【作者】 杨锦；

【导师】 巩长旸；

【作者基本信息】 四川大学 ， 药剂学， 2022， 博士

【摘要】 研究背景与目的恶性肿瘤是一种威胁人类生命健康的重大疾病,已成为沉重的全球疾病负担。作为肿瘤治疗的主要手段,手术切除、化疗和放射治疗发挥了重要的临床作用,但仍然存在一定的治疗局限性和不良反应,疗效并不理想,亟待开发针对肿瘤治疗的新策略。恶性肿瘤的发病机制十分复杂,往往涉及多个基因突变和多条信号通路的改变。基因治疗能够从基因组源头入手,通过替代、增强或阻断特定基因表达以达到治疗疾病目的,已逐渐发展为继传统疗法后极具潜力的抗肿瘤方法。随着基因工程的进步和生物技术的革新,先进的基因编辑技术正在改变着基因治疗的格局,为精准调控肿瘤治疗关键靶点提供了新的手段。成簇规则间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)是近年来兴起的第三代基因编辑技术,其通过单向导RNA(Single-guide RNA,sg RNA)引导Cas9核酸酶切割目的DNA以达到插入、敲除或矫正基因等多种基因编辑目的,具有特异性好、编辑效率高及操作性强等优势,为肿瘤基因治疗提供了更加强大而简便的基因组编辑工具。然而,将CRISPR/Cas9系统递送到体内靶细胞发挥作用的过程中还面临着复杂的生理病理屏障,这极大地阻碍了CRISPR/Cas9的高效递送与精准激活。此外,CRISPR/Cas9系统在其他正常组织的非特异性分布也可能带来严重且不可预估的副作用。因此,如何实现CRISPR/Cas9系统体内的高效靶向递送和特异性激活是其抗肿瘤应用中迫切需要解决的难题。近年来,新兴的非病毒递送载体因其免疫原性低、稳定性好和功能多样化等优点成为可替代病毒载体的新选择,被广泛用于CRISPR/Cas9系统的体外及体内递送。然而,普通的非病毒载体往往无法有效应对体内动态变化的生理病理环境而导致递送效率低、靶向性差及治疗相关毒性增加等问题。同时,CRISPR/Cas9系统需入核启动基因编辑功能的独特工作机制也对递送载体提出了更高的要求,亟待开发兼具高装载能力、特异靶向性、高递送效率、准确核定位及低毒性的新型非病毒递送系统以提高体内CRISPR/Cas9编辑的高效性、精准性与安全性。因此,本文聚焦CRISPR/Cas9基因编辑系统在体内靶向递送及抗肿瘤应用的前沿与难点问题,采用稳定性好且灵活度高的CRISPR/Cas9质粒系统作为基因编辑工具,针对恶性肿瘤的关键治疗靶点设计并构建CRISPR/Cas9基因编辑系统。同时结合肿瘤微环境的特点,合理设计并开发两类新型肿瘤微环境级联响应的CRISPR递送系统,通过分步自组装策略装载CRISPR/Cas9,以期克服体内层级递送屏障,实现CRISPR/Cas9高效、精准、安全的体内递送与激活,从而突破当前肿瘤免疫治疗和光动力治疗(Photodynamic therapy,PDT)困境,达到安全高效的抗肿瘤治疗目的。研究方法首先,以酮缩硫醇为连接链交联低分子量聚乙烯亚胺(Polyethyleneimine,PEI 1.8K)制备得到活性氧(Reactive oxygen species,ROS)敏感性聚乙烯亚胺衍生物(ROS-sensitive polyethyleneimine derivative,PR)作为内核材料;以透明质酸(Hyaluronic acid,HA)为外壳骨架,并进一步通过酰胺化反应和点击化学反应接枝RGD-MMP多肽和聚乙二醇(polyethylene glycol,PEG)以制备得到多功能外壳材料(HA-RGD-MMP-PEG polymer,HRMP)。随后通过核磁共振氢谱(~1H nuclear magnetic resonance,~1H-NMR)和傅里叶变换红外光谱(Fourier Transform Infra-Red,FTIR)对产物进行结构表征以验证是否合成成功。接着,通过琼脂糖凝胶电泳实验考察PR的质粒压缩能力,并进一步利用静电相互作用制备复合物Core和可程序性解锁的纳米套娃CRISPR系统(Programmable unlocking nano-matryoshka-CRISPR system,PUN),同时考察其在高浓度金属基质蛋白酶(Matrix metalloproteases,MMPs)、透明质酸酶(Hyaluronidase,HAase)或ROS处理前后粒径、电位和形貌等变化。然后,对PUN的体外细胞毒性和溶血程度进行研究,并考察其在细胞水平上的递送效率及转运机制,包括细胞摄取实验、溶酶体逃逸实验、细胞转染实验和肿瘤球穿透实验等。接下来,以PX333质粒为载体构建并优化得到可同时高效编辑程序性死亡配体-1(Programmed cell death ligand-1,PD-L1)和蛋白络氨酸磷酸酶N2(Protein tyrosine phosphatase N2,PTPN2)的CRISPR/Cas9质粒系统,随后将其装载于PUN并考察其调控PD-L1和PTPN2蛋白表达水平的情况。进一步地,考察PUN在体内的转染效率并通过小动物活体成像系统监测PUN在体内的分布情况。随后,构建B16-F10黑色素瘤小鼠皮下移植瘤模型以考察PUN的体内抗肿瘤疗效,并通过小鼠体重监测、免疫组化分析和血生化分析等方法进行体内安全性评价。同时,通过分析治疗后小鼠肿瘤免疫微环境的改变以评价体内免疫激活水平。最后,在荷瘤小鼠模型中考察PUN诱导体内免疫记忆效应的作用。在进一步的研究中,我们以上述PR为内核材料,以二硫键为连接键将二氢卟吩e6(Chlorin e6,Ce6)修饰在HA骨架上以制备得到外壳HA-Ce6,通过核磁共振氢谱法和傅里叶变换红外光谱法对两者进行结构表征,同时对载体材料的细胞毒性进行考察。此外,通过分析HA-Ce6的荧光光谱和紫外可见光谱以考察其光学特性。接着,以PX330质粒为载体构建并筛选出精准靶向肿瘤缺氧诱导因子(Hypoxia-inducible factor-1α,HIF-1α)的CRISPR/Cas9质粒系统,利用分步自组装方式制备得到PR/p DNA和多重肿瘤微环境刺激响应型的共递送纳米系统(Multiple stimuli-responsive nano CRISPR,Must-nano),并考察其对质粒的装载能力和光降解保护能力。进一步地,通过分别考察Must-nano在高浓度HAase、谷胱甘肽(Glutathione,GSH)或ROS条件下粒径、电位和形貌的变化及Ce6释放情况以评价其多重敏感性。然后,在体外考察Must-nano介导ROS生成和GSH消耗水平,同时利用流式细胞仪、激光共聚焦扫描显微镜检测Must-nano的体外细胞摄取水平、胞内解组装行为、线粒体靶向作用、溶酶体逃逸、细胞核定位及细胞转染效率。在体外抗肿瘤作用研究中,分别采用细胞毒性实验、细胞凋亡实验和细胞死/活检测等评价Must-nano的体外光动力疗效;另外考察常氧/乏氧条件下不同处理后细胞的活力、侵袭和迁移能力及相关蛋白表达水平的变化。在体内抗肿瘤疗效的研究中,构建HCT-116结直肠癌小鼠皮下移植瘤模型并通过小动物活体成像技术考察Must-nano的体内分布情况,随后采用静脉给药方式对荷瘤小鼠进行治疗,并通过对肿瘤组织的免疫组化分析等进一步阐明抗肿瘤机制。最后,通过监测小鼠体重、血生化指标和组织病理改变等对Must-nano在体内应用的潜在系统毒性进行评价。