CRISPR/CAS9 Delivery By Nir-responsive Biomimetic Nanoparticles For Targeted HBV Therapy

Background:Globally,more than 300 million persons worldwide are chronically infected with hepatitis B virus（HBV）,which is associated with about 1million annual deaths as a result of liver diseases.Currently,the approved HBV therapies are mainly based on interferons（IFNs）and nucleoside analogs（NUCs）.However,lengthy treatment is associated with drug resistance mutations and a risk of Hepatic Fibrosis/Cirrhosis.Moreover,these drugs cannot target or eliminate HBV covalently closed circular DNA（ccc DNA）.Complete elimination of HBV ccc DNA is key to the complete cure of hepatitis B virus infection.Currently,studies on HBV ccc DNA targeted therapy are focused on two aspects:i.Inhibiting the formation of ccc DNA.ii.Elimination of ccc DNA.Studies have shown that the CRISPR/Cas9 system can directly destroy HBV ccc DNA,which provides a completely new strategy for the complete cure of hepatitis B.However,a CRISPR/Cas9 delivery system with low immunogenicity and high efficiency has not yet been established.Moreover,effective implementation of precise remote spatiotemporal operations in CRISPR/Cas9 is a major limitation.Objective:1.To prepare and characterize the NIR-responsive biomimetic nanoparticles（UCNPs-Cas9@CM）,which will be used for HBV therapy.2.To verify the properties of NIR-responsive biomimetic nanoparticles,and provide the basis for further HBV therapy both at cellular and animal levels.3.To study the therapeutic effect of UCNPs-Cas9@CM on HBV in vitro.4.To study the therapeutic effect of UCNPs-Cas9@CM on HBV in vivo.Methods:1.The Na YF₄:Yb/Tm/Ca@Na YF₄:Nd/Yb core/shell upconversion nanoparticles（UCNPs）were successfully prepared by solvothermal method.Water-soluble carboxyl-functionalized nanoparticles（UCNPs-PAA）were obtained after treatment with PAA.The UCNPs-PAA were conjugated with avidin proteins through amide bonds to synthesize UCNPs-Avidin.Detection of the Cas9/sg RNA mediated cleavage of HBV ccc DNA in vitro to screen out the sg RNA with the highest splicing efficiency.Due to the strong affinity between avidin and biotin,PCB could easily band with UCNPs-Avidin to form UCNPs-Avidin/PCB nanoparticles,which were covalently conjugated with Cas9 proteins and incubated with sg RNA to generate the inner core of the biomimetic nanoparticles（UCNPs-Cas9）.Cell-membrane-derived vesicles（CMs）of the HBV replication liver cancer cell models were extracted,and UCNPs-Cas9 were cloaked with CMs by extrusion through polycarbonate membranes to obtain the final desired biomimetic nanoparticles UCNPs-Cas9@CM.The morphology,particle size,Zeta potential,fluorescence spectrum,UV-Vis absorption spectrum,and cell membrane protein and other material characterization of UCNPs-Cas9@CM were detected.2.q RT-PCR was used to determine the m RNA expression levels of inflammatory factors.Then the particle size of UCNPs-Cas9@CM in PBS solution was detected for 7 consecutive days to evaluate its colloidal stability.Immune escape and homotypic target of UCNPs-Cas9@CM were detected by confocal imaging and Flow cytometry analysis at the cellular level.The Cas9/sg RNA release was investigated by using an 808 nm wavelength laser.3.The NIR-responsive photothermal property was firstly evaluated,and CCK-8 method was used to detect the cytotoxicity assessment of UCNPs-Cas9@CM.T7E1 assay was used to determine whether UCNPs-Cas9@CM could effectively disrupt HBV ccc DNA in cell models.Cells were treated with PBS and UCNPs-Cas9@CM respectively,and the changes of HBV ccc DNA,HBV pg RNA,HBV DNA,HBs Ag and HBe Ag were detected.Sanger sequencing was then used to detect whether UCNPs-Cas9@CM had off-target effects in the process of clearing HBV in vitro.4.UCNPs-Cas9@CM was prepared by extracting mouse hepatocyte membrane in in vivo experiment.The homing ability of UCNPs-Cas9@CM in vivo was firstly verified by bioluminescence imaging and ICP-MS.Next,we performed a haemolysis assay to assess the safety of UCNPs-Cas9@CM.HBV-Tg mice were randomly divided into three groups,then treated with PBS,UCNPs-Cas9 and UCNPs-Cas9@CM,respectively.Their body weights were monitored every 2 days.Mice were