Design Of Highly Efficient Delivery CRISPR-Cas9 RNP Engineered Extracellular Vesicles And Anti HSV-1 Research

BACKGROUNDHerpes simplex virus(HSV)is a neurotropic virus that is mainly divided into two subtypes:type 1(HSV-1)and type 2(HSV-2).HSV-1 mainly establishes the incubation period in the trigeminal ganglion,and it can lurk in the body for a long time to cause latent infection,which is prone to relapse when the body has low immunity.Current antiviral therapies such as acyclovir and valacyclovir can effectively limit virus replication in epithelial cells to alleviate clinical symptoms,but they cannot cure infection and are ineffective for eliminating the latent virus pool in neurons.HSV has even developed and is resistant to all available antiviral drugs.At the same time,vaccination with prophylactic vaccines may be difficult to develop due to the recurrence of HSV in humoral and cell-mediated immune individuals.Based on the above problems and the emergence of drug-resistant strains,it is necessary to develop a new strategy for the treatment of HSV.Genomic engineering HSV represents a promising antiviral therapy capable of directly editing or destroying the genetic material of the virus.CRISPR(clusters of regularly spaced short palindromic repeats)is an immune protection system existing in bacteria.Subsequently,a new gene editing system,CRISPR/Cas9(Crispr-associated protein 9),was developed.Compared with ZFN system and TALEN system,CRISPR/Cas9 has wider application range,higher gene editing efficiency and lower off-target effect.Due to its simple construction and low cost,it is more suitable for us to use as a tool for anti-HSV-1 treatment.However,Cas9 protein is a very large molecule(～160 kDa)and it is difficult to enter the cells through the cell membrane.Therefore,we need to use an effective delivery tool for Cas9 to work in vivo.Extracellular vesicles(EVs)are membrane-derived vesicles released by cells to the extracellular space,playing an important role in intercellular communication and involved in the regulation of a series of biological processes.EVs,which is widely present in cell culture supernatants and various body fluids,is becoming a new diagnostic tool,a new therapeutic method,and a delivery tool.EVs can be loaded with therapeutic drugs,RNAs,protein,and so on,for delivery to target cells.At the same time,EVs also has the advantages of crossing biological barrier,good biocompatibility,and being capable of genetic engineering transformation.Therefore,we hope to combine CRISPR-Cas9 with EVs for anti-HSV studies.METHODS1.The activity and delivery efficiency of EVs were verified by the construction of the Cre/LoxP recombinant enzyme system.2.The enrichment of EVs modified by Fc/Spa and PTGFRN was studied.3.EVs was characterized by Western blotting,TEM and NTA.4.Laser Confocal detection was performed to detect the escape of EVs in cell lysosomes.5.The packaging efficiency of SpCas9 and sgRNA in EVs was verified by protein quantification and RT-qPCR.6.Western blotting,RT-qPCR,plaque assay and T7EI enzyme were used to verify the anti-HSV-1 effect of engineered EVs in vitro.7.EVs were modified by RVG to verify its targeting ability.8.Engineered EVs was used for toxicity evaluation in vitro and in vivo.Engineered EVs was used to investigate in vivo activity and antiviral effects in infected models by establishing the herpes simplex virus type I infection model.RESULTS1.PTGFRN-Fc can enrich Cre-Spa into EVs.2.Fc/Spa system can promote the packaging of Cas9/sgRNA RNP in EVs.3.EVPTGFRN-Fc/SpCas9-Spa/sgUL29 can effectively deliver Cas9/sgRNA RNP against HSV-1 infection.4.EVs modified by RVG had the function of nerve targeting.5.Engineering EVs can’t produce toxic effects in vivo and in vitro.6.EVRVG-PDGFRN-Fc/SpCas9-Spa/sgUL29 can exert anti-HSV-1 effect in vivo.CONCLUSIONSIn this study,we fused the human Fc domain to the intracellular domain of PTGFRN and anchored it to the EVs membrane,while the B domain of Spa protein fused to SpCas9 to form the Fc/Spa system,and the interaction between the two enriched Cas9 RNPs into EVs.At the same time,we combined sgRNA targeting the HSV-1 genome with Cas9 and packaged it in EVs to fight HSV-1 infection.The results showed that engineered EVs can significantly improve the survival rate of mice and reduce the viral load in mouse tissues.In addition,EVs can target the trigeminal nerve after modification with RVG,making Cas9/sgRNA RNPs exert significant antiviral efficacy therein.Our study provided a platform for the efficient delivery of SpCas9 based on engineered EVs with potential therapeutic value for HSV infection and other neurological diseases.