研究结果针对目前免疫检查点阻断疗法(Immune checkpoint blockade,ICB)存在特异性缺乏、作用短暂、阻断不完全以及肿瘤细胞免疫应答低等问题,我们设计并构建了一种具有多靶点高效编辑功能的CRISPR/Cas9质粒系统,并利用合成的多功能靶向递送载体与质粒的分子自主装原理,构建出粒径均一、稳定性良好的递送系统PUN。通过对材料和递送系统的表征与探索性研究,结果表明PUN是一个低毒安全、可高效装载大尺寸CRISPR/Cas9质粒且具有多功能化修饰的纳米递送系统。在体外模拟的肿瘤微环境条件下,PUN可响应多重肿瘤刺激信号发生尺寸缩小、电荷反转和结构崩解等变化,表现出微环境刺激增强的细胞摄取水平、溶酶体逃逸、胞内控释、细胞核定位、转染效率和肿瘤球深层穿透的能力。在B16-F10黑色素瘤小鼠皮下移植瘤模型中,PUN展现出显著性的抗血液干扰、肿瘤特异性靶向及长效肿瘤蓄积能力。体内抗肿瘤研究进一步表明,PUN可以借助肿瘤微环境级联解锁性能以突破体内多重递送屏障,精准高效地将CRISPR/Cas9递送到靶细胞中并激活其高效的多靶点基因编辑功能,有效确保了PD-L1下调介导的PD-1/PD-L1通路阻断效应与PTPN2敲除调控的干扰素-γ依赖性肿瘤细胞免疫增敏作用的同步开启。由此,PUN有效重塑了肿瘤高度抑制的免疫微环境,级联激活体内适应性抗肿瘤免疫响应并诱导长期的免疫记忆效应,最终达到了安全且高效抑制小鼠恶性黑色素瘤生长与复发的目的。本部分的研究为当前的ICB疗法提供了一种安全高效、彻底持久且高响应性的抗肿瘤免疫治疗新策略,也为临床肿瘤免疫治疗的发展注入了新动力。除了基于CRISPR/Cas9的单一疗法,发展多途径协同的抗肿瘤策略将有望进一步拓展CRISPR/Cas9在恶性肿瘤治疗中的应用前景。基于肿瘤光动力疗法面临光敏剂缺乏肿瘤组织选择性、肿瘤细胞固有的抗氧化防御机制和乏氧相关的治疗抵抗性等重大挑战,我们成功构建了可精准靶向HIF-1α的CRISPR/Cas9质粒系统,并通过静电相互作用将其与低毒性的多功能载体自组装形成多重肿瘤微环境刺激响应型的共递送纳米系统Must-nano。体外研究表明,Must-nano具有良好的水分散性、均一的粒径和带负电的表面电位,其可以借助核壳结构的空间阻隔效应,同时兼容理化性质和光敏特性各异的CRISPR/Cas9质粒和光敏剂Ce6,并保护携载的质粒免受酶降解和光化学损伤。通过对Must-nano多重刺激响应性的评价及体内外递送效率的考察,发现其在递送过程具有敏锐的生物感知与级联响应特性,可按序表现出长效血液循环、精准肿瘤靶向、靶细胞识别与内化、高效胞内转运、定点释放及亚细胞器定位等功能,由此成功克服多级递送屏障,大幅度提高CRISPR/Cas9质粒与光敏剂空间异步递送的效率与靶向性。CRISPR/Cas9与PDT协同疗效研究表明,Must-nano可以有效消耗肿瘤细胞内高水平GSH而削弱细胞异常的抗氧化能力,显著增强PDT作用的ROS损伤效力,诱导明显的细胞凋亡;另一方面,CRISPR/Cas9的精准激活彻底破坏了肿瘤细胞固有的乏氧防御机制,有效降低肿瘤细胞增殖、侵袭和迁移能力,进而极大地提高了肿瘤细胞对PDT治疗的敏感性,最终在荷瘤小鼠模型中实现了协同增强且安全低毒的抗肿瘤疗效。本部分研究为突破当前PDT治疗困境提供了新的解决方案,也为CRISPR/Cas9基因编辑治疗协同多种疗法增强体内抗肿瘤治疗提供了新的思路。结论综上所述,本文针对CRISPR/Cas9体内精准编辑和抗肿瘤治疗应用的关键挑战,以CRISPR/Cas9质粒系统作为基因编辑工具,开发了两种新型肿瘤微环境级联响应的CRISPR递送系统,通过级联响应的递送策略逐级击破体内多重生理病理屏障,实现了CRISPR/Cas9基因编辑系统的高效靶向递送与精准激活,分别在恶性肿瘤的免疫治疗和基因/光动力协同治疗中取得了显著性的疗效。本文所开发的肿瘤微环境级联响应型CRISPR递送系统和肿瘤治疗策略,为攻克恶性肿瘤免疫治疗和光动力治疗中的难题提供了新的途径与手段,也为拓展CRISPR/Cas9编辑技术在多途径协同的抗肿瘤治疗的应用引入了新的思考。更多还原

【Abstract】 Background and objectiveMalignant tumor is a major life-threatening disease and has become a heavy global burden of disease.As the major therapies of tumor treatments,surgery,chemotherapy and radiotherapy have played an important role in clinic,but they still have their own limitations and side effects,resulting in unsatisfactory treatment outcomes,so it is urgent to develop new strategies for tumor therapy.The pathogenesis of malignant tumors is very complex,which involves mutations in multiple genes and alterations in multiple signal pathways.Gene therapy can effectively treat diseases from genetic resources by replacing,enhancing or blocking the specific genes,and has developed into a potential therapeutic method after traditional tumor therapies.With the progress of genetic engineering and the innovation of biotechnology,advanced gene-editing technologies are changing the gene therapy landscape and providing new means for the precise regulation of key targets in tumor therapy.The clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(CRISPR/Cas9)has emerged as the third-generation genome-editing technology that utilizes Cas9 nuclease to cleave the target DNA under sg RNA guidance for various genome editing purposes such as adding,eliminating or correcting genes.Depending on advantages including good specificity,high editing efficiency and excellent operability,CRISPR/Cas9 provides a more potent and convenient genome-editing tool for tumor gene