Date: 22nd June 2021
Emerging genetic engineering technologies are transforming modern life science, and are having a major impact in research, medicine, industrial biotechnology and agriculture. However, the methods used to date to insert genes into the genome are largely non-specific, meaning that scientists lack control of which cells receive the gene transfer. Now, a new technology has been developed that transfers genetic information into native cells upon illumination with cell-compatible red light, allowing adjustable and spatially resolved gene transfer down to single cell resolution.
Most clinical trials to date have employed viral vectors for the delivery of genetic material as they have high efficacy and prolonged transgene expression. However, despite its immense clinical potential, many clinical trials result in less than optimal outcomes, predominantly due to poor virus retention in the target tissue, neutralisation by the host’s immune system and off-target effects. These issues highlight a unmet clinical need for the development of innovative approaches to viral gene delivery. Whilst, recent advances have introduced a measure of specificity, such as the addition of biomaterials, electric fields or ultrasound to name a few, others have turned to light-controlled gene delivery technologies for spatial control. These types of gene therapy are starting to filter into the clinic however, there is still room for improvement as cytotoxic light is often used, both target and off-target cells take up the virus before optical control, or the host cells have to be pre-engineered to express photoreceptors.
Now, researchers at the University of Freiburg, Germany, led by Wilfred Weber and Maximilian Hörner, have engineered an adeno-associated viral (AAV) vector system that transferred genetic information into native target cells upon illumination with cell-compatible red light – creating an optical remote control for gene transfer. The system impressively allowed for adjustable and spatially resolved gene transfer down to a single-cell resolution.
To start, the team modified an AAV vector commonly used in the clinic, and mutated the virus so it could no longer recognise its natural receptor. In addition, they engineered the virus, now called OptoAAV, to display the phytochrome-interacting factor 6 (PIF6) from the plant Arabidopsis thaliana. In nature, PIF6 binds to another protein called PhyB when PhyB is illuminated with red light – so the team also modified PhyB giving it additional functionality to bind to target human cells. In theory, upon the application of OptoAAV and PhyB to cells, PhyB will bind to the target cells, then once a selected cell is illuminated, OptoAAV binding is initiated, and the target gene is then transduced into the cell.
To test the theory, the team applied the system to cells in culture. They found that cells were transduced in a light-dependant manner and the percentage of transduced target cells could be precisely fine-tuned by adjusting the illumination intensity and/or period. Furthermore, the system worked in a range of tumour cell lines.
Next, the team wanted to investigate the spatiotemporal control of transduction. In order to do this they incubated the cells with PhyB, then added an OptoAAV carrying one of two fluorescent reporters, green fluorescent protein (GFP) or Scarlet, and illuminated the cells using a photomask. Cells in the illuminated area fluoresced whilst, non-illuminated cells were not transduced and therefore did not express the fluorescent markers. Sequential exposure to the two reporter OptoAAvs using two different photomasks, showed regions of cells expressing both reporters in areas exposed to the light by both masks, and areas only expressing one reporter where illumination was restricted by one of the photomasks. This approach enabled the spatially resolved transduction of cells with two different transgenes, and could likely be expanded to additional transgenes.
Whilst there was respectable resolution using the photomasks, the team wanted to resolve this yet further – down to the single cell level. To address this, they used a conventional confocal microscope with the appropriate laser as the illumination source with the OptoAAV system. In 60% of the experiments they were able to transduce a single illuminated cells, and follow that cell for 48 hours.
The team here have designed a new OptoAAV technology that enabled the spatiotemporally resolved and light dose-dependant selective transduction of cells using low-intensity red light. The systems offers flexibility, such that it can be customised to target different cell types by switching adapter proteins, and the AAV can be loaded with different therapeutic agents/genes.
The team envisage the technique could allow the perturbation of biological processes at the single cell level, increasing our knowledge of how cells communicate for example to control development or regeneration of organs, to help us understand the applications of cell heterogeneity, and has potential to drive more precise biomedical applications.
The next milestone for OptoAAV will be in vivo testing. Optogenetics have already been tested in a blind patient, where retina cells were genetically altered to produce light-sensitive proteins, partially restoring sight. Here light-stimulating goggles were used, but specific cells were not transduced. However, such applications might benefit greatly from increased transduction specificity. Others have coupled bacterial injection systems with a light-controlled molecular switch, enabling delivery of cargo proteins in cells with spatial and temporal resolution albeit not at single cell resolution. Although the OptoAAV system is just at the start of its journey, it has great potential to accelerate specificity of vector delivery for a wide range of biomedical applications from neuroscience to cancer treatments.
For more information please see the press release at the University of Freiburg
Hörner, M., Jerez-Longres, C., Hudek, A., Hook, S., Yousefi, O.S., Schamel, W.W.A., Hörner, C., Zurbriggen, M.D., Ye, H., Wagner, H.J., et al. (2021). Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors. Science Advances 7, eabf0797.