View source for Sintetična optogenetska naprava na osnovi BRET za pulzirajočo ekspresijo transgena, ki omogoča glukozno homeostazo pri miših

== INTRODUCTION ==

Gene expression patterns are important for understanding protein function, biological signaling pathways and cellular responses to external and internal stimuli. Transcription factors and many other regulatory proteins are regulated in a pulsatile fashion, leading to a variety of phenotypic consequences, ranging from stress response to differentiation and signaling.[2]
Pulsing has been observed in many types of proteins. In alternative bacterial sigma factors, but also in mammalian tumor suppressors like p53. Mammalian cells exhibit many pulsatile factors. The stress response pathways mediated by p53, which controls the DNA damage response pulses, just as nuclear factor κB which is involved in immune responses. [1] Iti s still unknown what functional capabilities can this pulsatile dynamic regulation provide for cells. Chemical inducers were used  to dynamically regulate transgene expression. Slow degradation and metabolisation of chemical inducers has been shown as not so good application for controlling the dynamics of regulators in most pulsing systems. Therefore, more precise light-inducible systems have been developed that allow control of gene expression. Pulsatile dynamic regulation of regulators is achieved. The limitation that occurs is  supplying adequate light intensity to activate these optogenetic systems. To solve this problem luminiscent opsins were developed ( directly coupling bioluminescent proteins to conventional light-sensitive opsins). BRET or bioluminescence resonance energy transfer is how luciferase substrates create bioluminiscence and activate opsins. After some time inactivation of opsins occurs and they regulate the cell activity in a pulsatile fashion. That is where the idea for transgene expression system that allows pulsatile regulation of gene expression both in vitro and in vivo came from. [2]
Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. [6]

== EXPERIMENT ==

In this experiment the development of the luminGAVPO, a luminsecent transcription factor is explained. LuminGAVPO enables pulsatile and quantitative activation of transgene expression in both mammalian cells and animals in a furimazine- or light irradiance-dependent manner. By controlling blood glucose homeostasis in type 1 diabetic mice its usefulness is tested. [2]

== RESULTS ==

Design and construction of the LuminON transgene expression system.
NanoLuc (Nluc) is a engineered luciferase from a deep sea shrimp, which is stable, bright, small and with sustained luminescence. It is used as luminiscence donor ( produces blue light) to activate LOV-based (The light-oxygen-voltage proteins) optogenetic modules. Nluc was fused to the N terminus of the mCherry-LOV2 fusion protein. When a protein was coexpressed with a mitochondria-anchored mVenus-Zdk protein, both mCherry and mVenus fluorescence accumulated at mitochondria under dark conditions. But the mCherry fluorescence readily diffused to the cytosol within 5 seconds after the addition of furimazine or exposure to external blue ligh. Blue light-inducible transgene expression systems based on Vivid were developed in which transcription factors bind to the promoter region ( to activate or repress gene transcription upon blue light exposure). The next step was to create a luminiscent transcription factor of a chimeric protein that consists of Nluc and a light-switchable transcription factor, GAVPO. Nluc-GAVPO fusions activated Gluc expression upon incubation with furimazine (FZ) or illumination with external blue light. On the other hand expression of Nluc and GAVPO saparately  yielded only a response to external blue light. Further studies showed also that Nluc-GAVPO fusions driven by a strong CMV promoter had higher activation levels than those driven by a weak SV40 promoter. . Since Nluc-GAVPO with NG configuration had moderate activation levels yet exhibited maximal induction ratio by furimazine, we termed it luminGAVPO (luminescent GAVPO), and the transgene expression system based on luminGAVPO was named the LuminON system.Based on the activation information “OR”-like logic gate is predicted for luminGAVPO-activated transgene expression using furimazine and external blue light as the two inputs. It was also noticed that higher concentrations of furimazine could produce brighter and more sustained luminescence, but Gluc expression slightly decreased when the furimazine concentration is higher than 5 μM. In addition, highly precise control of transgene expression was achieved by combining the furimazine concentration and light irradiance. Another assumption was made, that induction by furimazine multiple times could increase the luminGAVPOmediated activation levels, considering that Nluc is resistant to autoinhibition by its catalytic byproducts. The results showed that Gluc expression increased with increasing round of induction by furimazine.