therapy.However,the complex pathophysiological barriers before CRISPR/Cas9 reaches the destination to exert functions will seriously hinder the efficient delivery and precise activation of CRISPR/Cas9 systems.Besides,the non-specific distribution of the CRISPR/Cas9 system in other normal tissues is prone to serious and unpredictable side effects.How to achieve efficient targeted delivery and site-specific activation of CRISPR/Cas9 in vivo is an urgent problem to be solved in antitumor applications.In recent years,emerging non-viral delivery vectors are considered potential alternatives to viral vectors due to their low immunogenicity,good stability and functional diversity,which have been widely used for CRISPR/Cas9 delivery in vitro and in vivo.However,conventional non-viral vectors are usually unable to effectively cope with the dynamically changing physiological and pathological environment in vivo,resulting in ineffective delivery,poor targeting and increased treatment-related toxicity.Meanwhile,the unique mechanism that CRISPR/Cas9 system needs to enter the nucleus to perform genome-editing function also puts forward higher requirements for delivery vectors.There is an urgent need to develop superior non-viral delivery systems integrating excellent loading capacity,specific targeting,high delivery efficiency,accurate nuclear localization and low toxicity to improve the efficiency,accuracy and safety of CRISPR/Cas9 mediated genome-editing in vivo.Focusing on the frontiers and difficulties in CRISPR/Cas9 targeted delivery and antitumor applications in vivo,we selected CRISPR/Cas9 in form of plasmid as the genome-editing tool due to its good stability and high flexibility,and further designed and constructed CRISPR/Cas9 genemo-editing systems targeting key therapeutic targets of malignant tumors.In view of the characteristics of tumor microenvironment,two types of hierarchically responsive tumor-microenvironment activated CRISPR delivery systems were rationally designed and developed and further separately loaded with CRISPR/Cas9 systems through a step-by-step self-assembly approach.It is expected that they could surmount the multistage delivery obstacles in vivo to achieve efficient,targeted and safe delivery of CRISPR/Cas9 as well as precise activation,thus breaking through the current predicament of tumor immunotherapy and photodynamic therapy(PDT),and eventually realizing significantly effective and safe antitumor efficacy.Research methodsFirstly,the reactive oxygen species(ROS)-sensitive polyethyleneimine derivative(PR)was synthesized by crosslinking low-molecular-weight polyethyleneimine(PEI1.8K)with a thioketal linker.Hyaluronic acid(HA)was used as the backbone of the shell,and further grafted with RGD-MMP peptide and polyethylene glycol(PEG)through amidation reaction and click reaction respectively,to prepare multifunctional HA-RGD-MMP-PEG polymer(HRMP).The final products were characterized by ~1H nuclear magnetic resonance(~1H-NMR)and Fourier Transform Infra-Red(FTIR)spectroscopy for verification.Then,the plasmid condensation capacity of PR was evaluated by an agarose gel retardation assay.Based on electrostatic