sacrificed on day 14 post-injection.Hematological parameters,HBV DNA,HBs Ag and HBe Ag levels in serum,as well as HBs Ag and HBc Ag levels in hepatocytes were detected.Major organs from mice in different groups were also obtained and fixed for histology analysis to assess the toxicity effects of UCNPs-Cas9@CM.To further analyze the excretion of UCNPs-Cas9@CM in vivo,excreted concentrations of lanthanide ion(Y³⁺)from fecal and renal excretions were measured using ICP-MS.Finally,genomic DNA extracted from mice livers was used to evaluate the off-target effects and PCR amplicons containing the off-target sites were validated by T7E1assays.Results:1.Three HBV-specific g RNAs were selected from published literature and the in vitro cleavage assay showed that sg RNA₁₇was better at targeting the HBV gene than other sg RNAs.TEM imaging showed that UCNPs-Cas9@CM had a typical core-shell structure with an outer shell of about 10 nm.SEM imaging revealed a regular spherical of UCNPs-Cas9@CM,which also had uniform size and good dispersion.The particle size of UCNPs-Cas9@CM was 133.34±2.31 nm,and the zeta potential was-26.34±1.32 m V.Protein ingredients of UCNPs-Cas9@CM were evaluated by coomassie brilliant blue staining and Western Blot analysis.Membrane proteins within the source cell membrane were widely retained,and there were bands corresponding to monomeric avidin and Cas9.2.The results showed that UCNPs-Cas9@CM had good biocompatibility,colloidal stability,negligible cytotoxicity,and possessed high target specificity and immune evasion ability.Additionally,Cas9/sg RNA was released by cleaving the PC linker under 808 nm laser irradiation,entered the nucleus with the aid of nuclear localization sequence（NLS）to perform gene editing.3.We used short interval irradiation（2 min break after 1 min irradiation）during the experiment to avoid these damages to cells caused by the NIR-responsive photothermal property.CCK-8 results showed that UCNPs-Cas9@CM had no significant effect on cell viability,and T7E1assay showed that UCNPs-Cas9@CM had good gene editing efficiency in vitro.We then assessed the first few putative off-target sites for target sequences in Homo sapiens.The off-target effect of UCNPs-Cas9@CM in vitro was negligible.HBV 3.5 kb RNA,intracellular HBV DNA,HBV ccc DNA and HBV viral antigens were significantly decreased after incubation with UCNPs-Cas9@CM,relative to the control group（PBS group）.4.In in vivo study,UCNPs-Cas9 were coated with mouse liver cell membrane fragments to form the UCNPs-Cas9@CM nanoparticles.Both ICP-MS analysis and bioluminescence imaging revealed that CMs-functionalized UCNPs-Cas9@CM mainly distributed in the liver,suggested that UCNPs-Cas9@CM had a good homing capability in vivo.There was no hemolysis after treatment with different UCNPs-Cas9@CM concentrations.Compared with the UCNPs-Cas9 group,the UCNPs-Cas9@CM group significantly decreased serum levels of HBV DNA,HBs Ag and HBe Ag,as well as the HBs Ag and HBc Ag levels in hepatocytes.However,there were no significant changes in various blood parameters,body weights and HE staining results of major organs.These results confirmed that UCNPs-Cas9@CM had good homotypic targeting,biocompatibility and properties without systemic toxic effects.Y³⁺levels steadily decreased over time in mice urine and feces,implying that UCNPs were efficiently excreted.In addition,T7E1 assay showed that no off-target effects were detected.Conclusions:1.UCNPs-Cas9@CM were successfully prepared with uniform size distribution and good dispersion,loaded with Cas9/sg RNA ribonucleoprotein complexes.The coated CMs retained the membrane proteins of the source cell membrane,which could efficiently target homologous cells.2.UCNPs-Cas9@CM had exhibited good biocompatibility,homotypic target and immune escape ability,which also exhibited good stabilities while the hydrodynamic diameter variation was almost constant over 7 days.UCNPs-Cas9@CM could effectively release Cas9/sg RNA under the excitation of 808 nm laser to play gene editing role.3.UCNPs-Cas9@CM showed good anti-HBV therapeutic effect in in vitro studies.4.UCNPs-Cas9@CM showed good anti-HBV therapeutic effect in in vivo studies.