Self-assembled Genome Editing Delivery Vectors Based On Natural Polymers

Tumor is currently the most serious threate to human health.Among diverse therapeutic strategies,gene therapy demonstrates great promise for tumor treatment.In gene therapy,safe and effective gene delivery vectors are of critical importance to deliver therapeutic genes to target cells to achieve therapeutic goals.In this study,a series of functional vectors for delivery of genome editing plasmids were designed and prepared.These vectors are composed of natural polymers and their derivatives,and inorganic compounds such as calcium carbonate and calcium phosphate.All the components of the vectors possess good biocompatibility,ideal biodegradability,non-immunogenicity and high safety.These gene delivery systems can efficiently deliver gene editing plasmids to targeted tumor cells for mediating genome editing,and thus reverse tumor cell deterioration.In the Chapter 1,the advances in gene delivery vectors and gene editing methods are briefly introduced,with the emphasizes on non-viral vectors for gene delivery,the strategies to enhance tumor cell uptake,and the gene therapy based on CRISPR/Cas9 gene editing technology.Chapter 2 focuses on a dual-targeting delivery system based on polymer/inorganic hybrid nanoparticles for effective genome editing and in situ detection on related protein expression.In the delivery system,the CRISPR-Cas9 plasmid for CDK11 knockout was encapsulated in the core of the delivery system composed of protamine sulfate,calcium carbonate and calcium phosphate by co-precipitation,and functional derivatives of carboxymethyl chitosan(biotinylated carboxymethyl chitosan with biotin ligands and aptamer incorporated carboxymethyl chitosan with AS1411 ligands)were decorated on the nanovector surface by electrostatic interactions to form the dual-targeting delivery system.Due to the tumor cell targeting capability of biotin and AS1411 ligands as well as the nucleus targeting of AS1411,the dual-targeting system can deliver CRISPR-Cas9 plasmid into the nuclei of tumor cells to realize highly efficient genome editing,resulting in a dramatic decrease(> 90%)in CDK11 protein,together with the significant down-regulation of other proteins involved in tumor development.Using the same vector,molecular beacons can be easily delivered to edited cell nuclei to in situ detect the m RNA level of related proteins and m RNA.The gene vector developed can realize effective genome editing and in situ detection on related protein expression simultaneously.Chapter 3 is focused on an aptamer and peptide functionalized delivery system for effective regulation of cell behaviors,prevention of tumor development and reversal of tumor immnosuppression by genome editing.In the delivery system,the CRISPR based gene editing plasmid to knockout CDK11 gene was complexed with protamine sulfate,and then the complex was decorated by a multi-functional outer layer composed of an endosomolytic peptide(KALA)and aptamer AS1411 incorporated carboxymethyl chitosan.The resultant multi-functional nanoparticles,which exhibit significantly enhanced delivery efficiency,can specifically deliver the plasmid into tumor cell nuclei owing to the favorable effects of KALA in cellular uptake and endosomal escape,together with the cancer cell and cell nucleus targeting capability of AS1411 ligands.The genome editing mediated by the nanoparticles leads to a dramatic decrease in CDK11 expression,which results in further modulation of cancer cells with significant down-regulation of the proteins(MMP-9 and VEGF)involved in tumor development and metastasis as well as up-regulation of the tumor suppressor protein p53.More importantly,the detection of immune-related proteins after genome editing shows that the significantly enhanced Fas,CD80,MICA,MICB,and HLA-1 expression and decreased CD47 and MUC1 expression,indicating the genome editing is favorable for reversal of tumor-induced immunosuppression and prevention of tumor development.Chapter 4 is concentrated on a dual aptamer-functionalized delivery system for effective reversal of tumor malignization.The nanovector core consisting of protamine and calcium carbonate entrapping CRISPR-Cas9 plasmid is decorated by aptamer incorporated heparin derivatives.Owing to a high affinity between MUC1 specific aptamer and mucin 1(MUC1)overexpressed in tumor cells as well as the interaction between AS1411 and nucleolin on the tumor cell surface and cell nuclei,the nanovector can target the nuclei of tumorous cells for knockout of tyrosine kinase2(PTK2)encoding focal adhesion kinase(FAK).Notably,the genome editing mediated by our delivery systems can effectively modulate cell behaviors and thus to reverse tumor malignization.The up-regulated p53,p16,p21,E-cadherin,CD80,MICA,MICB and Fas,together with down-regulated MMP-9,Vimentin,VEGF,TGF-?,CD47 and CD133 in genome edited cells indicate that the genome editing system can inhibit cancerous cell growth,prevent tumor invasion and metastasis,reverse tumor-induced immune suppression,and inhibit cancer stemness.More importantly,the edited cells can maintain the modulated cellular function after succeeding subcultures.Chapter 5 focuses on a peptide and aptamer decorated multi-functional delivery system and the mechanism of cell apoptosis induced by genome editing.In the delivery system,the Cas9/sg RNA plasmids for FAK knockout were compacted by protamine in the presence of calcium ions to form nano-sized cores,which were further decorated by NLS peptide conjugated alginate and AS1411 aptamer conjugated alginate.With the help of the nuclear location signal(NLS)peptide and AS1411 aptamer with specific affinity for nucleolin in tumor cell membrane and nuclei,the delivery vector can specifically deliver the plasmid to the nuclei of tumorous cells for knocking out protein tyrosine kinase 2(PTK2)gene to down-regulate focal adhesion kinase(FAK).The tumor cell apoptosis induced by genome editing is mitochondrial-dependent.In addition,FAK knockout results in negative regulation on PI3K/AKT signaling pathway.Meanwhile,favorable modulation on various proteins involved in tumor progression can be realized by genome editing.The enhanced E-cadherin and decreased MMPs,Vimentin and VEGF imply the desirable effects of genome editing on suppression of tumor development.Wound healing and transwell assays confirm that the genome editing system can suppress tumor invasion and metastasis in edited cells efficiently.