Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery ACS Publications does not have a subscription to this publication. Please contact your librarian to recommend that your institution subscribe to this publication. This paper published ASAP on 11/30/15. The Acknowledgment section was modified and the revised version was reposted on 1/26/16. Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic ( can be reversed with far-red light, wt AAV2 and VNP-PIF6 in the presence of PhyB650-NLS do not exhibit increased nuclear localization, gene expression by VNP-PIF6 is induced by red light in cells expressing PhyB-NLS, cell metabolic activity assay indicates moderate toxicity induced collectively by VNP-PIF6, PhyB, PCB, and light, cell metabolic activity assay for additional cell lines, light-activatable viral gene delivery in three other cell lines, gene delivery by VNP-PIF6 begins to decline at high red light flux, virus-nucleus colocalization statistics for additional cell lines. ( Intracellular distribution of VNP-PIF6 4 h after transduction in HeLa cells exposed to far-red light. Blue: cell nuclei. Red: VNP-PIF6. ( Intracellular distribution of VNP-PIF6 4 h after transduction in HeLa cells exposed to red light. Blue: cell nuclei. Red: VNP-PIF6. ( Source.