interaction,the complexes named Core and programmable unlocking nano-matryoshka-CRISPR system(PUN)were prepared and their changes in particle size,zeta potential and morphology after incubation with matrix metalloproteases(MMPs),hyaluronidase(HAase)or ROS were investigated.Next,we detected the cytotoxicity and hemolytic toxicity of PUN in vitro,and further studied its delivery efficiency and transport mechanism at the cellular level,including cellular internalization assay,endosomal escape experiment,gene transfection and tumor penetration,etc.Using PX333 plasmid as the vector,we constructed and optimized a CRISPR/Cas9 system that can simultaneously and efficiently target programmed cell death ligand 1(PD-L1)and protein tyrosine phosphatase N2(PTPN2)and then loaded it into PUN for studies on the regulation of PD-L1 and PTPN2 protein expression.Furthermore,we investigated the transfection efficiency of PUN in vivo and monitored its biodistribution with the IVIS Lumina imaging system.Subsequently,we established a subcutaneous B16-F10 melanoma cancer model to study the in vivo anti-tumor activity of PUN,and further assessed the systemic toxicity by monitoring the mice body weight,immunohistochemistry analysis and blood biochemistry.Meanwhile,the changes of tumor immune microenvironment in mice after different treatments were also investigated for immune response evaluation.Finally,the immune memory effects induced by PUN were verified on a B16-F10 tumor-bearing mouse model.In further research,the aforementioned PR was served as the core material.The chlorin e6(Ce6)was conjugated on the HA backbone with disulfide bonds as the linker to prepare the shell HA-Ce6.The structures of PR and HA-Ce6 were characterized by~1H-NMR and FTIR,and their cytotoxicity in vitro was also investigated.In addition,the optical properties of HA-Ce6 were determined by fluorescence intensity spectra and ultraviolet visible absorption spectra.Next,we constructed and screened the optimal CRISPR/Cas9 plasmid targeting hypoxia-inducible factor-1α(HIF-1α)using the PX330 vector,and then prepared PR/p DNA and multiple stimuli-responsive nano CRISPR(Must-nano)through a step-by-step self-assembly approach.Their DNA compression capacity and photodegradation protection ability to plasmid were further evaluated.Furthermore,the multiple-stimuli responsiveness of Must-nano was evaluated by examining the changes in particle size,zeta potential and morphology under high concentration of HAase,glutathione(GSH)or ROS,as well as investigating Ce6 release behavior.Both ROS generation ability and GSH scavenging capacity of Must-nano were assessed in vitro.At the same time,cellular uptake level,disassembly behavior,mitochondrial targeting ability,endosomal escape,nuclear localization and transfection efficiency of Must-nano were investigated using flow cytometry or confocal laser scanning microscopes.For in vitro anti-tumor effect,cytotoxicity measurement,apoptosis and LIVE/DEAD assay were performed to determine photodynamic efficacy induced by Must-nano.Besides,the viability,invasion and migration abilities and related protein expression of cells treated with different treatments were