Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction

Abstract

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging–uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

Materials promoting viral gene delivery

“…89,90 Further, genetically modified viruses can enhance TE, 91,92 broaden the tropism, 63,93 enable fluorescent imaging, 85 escape neutralizing antibodies, 94,95 generate stimuli responsive vectors, 96 e.g. by light 97,98 or enzymes, 99 and target cells. 100,101 Comprehensive recent reviews on bioengineering AAV can be found elsewhere.…”

Viral vector platforms within the gene therapy landscape

“… 324 , 325 Such lentiviral vectors are being continuously developed and optimized, and could enjoy a broader application in future gene therapies. In addition, the development of safer vectors via photo-switchable non-canonical amino acids to regulate transgene expression in a spatial and temporal manner, 326 also represent the next generation of lentiviral vectors.…”

Spatially Defined Gene Delivery into Native Cells with the Red Light‐Controlled OptoAAV Technology

“…To externally control transgene delivery, light is the optimal stimulus as it can be easily applied with high spatiotemporal precision in a reversible manner. As existing technologies for light‐controlled viral gene delivery were hampered by the need for cytotoxic ultraviolet (UV) light (Goater et al., 2000; Ito et al., 2004; Pandori & Sano, 2000; Pandori et al., 2002; Ulrich‐Vinther et al., 2002; Wang et al., 2019), the required uptake of viral vectors to both target and off‐target cells before optical control (Bonsted et al., 2006; Bonsted, Hogset, Hoover, & Berg, 2005; Goater et al., 2000; Gomez, Gerhardt, Judd, Tabor, & Suh, 2016; Hagihara et al., 2020; Hogset et al., 2002; Ito et al., 2004; Tahara et al., 2019; Ulrich‐Vinther et al., 2002; Wang et al., 2019), or the requirement for genetic pre‐engineering of the target cells to express the photoreceptor (Gomez et al., 2016), we recently published the OptoAAV technology that overcomes these limitations (Hörner et al., 2021). The OptoAAV system allows the spatially resolved delivery of transgenes into native target cells upon illumination with low‐intensity red light and is discussed in more detail in the section Background Information.…”