning of adeno-associated virus (AAV) capsid libraries in animal tissue. Neuron- and glial-specific selection of an AAV9 library in mouse brain identified several AAV variants with unprecedented brain and spinal cord transduction.