Pulsatile activation of transgene expression mediated by luminGAVPO.
Furimazine-induced transgene expression by luminGAVPO would display a pulsatile profile ( activation followed by inactivation). After furimazine induction, high levels of Gluc mRNA and protein production were observed. The results showed that the Gluc synthesis reached a maximum m at ~4–6 h after furimazine induction and then sharply decreased. The medium has also been changed to remove the residual furimazine at 4 h after induction. Therefore, it can be seen that the majority of the furimazine had been consumed within 4 h after induction. It can be concluded that the pulse amplitude and duration of transgene expression were highly dependent on the dose of furimazine. Lower doses of furimazine exhibited shorter durations and smaller amplitudes of pulsatile activation. 

Pulsatile activation of transgene expression in mice.
These results in vitro needed to be proved in vivo. Therefore, luminGAVPO and mCherry reporter plasmids were transfected into the livers of mice.[2] This has been done using a hydrodynamic procedure, which is a rapid injection of a large volume of solution into a vasculature to facilitate substance transfer into parenchyma cells. [3]
Mice were administrated furimazine via tail intravenous (IV), intraperitoneal (IP) or intragastric. mCherry fluorescence was observed in the livers of these mice. After the measured time, the results showed that Gluc expression reached a maximum level within 4 h after furimazine induction and decreased after that. It can be concluded that the half-life of Gluc in the bloodstream is shorter than in the culture medium, and a lower dose of furimazine exhibited shorter durations and smaller amplitudes of Gluc expression in the bloodstream. After all these results demonstrate that the LuminON system can be used for pulsatile and reversible activation of transgene expression in vivo. [2]

Pulsatile expression of insulin to enhance blood glucose homeostasis in T1D mice.
Insulin is a hormon synthesized by the β-cells of pancreas and is a regulator of metabolism because it maintains glucose homeostasis. [5]
This pulsatile secretion of insulin may match the expression profile mediated by luminGAVPO. HEKFUR-Gluc-P2A-mINS cells were engineered for luminGAVPO-mediated insulin production. [2]
luminGAVPO and UASG-TATA-Gluc-P2A-mINS expression cassettes were integrated into HEK293T cells using the Sleeping Beauty transposon system, which is a promising technology platform for gene transfer in vertebrates. [2,4]
After that clones with the highest Gluc expression were microencapsulated into coherent, semipermeable and immunoprotective alginate-poly-(L-lysine)-alginate beads that allow free diffusion of substances with low molecular weight. T1D mice receiving the microencapsulated cells were treated with 5 mg/kg furimazine, and their blood glucose levels were measured after 3 h. The results showed that , a higher dose of furimazine resulted in more significant restoration of blood glucose and prolonged the maintenance of glucose homeostasis. The results showed the potential in the oral administration of furimazine instead of traditional intramuscular injection of insulin before and after meals. [2]

== DISCUSSION ==

Nluc is an ideal luminescence donor to activate LOV protein-based optogenetic systems via BRET, because it has the advantages of high brightness, no requirement of ATP and resistance to autoinhibition by its catalytic byproducts and perfectly matches the absorption spectra of the light-sensitive LOV proteins. In this study BRET-based transgene expression system was used fusing Nluc to a light-switchable transcription factor. Induction by furimazine caused activation of the proximal VVD domain in luminGAVPO, resulting in dimerization of luminGAVPO and binding of luminGAVPO to its cognate promoter, thereby initiating transcription of the target gene. The luminGAVPO dimer gradually dissociates from the promoter, leading to inactivation of transcription, after the furimazine is consumed. That is why this transgene expression is called pulsatile. LuminON system was also used to control the pulsatile and repetitive expression of insulin to enhance blood-glucose homeostasis in T1D mice, which could prevent the hypoglycemia. To conclude, BRET-based optogenetic device developed in this study can be used to precisely control the dynamics of key signaling proteins in a pulsatile fashion both in vitro and in vivo. [2]

== LITERATURE ==

[1] Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193-1200. doi:10.1126/science.1239999 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100686/

[2] Li, T., Chen, X., Qian, Y. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun 12, 615 (2021).https://www.nature.com/articles/s41467-021-20913-1#citeas

[3] Bonamassa B, Hai L, Liu D. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res. 2011;28(4):694-701. doi:10.1007/s11095-010-0338-9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064722/

[4] Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. doi: 10.1038/ng.343. Epub 2009 May 3. PMID: 19412179. https://pubmed.ncbi.nlm.nih.gov/19412179/

[5] Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25-53. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934755/

[6]Kobayashi, H., Picard, LP., Schönegge, AM. et al. Bioluminescence resonance energy transfer–based imaging of protein–protein interactions in living cells. Nat Protoc 14, 1084–1107 (2019). https://doi.org/10.1038/s41596-019-0129-7