investigated under normoxia or hypoxia,respectively.As for in vivo antitumor therapy,we established the HCT-116 subcutaneous xenograft mouse model and study the in vivo biodistribution of Must-nano via real-time fluorescence imaging.Subsequently,HCT-116 tumor-bearing mice were treated by intravenous administration.The antitumor mechanism was further elucidated by immunohistochemical analysis of tumor tissues.Finally,the body weight fluctuation,blood biochemical indexes and histopathological changes of mice were detected for the biocompatibility evaluation of Must-nano.ResultsAiming at the dilemma of immune checkpoint blockade(ICB)therapy including non-specificity,transient and incomplete blockade and low immune response,we designed and constructed a multi-targeted CRISPR/Cas9 plasmid system with high genome-editing ability and further facilitated it to self-assemble with synthesized multifunctional targeted delivery vector to fabricate PUN with uniform particle size and good stability.The in vitro characterization and systematic exploratory on the vector and delivery system revealed that PUN was capable of low toxicity,excellent large-scale CRISPR/Cas9 plasmid compression ability and versatile modification properties.Under the simulated tumor microenvironment in vitro,PUN can undergo changes such as size reduction,charge reversion and structural disintegration in response to multiple tumor stimuli,thus exhibiting enhanced cellular internalization,endosome escape,site-specific payload release,nuclear localization,excellent transfection efficiency and deep tumor penetration.In a subcutaneous B16-F10 melanoma cancer model,PUN showed significant anti-blood interference,specific tumor targeting and long-lasting tumor accumulation ability.In vivo anti-tumor studies further demonstrated that PUN can overcome sequential delivery barriers relying on its programmable unlocking performances,thereby accurately and efficiently delivering CRISPR/Cas9 into destinations and then activating highly effective multiple gene-editing functions.This ensured that the intervention of PD-1/PD-L1 pathway induced by PD-L1 knockdown as well as the sensitization effect on interferon-γmediated by PTPN2 deletion occurred simultaneously in the tumor.As a result,PUN effectively reshaped the tumor immunosuppressive microenvironment to elicit cascade amplified adaptive anti-tumor immune responses in vivo and induced long-term immune memory effects,eventually achieving the goal of significantly inhibiting the growth and recurrence of malignant melanoma with minimal toxicity.The research provides a new safe,efficient,thorough,durable and highly responsive antitumor immunotherapy strategy for current ICB therapy,and also boosts the development of clinical tumor immunotherapy.In addition to CRISPR/Cas9-based monotherapy,the development of multi-approach synergistic antitumor strategies is expected to further expand the application prospects of CRISPR/Cas9 in malignancy therapy.Based on the major challenges of PDT including the nonspecificity of photosensitizers,inherent antioxidant defense mechanisms and hypoxia-related treatment resistance of tumor cells,we constructed