An optogenetic system for nuclear translocation of viral nanoparticles and tunable gene delivery

The use of viruses to deliver nucleic acids for various applications, ranging from fundamental biological studies to clinical translation, is an active area of research recently fueled by the European approval of an adeno-associated virus (AAV) based product for human use. AAV is a promising vector able to deliver genes, parts of genetic circuits, RNAi, and most recently CRISPR/Cas. However, in vivo viral gene delivery still suffers from low gene expression at the target site and leaky off-target expression.

With the hopes of improving targeting and efficiency of gene delivery, vectors have been designed to be stimulus-responsive against a variety of inputs, including pH, temperature, and enzyme concentration in the extracellular environment. Although promising, engineering vectors to be responsive to exogenous stimuli, rather than endogenous inputs, may render the delivery process more controllable both temporally as well as spatially.

Additionally, mainly extracellular steps of the gene delivery process have been manipulated so far. Once inside the cell, gene vectors are challenged by several obstacles like sequestration in endosomes, ubiquitination and clearance by autophagosomes, cytosolic trafficking, and perinuclear accumulation. This makes nuclear localization a formidable rate-limiting step and a major determinant of effective gene delivery.

To overcome this problem, we have engineered AAV controllable by externally applied light. The virus is genetically engineered to display surface-exposed Phytochrome interacting factor 6 (PIF6) moieties capable of binding to Phytochrome B (PhyB). In HeLa cells constitutively expressing PhyB tagged with a C-terminal nuclear localization sequence (NLS), we are able to exert control over virus nuclear localization and gene delivery by varying the intensity, duration, and wavelength of light. Red light activates PhyB-NLS to bind to AAV-PIF6 and promote nuclear accumulation, and far-red light deactivates PhyB. A transfer function has been established relating the ratio of red:far-red light to the overall gene delivery by the virus, and spatial control of light is being investigated to enable patterned gene expression.

Combining optogenetic tools with viral gene delivery can lead to the development of predictable and controllable delivery platforms, which will be broadly useful in gene therapy and mammalian synthetic biology research. Other light-sensitive proteins such as the Light-Oxygen-Voltage (LOV) domains are also under investigation for use in gene delivery. Future applications include organized gene expression from regulated patterns of light in 2D and in 3D biomaterials for regenerative medicine applications.