Brain-Targeted Delivery Of Cas9 Ribonucleoprotein For In Vivo Glioblastoma Therapy

Glioblastoma(GBM)is a common central nervous system tumor with high morbidity and mortality.Advancement in the conventional treatment consisting of surgery,radiotherapy,and chemotherapy in clinics has not significantly improved the survival rate of GBM patients,and therefore new therapeutic approaches are urgently needed.In recent years,genome editing provides new solutions for cancer treatment by editing DNA sequences or regulating gene expression.In this context,CRISPR/Cas9 is one of the simplest and most precise genome editing mechanisms employed till date.The cluster-like regular short palindrome repeat sequence CRISPR can completely delete a gene from the genome,and hence treating the diseases effectively.Cas9/sg RNA ribonucleoprotein(Cas9-RNP)is composed of Cas9 protein and guide RNA(sg RNA).However,the poor in vivo stability,lower blood-brain barrier(BBB)penetration,and lack of tumor targeting have restricted the applications of Cas9-RNP for GBM treatment.Thus,the design and development of safe and effective Cas9-RNP brain targeted delivery system is highly desired for GBM gene therapy.In the current thesis,we developed angiopeptide-2 modified guanidininium and fluorine functionalized PEG polymer,which can effectively complexed with the Cas9-RNP through the guanidine and fluorine moieties.These interactions enable the effective incorporation of Cas9-RNP,and consequently achieve a multifunctional nanomedicine(Ang-NP@Cas9/sg RNA).The developed nanomedicine bestowed Cas9-RNP high stability in the blood-stream,without any loss of activity.In addition,the presence of fluorine groups facilitated Cas9-RNP nanomedicine to effectively escape endosomes.Moreover,Ang-NP@Cas9/sg RNA is capable of crossing BBB and could actively target GBM due to the angiopep-2 mediated BBB traversing ability and and recognizing low-density lipoprotein receptor(LRP)on the surface of BBB endothelial cells and gliomas.Characterization by dynamic light scattering(DLS)and zeta potential showed that the Ang-NP@Cas9/sg RNA nanomedicines had a hydrodynamic diameter of 149 nm and a surface potential of 8.3 m V.Transmission electron microscopy(TEM)results showed that Ang-NP@Cas9/sg RNA nanomedicines had a spherical morphology with uniform size distribution.Importantly,the circular dichroism chromatograph evidenced that,the encapsulation of Cas9 by our synthesized polymers did not affect its activity.The in vitro performance of Ang-NP@Cas9/sg RNA nanomedicines at cellular level was evaluated against U87 MG cell lines.For cellular uptake analysis,confocal and flow cytometry were conducted,where the Ang-NP@Cas9/sg RNA nanomedicines demonstrated effective uptake ability against U87 MG cells versus its non-targeted counterpart(NP@Cas9/sg RNA).Meanwhile,due to the intervention of fluorine groups,the Ang-NP@Cas9/sg RNA nanomedicines can efficiently escape from the endosome and subsequently entering cellular nuclei successfully.MTT experimental results displayed that Ang-NP@Cas9/sg RNA nanomedicines had excellent biocompatibility.In vitro gene editing results revealed that Ang-NP@Cas9/sg RNA achieved 32% Indel by targeting Polo-like kinase 1(PLK1)gene for gene editing.Moreover,Ang-NP@Cas9/sg RNA reduced 67% PLK1 protein expression,while,non-Ang NP@Cas9/sg RNA and non-PLK1 specific Ang-NP@ Cas9/sg Scr controls achieved 41% and 11% PLK1 reduction,respectively tested by western blotting.Since,PLK1 gene is related to cell proliferation,while reducing the expression of PLK1 protein will promote cell apoptosis,which was confirmed from the apoptosis experiments,where Ang-NP@Cas9/sg RNA induced 37.2% cellular death.Pharmacokinetic study in mice verified that Ang-NP@Cas9/sg RNA has longer blood circulation with a half-life of 40 min,that is much higher than free Cas9-RNP(11 min only).Ang-NP@Cas9/sg RNA nanomedicine effectively crossed BBB and efficiently accumulated at the brain tumor due to the Angiopep-2 targetability,as confirmed by the biodistribution evaluations in orthotopic GBM mice model,Lastly,in vivo therapy results demonstrated that Ang-NP@Cas9/sg PLK1 successfully suppressed tumor growth,while significantly improved medium survival time of nude mice bearing orthotopic GBM brain tumor up to 40 days,compared to PBS(18 days),Ang-NP@Cas9/sg Scr(22 days),and NP@Cas9/sg RNA(27 days)as controls respectively.Histological analysis further speculated that,Ang-NP@Cas9/sg PLK1 nanomedicines can effectively inhibit the proliferation of tumor cells and promote tumor tissue apoptosis and reduced the expression PLK1 protein up to 66.7% in tumor tissue western blotting test.Notably,Ang-NP@Cas9/sg PLK1 nanomedicines induced no toxicity and side effects in heart,liver,spleen,lungs,and kidneys,as confirmed through histological analysis in vivo.In summary,we developed an angiopep-2 decorated,guanidinium and fluorine factionalized polymeric nanoparticle for delivering Cas9/sg RNA RNP to treat GBM.The established Ang-NP@Cas9/sg RNA nanomedicine has promising stability in blood stream,could effectively cross BBB to target GBM through angiopep-2 functionality,as well as,quickly escape endosome,thereby exerting its gene editing performance.In vitro and in vivo experiments showed that,Ang-NP@Cas9/sg RNA nanomedicine has good genome editing functionality and could provide a novel therapeutic approach for the treatment of GBM in clinics.