a CRISPR/Cas9 plasmid system targeting HIF-1α and further developed a novel multiple stimuli-responsive co-delivery nanoplatform Must-nano through electrostatic interaction-mediated self-assembly of CRISPR/Cas9 and low-toxic multifunctional vector.In vitro studies proved that Must-nano possessed good water dispersity,uniform particle size and negatively charged surface potential.Must-nano can realize compatibility of CRISPR/Cas9 plasmids and photosensitizers with distinct physicochemical and photosensitive properties by virtue of spatially segregated core-shell structure and further protect the loaded CRISPR/Cas9 from enzymatic and photochemical damage.Based on the evaluation of multiple responsiveness and delivery efficiency in vitro and in vivo,we confirmed that Must-nano has agile biological perception and hierarchical response capability.It can orderly exhibit prolonged blood circulation,precise tumor recognition,specific cellular internalization,effective intracellular trafficking,site-specific release and subcellular organelle localization during the delivery process,thereby successfully surmounting multiple delivery barriers and greatly improving the efficiency and targeting of spatially asynchronous delivery of CRISPR/Cas9 and photosensitizers.The synergistic antitumor effect of CRISPR/Cas9 and PDT demonstrated that Must-nano could effectively consume intracellular high-level GSH to attenuate the abnormal antioxidant capacity of cells,thus significantly enhancing PDT-mediated ROS damage,resulting in effective cell apoptosis.On the other hand,the precise activation of CRISPR/Cas9 completely destroyed the inherent hypoxia-related resistance mechanism,effectively inhibited the proliferation,invasion and migration abilities of tumor cells,further greatly promoted tumor susceptibility to PDT,thus eventually achieving a synergistically enhanced antitumor effect with negligible toxicity in the tumor-bearing mouse model.This work not only provides a novel strategy to break through the dilemma of PDT,but also paves a new way for CRISPR/Cas9 to synergize multiple therapeutics to enhance antitumor therapy in vivo.ConclusionsIn summary,addressing the key challenges of in vivo precise activation and antitumor applications of CRISPR/Cas9,we constructed CRISPR/Cas9 plasmid systems as the genome-editing tool,and further developed two types of novel hierarchically responsive tumor-microenvironment activated CRISPR delivery systems.Taking advantages of the cascade-responsive delivery strategy,they can successfully overcome complex pathophysiological barriers in vivo in a step-by-step manner,thereby realizing the efficient targeted delivery and precise activation of CRISPR/Cas9 and resulting in significant treatment outcomes in tumor immunotherapy and gene/PDT synergistic therapy,respectively.The hierarchically responsive tumor-microenvironment activated CRISPR delivery systems and antitumor strategies developed in this dissertation provide new ways for overcoming dilemmas of tumor immunotherapy and PDT,and bring new insights for expanding the application of CRISPR/Cas9 technology in multiple-approach synergistic antitumor therapies.更多还原