Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery

Abstract

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 Arabidopsis thaliana 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 ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.

At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior

“…For example, the gene delivery efficiency of adeno-associated virus was increased sixfold and made light tunable by a Pif-bound viral capsid whose nuclear translocation was enhanced by a nuclear localization signal–tagged PhyB (93). A range of strategies have also been developed to control intracellular membrane trafficking (64, 94–96) to study protein secretion, endocytosis, and intracellular protein trafficking.…”

Stimulus-responsive viral vectors for controlled delivery of therapeutics

“…For example, Gomez et al rendered the AAV transduction process responsive to an externally applied light stimulus by using the light-activatable protein Phytochrome B (PhyB) and its binding partner phytochrome interacting factor (PIF) [25,103]. PIF was genetically inserted into the AAV capsid and target cells were made to express the PhyB protein tagged with a nuclear localization signal (NLS).…”

Light‐Patterned RNA Interference of 3D‐Cultured Human Embryonic Stem Cells

“…[15][16][17] The light-based optogenetic field has answered real biological questions [16] and developed tools for light-controlled genome editing and gene transfection. [18][19][20][21][22] However, these approaches rely largely on the visible light excitation sources and the construction of complex protein fusions via viral transfection. Other alternatives use photocaged small molecules and biopolymers for light-dependent gene regulation, [4,15,[23][24][25][26] which are limited by cellular delivery hurdles and the use of low tissue-penetrating UV-vis light.…”