The Role Of GSS Targeted Gene Editing Therapy Combined With Radiotherapy In The Treatment Of Glioma

BackgroundGlioma is the most common malignant tumor of the central nervous system,and glioblastoma（GBM）is one of the most malignant aggressive diffuse tumor among them,with an average annual incidence of more than 250 thousand.At present,the main treatment methods for GBM patients are still surgical resection combined with radiotherapy and chemotherapy,but the prognosis is extremely poor,with the median survival time of patients<14.6 months,and the 5-year survival rate is only 5.6%.Due to the blood-brain barrier,treatment options other than radiotherapy have limited effect on overall survival.Therefore,radiotherapy plays an extremely important role in the treatment of GBM.Postoperative radiotherapy can reduce the recurrence rate of glioma.For glioma that cannot be resected surgically,radiotherapy can also be used to control tumor growth.However,the resistance of GBM to radiation is one of the main cause of poor prognosis.Therefore,it has become an urgent task to find a new target to enhance the therapeutic effect and prolong the survival of glioma patients with radiosensitization.AimsThe CRISPR/Cas9 library was used to screen the lethal genes in combination with GBM radiotherapy,and the function and mechanism of this target mediating radiotherapy resistance were systematically explored.Based on this target,new strategies of gene editing technolo Gy in GBM therapy were explored.Methods1.GBM cells were infected with genome-wide CRISPR/Cas9 screen,and GBM models were constructed in situ for in vivo screening.The targets of GBM radiotherapy resistance were analyzed by library sequencing.2.Radiotherapy resistance GBM cells were constructed,the cells were subjected to RNA-seq,and intersection of RNA-seq results and library results was used to preliminarily identify GSS as the key gene of radiotherapy resistance through data analysis and experiments.3.The stable GSS knockdown cell line and the model of radiotherapy resistance to PDX were constructed,and the relationship between the expression level of GSS and radiotherapy sensitivity was determined by in vitro and in vivo experiments,such as immunofluorescence,Western Blot,CCK-8,and cell colonies assay.4.Explore the clinical correlation between GSS and glioma by analyzing public database data and GBM tissue chip;5.Immunofluorescence and RNA-seq assay were used to analyze the activation of iron death pathway by GSS-mediated radiotherapy sensitization.6.The association between radiation and ferroptosis in GBM cells was verified by database analysis,flow cytometry,transmission electron microscopy,CCK-8 assay and other methods.7.The mechanism of GSS in glioma radiotherapy was elucidated through immunohistochemistry,WB,metabolomics analysis and other experimental systems;8.Loading Cas9/sgGSS complex into extracellular vesicles targeting gliomas and testing its effect on radiotherapy sensitivity in multiple in situ glioma models.Results1.The genome-wide CRISPR/Cas9 screen was successfully screened in vivo,and the radiotherapy sensitive genes were obtained by data analysis;2.The radiotherapy resistant cell line was successfully constructed,and the target GSS was obtained by the intersection of RNA-seq and library results.3.Stable knockout GSS cell lines and radiotherapy resistance PDX models were successfully constructed.Immunofluorescence and WB experiments proved that GSS protein was highly expressed in radiotherapy resistance models;CCK-8 and cell cloning experiments proved that GSS deletion enhanced radiotherapy effect;4.Database data and tissue chips showed that upregulated GSS expression led to poor prognosis;5.Immunofluorescence and RNA-seq results showed that GSS-mediated radiosensitization was not related to DNA damage repair,but to ferroptosis pathway.6.After radiation,the increase of lipid-derived ROS was detected by flow cytometry.Transmission electron microscopy,CCK-8 test and database analysis showed that ferroptosis of GBM cells was induced by radiation.7.During radiotherapy,GSS knockout inhibited GSH synthesis.On the one hand,GPX4activity was reduced,leading to lipid peroxide accumulation;On the other hand,it reduces the discharge efficiency of Fe²⁺,increases the accumulation of unstable iron pools,causes Fenton reaction,produces excessive ROS,and aggravates the occurrence of ferroptosis.8.Ang/TAT-sgGSS-EV was constructed and verified in LN229 in situ model and GSC in situ model to effectively interfere with GSS expression.Combined radiation treatment significantly enhanced radiotherapy sensitivity,which proved the success of in vivo gene editing therapy strategy.Conclusion1.Through in vivo pressure screening of the radiotherapy combined lethal gene set through CRISPR/Cas9 library,transcriptional sequencing results of the radiotherapy combined resistance cell lines showed that GSS played a key role in the radiotherapy resistance process of glioma.2.The online database data,tissue chip and clinical sample system confirmed that the expression level of GSS was closely related to the grade and stage of glioma patients and poor prognosis.The higher the grade of glioma,the higher the expression level of GSS,the worse the prognosis.3.According to cytological experiments,molecular biology experiments and database data analysis,the mechanism of radiotherapy resistance mediated by GSS is closely related to ferroptosis.Through the synthesis of reducing GSH,GSS enhances the activity of GPX4,thereby reducing the lipid peroxide,reducing the accumulation of Fe²⁺,and inhibiting Fenton reaction,thus inhibiting ferroptosis.4.In LN229 in situ glioma model and GSC in situ glioma model,Ang/TAT-sgGSS-EV has high targeting efficiency and strong gene editing ability,which can realize in vivo targeted intervention of GSS.Combined with radiotherapy,ANG/Tat-Sgg SS-EV can significantly inhibit the development of glioma,which has a wide range of clinical application prospects.