Optogenetic Controlled Transgene Expression System For Controllable Gene Editing And Tumor Therapy

"Gene editing" and "tumor therapy",as two research hotspots in the current field of life science,there are some scientific problems that need to be solved urgently.In the field of gene editing,the off-target effects of CRISPR-Cas9 gene editing technology caused by the uncontrollable expression of Cas9 may lead to serious and immeasurable side effects.Therefore,the applications of CRISPR-Cas9 have been greatly limited.In the field of tumor therapy,surgical resection,chemotherapy and radiotheray are still the most recommended method for tumor therapy,but it has been criticized for its low cure rate and serious side effects.However,although the emerging tumor immunotherapy can produce good therapeutic effects,it cannot be applied in clinic on a large scale due to the over-activation of immune system caused by its uncontrollability.In short,these two hot research areas are in urgent need of precision control technology to solve the security problems caused by uncontrollability.Therefore,it is of great significance to develop controllable gene editing technology and novel tumor therapy to solve the above problems.Optogenetics combines optical technology and genetic technology to control the behavior of cells precisely.This technology is widely used in biomedical fields with its unique advantages,including non-invasive and spatiotemporal specificity.In response to the above scientific problems,merging synthetic biology design concept with optogenetic technology,we use the far-red light-controlled gene expression system(FRL-v2)that we have reported,on the themes of “Construction of a far-red light-controlled split-Cas9 gene editing system and its application research in vivo”and “Far-red light-controlled novel tumor immunotherapy for preventing tumor recurrence and metastases after the surgical removal” respectively to conduct controlled gene editing and tumor therapy research.1.Construction of a far-red light-controlled split-Cas9 gene editing system and its application research in vivo.(1)A far-red light-controlled split-Cas9 gene editing system(FAST)was designed,constructed and optimized.The experimental results showed that FAST system could induce editing of multiple genomic loci in diverse human cell lines mediated by nonhomologous end joining and homology directed repair.(2)Characterization of the gene editing performance of the FAST system were studied in depth.The experimental results showed that the FAST system showed precise and spatiotemporal gene editing with illumination intensity-and duration-dependent,and almost no cytotoxicity.The potential off-target site of BMP1 were determined through T7E1 assay showed that the FAST system can significantly reduce the off-target effect compared with the full-length Cas9.(3)The ability of the FAST system to control gene editing in vivo was studied in three different mouse models.In subcutaneous transplantation mouse model,the advantages of FAST system in gene editing in vivo were verified preliminarily.In the td Tomato reporter transgenic mouse model,it was verified that the FAST system could edit the genes of somatic cells in the internal organs of mice with low intensity far-red LED illumination.In the xenograft tumor mouse model,the FAST system was verified to be able to knock out the oncogene PLK1 in the tumor and significantly inhibit tumor growth.2.Far-red light-controlled novel tumor immunotherapy for preventing tumor recurrence and metastases after the surgical removal(1)far-red light controlled immune regulatory gene circuit was designed,constructed.The engineered stable cell lines were screened that can secrete IFN-?,TNF-? and IL-12 under far-red light illumination,and the induced cytokine secretion was up to 200 times under FRL.(2)Long-term cytokine secretion capacity and characterization of the far-red light controlled immune regulatory engineered cells(IRECs)were tested.The transplantation carrier(hydrogel)containing IRECs was able to secrete cytokines in a controlled manner for up to a month in vitro culture environment,and showed illumination intensity,illumination time dependence and reversibility.(3)The anti-tumor effect of the far-red light controlled novel tumor immunotherapy in melanoma mouse model were studied.Through monitoring the tumor volume and survival rate of mouse,indicating the significant anti-tumor effect,and the survival rate of mice reached 100% within 60 days after surgery.(4)The internal mechanism of the anti-tumor effect was studied.Serum levels of IFN-?,TNF-?,IL-12,CXCL10,IFN-? and IFN-?,which are related to anti-tumor function,were measured.The levels of these cytokines were significantly increased in the far-red light group.Flow cytometry was used to detect NK cells and T cells in peripheral blood and spleen,and it was found that the proportion of NK cells and T cells in the far-red light group increased,and the proportion of activated cells in both of them also increased significantly.(5)The safety was evaluated.No significant difference was found between the mice in the treatment group and the untreated group by blood routine and serum biochemical index test,thus proving that the therapy was safe and reliable.Collectively,we develop a controllable CRISPR-Cas9 gene editing system and a novel tumor immunotherapy by using the light-controlled gene expression system.First,a far-red light controlled split-Cas9 gene editing system was developed to solve the off-target effect caused by the uncontrollability of CRISRP-Cas9 technology and achieve spatiotemporal specificity accurate gene editing.This research provides a new controllable gene editing tool suitable for in vivo application,and further extends toolbox of CRISPR-Cas9 gene editing,which can be used for basic research of gene function and the treatment of human genetic diseases,like genetic disease,tumor and so on,has great potential value in clinical translational application.Secondly,a far-red light controlled immune regulatory engineered hydrogel containing engineered cells was developed to achieve a novel tumor immunotherapy by combination of tumor surgery therapy and the local tumor immunotherapy and to solve the problem of tumor recurrence and metastases after the surgical removal.It can improve cure rate of tumor resection,at the same time,can avoid excessive activation of the immune system of tumor immunotherapy due to uncontrollable.This research provides assurance for the cure rate of surgical resection therapy for tumors,provides new ideas for cytokine immunotherapy,local immunotherapy,and expands the tumor therapy toolbox.This paper also provides a new exploration and a new direction for the application of optogenetics in biomedical field.