Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

Zárate, Rafaella V.; Arancibia, Duxan; Fernández, Anllely; Signorelli, Janetti; Larrondo, Luis F.; Andrés, Marı́a Estela; Zamorano, Pedro

doi:10.1016/j.ejbt.2021.03.006

Cited by 2 publications

(6 citation statements)

References 34 publications

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“…The OPTO BLUE system seems to maintain this dynamic since it can increase the expression of the GOI after 2 h of induction, which becomes significant after 8 h of illumination. This expression keeps increasing as the light induction is maintained, similar to previous reports [14,21]. Furthermore, we observed that the expression of EGFP could be regulated by light intensity.…”

Section: Discussionsupporting

confidence: 91%

“…For that purpose, we have adapted the Light-On system to a single lentiviral platform that has shown to be very reliable and efficient in transferring genetic information in HEK293-T cells. We previously developed a lentiviral Light-On platform that relies on the co-transduction of two viral particles to regulate gene expression effectively [21]. Although this system is appropriate in an experimental setting in cell biology research, it is not practical for applications in a therapeutic situation, due to the difficulty of transducing two viral particles to the same target cell, where the insertion of the expression cassettes of the GAVPO transactivator and the regulated light-induced UAS expression cassette with the GOI is required.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, we optimized and adapted the LightOn system to a lentiviral platform [21]. Although this system allows for the blue light-inducible expression of a GOI in mammalian cells, it is a dual system based on two lentiviral transfer plasmids, the pFUG-1D/2A-HA-GAVPO-W and pF-UAS(s)-mCherry-W plasmids, containing the two main components of the LightOn system: the light-switchable transcription factor GAVPO and the UAS inducible promoter to which it binds, which contains five consecutive repeats of the upstream activating sequence of Gal4 (5xUASG) fused to a TATA sequence of the minimal promoter E1b (5xUASG-TATA).…”

Section: Design and Construction Of The Opto-blue Systemmentioning

confidence: 99%

“…We previously optimized and adapted the LightOn system to a lentiviral platform [21]. Lentiviruses allow the integration of genetic material into the host-cell genome so that genetic incorporation remains stable over time [22].…”

Section: Introductionmentioning

confidence: 99%

“…In our previous work, we constructed two lentiviral plasmids bearing the two components of the LightOn system: (i) one that expresses GAVPO under the Ubiquitin constitutive promoter; and (ii) a second plasmid that expresses a gene of interest (GOI) driven by the UAS inducible promoter. We demonstrated the functionality of this system through the blue light-induced expression of the cerebral dopamine neurotrophic factor and mCherry by transfection and transduction of mammalian cell lines and neurons [21]. As the LightOn lentiviral system is based on two lentiviruses, its efficient operation requires the co-transduction of the target cells in the tissue where the regulation of gene expression is desired.…”

Section: Introductionmentioning

confidence: 99%

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OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression

Arancibia,

Pol,

Vargas-Fernández

et al. 2023

IJMS

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Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Design and Construction Of The Opto-blue Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression

Arancibia,

Pol,

Vargas-Fernández

et al. 2023

IJMS

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Optogenetics and Targeted Gene Therapy for Retinal Diseases: Unravelling the Fundamentals, Applications, and Future Perspectives

Kulbay,

Tuli,

Akdag

et al. 2024

JCM

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With a common aim of restoring physiological function of defective cells, optogenetics and targeted gene therapies have shown great clinical potential and novelty in the branch of personalized medicine and inherited retinal diseases (IRDs). The basis of optogenetics aims to bypass defective photoreceptors by introducing opsins with light-sensing capabilities. In contrast, targeted gene therapies, such as methods based on CRISPR-Cas9 and RNA interference with noncoding RNAs (i.e., microRNA, small interfering RNA, short hairpin RNA), consists of inducing normal gene or protein expression into affected cells. Having partially leveraged the challenges limiting their prompt introduction into the clinical practice (i.e., engineering, cell or tissue delivery capabilities), it is crucial to deepen the fields of knowledge applied to optogenetics and targeted gene therapy. The aim of this in-depth and novel literature review is to explain the fundamentals and applications of optogenetics and targeted gene therapies, while providing decision-making arguments for ophthalmologists. First, we review the biomolecular principles and engineering steps involved in optogenetics and the targeted gene therapies mentioned above by bringing a focus on the specific vectors and molecules for cell signalization. The importance of vector choice and engineering methods are discussed. Second, we summarize the ongoing clinical trials and most recent discoveries for optogenetics and targeted gene therapies for IRDs. Finally, we then discuss the limits and current challenges of each novel therapy. We aim to provide for the first time scientific-based explanations for clinicians to justify the specificity of each therapy for one disease, which can help improve clinical decision-making tasks.

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Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

Cited by 2 publications

References 34 publications

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression

Optogenetics and Targeted Gene Therapy for Retinal Diseases: Unravelling the Fundamentals, Applications, and Future Perspectives

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