Novel Self-Assembled Delivery System For CRISPR Transcriptional Activation Mediated Cancer Therapy

Cancer is a kind of malignant disease which is more difficult to be cured and seriously threatens human life and health.Conventional tumor therapeutics mainly include surgery,chemotherapy and radiation therapy.Although surgery can remove tumors in early stage,it has important limitations for advanced tumors with distal metastases and is prone to tumor recurrence after surgical treatment.Chemotherapy and radiation therapy have greatly improved the survival rate of cancer patients.However,the treatments are often accompanied by significant side effects,due to the lack of tumor-targeting capability.The limitations of this strategy are even more obvious for patients in advanced stages.Therefore,there is an urgent need to explore new methods to improve tumor treatment effects and patient prognosis.With the rapid development of life sciences in recent years,gene therapy has developed into a promising strategy because it can achieve safe and effective treatment through the regulation of gene functions.In the process of gene therapy,gene regulatory tools with rich function play the key role,laying the foundation for achieving better anti-tumor effect.The CRISPR/Cas9 system is derived from the immune system of bacteria and archaea to fight against exogenous nucleic acid materials such as DNA and plasmids that invade cells.In recent years,it has rapidly developed into a new generation of gene editing technology with its advantages of easy operation,short time consumption and high efficiency,which has greatly promoted the progress of biomedical field.Furthermore,the capabilities of this system are being updated to become a revolutionary tool for cancer gene therapy.Among them,CRISPR activation system(CRISPRa system),which is based on CRISPR/Cas9 system,is different from the negative regulation mode of traditional gene therapy.CRISPRa system contains inactivated Cas9 protein(catalytically dead Cas9)and transcriptional activation functional unit,which can play the positive regulation function of up-regulating endogenous gene expression under the guidance of sgRNA.The advantages of CRISPRa system in gene therapy include(1)without DNA double strand breaks,(2)strong specificity and precise regulation of gene targets,(3)upregulate of endogenous gene expression without restriction of gene size and epigenetic silencing.The transcription activation of CRISPRa system provides a new strategy for precise tumor treatment.However,the efficient delivery of CRISPRa system in vivo remain challenges.The application of CRISPRa technology remains at cellular level.So far,there are few reports about the application of CRISPRa system for in vivo tumor treatment.With the rapid development of nanotechnology and biomaterials,non-viral vectors have rapidly developed into ideal delivery systems with the advantages of good tumor targeting,low immunogenicity,functional diversity and easy scale-up production.A variety of carriers have been studied for CRISPR delivery.Despite these studies displayed promising results,all the approaches have been designed and explored for CRISPR/Cas9 mediated genome editing.Inducing cell phenotypic changes that achieve antitumor therapeutic effects in vivo via CRISPR-based transcription activation required more optimized intratumor delivery efficiency which are remains elusive.To address the issue of CRISPRa targeted delivery in vivo,we proposed a programmed multi-stage response strategy with core-shell structure to conquer a series of physiological barriers during delivery process and achieve tumortargeted delivery and CRISPRa system mediated efficient transcriptional activation of endogenous genes.In this study,we constructed two novel self-assembled non-viral delivery platforms for efficient delivery of CRISPRa plasmid to achieve precise gene therapy and immunotherapy for cervical cancer and malignant melanoma respectively.In addition,the anti-tumor mechanism was investigated.First,in order to achieve efficient delivery of CRISPRa systems in vivo,carriers need to have the ability to efficiently load CRISPRa plasmids and the function to overcome the physiological barriers in vivo.Therefore,we constructed programmable hierarchical-responsive self-assembling gene vector with core-shell structure and designed a novel CRISPRa plasmid system that can express both sgRNA and dCas9.The characterization data showed that the nanoparticle has a stable structure and homogeneous particle size.The delivery system can promote the cell uptake and transfection efficiency of tumor cells which benefit from the programmable hierarchical responsiveness triggered size reduction and charge reversal in the simulated tumor microenvironment.Meanwhile,the efficient transfection efficiency enables the CRISPRa system to perform powerful capability of transcriptional activation,which can significantly activate the gene targets expression of different sizes and types in tumor cells.After intravenous injection,the delivery system can programmatically break through multiple physiological barriers in vivo to deliver CRISPRa system into tumor cells.It is demonstrated that the treatment in tumor models can induce apoptosis through transcriptional activation of endogenous gene targets,thereby inhibiting tumor growth.The novel programmable hierarchical-response delivery strategy can substantially reduce the impact of multiple physiological barriers during the delivery process,leading to high performence of CRISPRa system-mediated tumor therapy through transcriptional activation of endogenous genes.The study open a new avenue for the new generation of tumor gene therapy which breaks the restrictions of gene size and type without genome alterations.In addition to the direct killing of tumor cells,the immune system,as an important mechanism for the body’s defense against foreign pathogens and abnormal cells,has brought sharp focus its function in tumor treatment.The activation of the immune system can promote the body to identify and remove cancerous cells through cellular immune mechanism,and the immune memory effect can effectively prevent tumor recurrence and metastasis in the long term Therefore,effective immune activation can better improve the therapeutic effect of tumor.Pyroptosis can activate the anti-tumor immune response and produce a long-lasting immune memory effect,showing great potential of anti-tumor recurrence and metastasis.However,the key gene target GSDME during the pyroptosis process is absent in most cancer types.Therefore,it is of great importance to develop a safe and precise strategy for the regulation and application of tumor pyroptosis.Effective manners of regulating tumor pyroptosis are still lacking.CRISPRa system has the ability of positive gene regulation,which can effectively activate the expression of endogenous gene targets and provides a new way for precise regulation of tumor pyroptosis.Thus,we next constructed a multistage responsive nanoplatform PCTC for the induction of tumor pyroptosis by systemic codelivery of a nucleic acid therapeutic(CRISPRa plasmid)and a chemotherapeutic drug cisplatin.We found that PCTC induced tumor pyroptosis not only effectively inhibited the growth of malignant melanoma,but also provide robust immune protection from recurrent and metastatic tumors,providing a new therapeutic strategy for the treatment of advanced melanoma.Through the in vivo targeted delivery of cisplatin and CRISPRa system targeting GSDME gene,caspase-3 pathway and GSMDE expression were activated simultaneously,achieving precise cascade regulation of tumor cells from apoptosis to pyroptosis.The induced pyroptosis kills tumor cells while reshaping the tumor immune microenvironment,activating robust anti-tumor immune response and long-term immune memory effects.In vivo results demonstrate that PCTC mediated tumor pyroptosis can effectively inhibit tumor recurrence and metastasis.The study of immune mechanism proved that PCTC mediated tumor pyroptosis could realize multiple boosting of cancer-immunity cycle.These steps include(1)tumor antigen and cytokine release,(2)DC maturation and antigen uptake,(3)T cell priming,(4)recognition of tumor cells by tumor specific T cells,(5)killing tumor cells,(6)generation of immune memory effect.In summary,we successfully constructed two innovative gene delivery system based on the self-assembly strategy of core-shell structure and CRISPRa plasmid for precise gene therapy and immunotherapy of tumor without genome changes.The antitumor mechanisms were further explored.The novel delivery systems and tumor treatment strategies developed in this study will provide theoretical basis and new ideas for the next generation of tumor therapy.