Nanocarrier-Mediated CRISPR/Cas9 Gene Editing For Intervention Of Macrophage And Dendritic Cells

Macrophages and dendritic cells are key components of the immune system,and are important cells against pathogen invasion and maintaining healthy homeostasis.Macrophages are critical effectors and regulators of inflammation and the innate immune response.Dendritic cells initiate and regulate the highly pathogen-specific adaptive immune responses,and are central to the development of immunologic memory and tolerance.Macrophages and dendritic cells have roles in the occurrence and development of various diseases.For example,tumor-associated macrophages can secrete a variety of cytokines and stimulate tumor cell migration,invasion,intravasation as well as the angiogenic response required for tumor growth.Macrophages recruited in adipose tissue of type II diabetes secrete pro-inflammatory cytokines,leading to insulin resistance.Acquisition of self-antigens by DCs and their subsequent presentation to self-reactive lymphocytes crucially contributes to both the initiation and the maintenance of autoimmune responses,leading to the occurrence of autoimmune diseases.Therefore,intervening in the function of macrophages and dendritic cells will contribute to the treatment of the disease.In this dissertation,with CRISPR/Cas9 technology and a drug delivery system based on cationic lipid-assisted PEG-b-PLGA polymeric nanoparticles(CLAN),we developed CLAN carried CRISPR/Cas9 nucleic acid drugs to target macrophages and dendritic cells,respectively.We explored to treat the related diseases by intervening in the function of macrophages and dendritic cells with CLAN.The main contents and conclusions of this dissertation are as follows:1.The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases.Macrophage-specific CD68 promoter-driven Cas9 expression plasmids were constructed and encapsulated in CLAN.The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo.More importantly,after further encoding a guide RNA targeting Ntn1(sgNtn1)into the plasmid,the resultant CLANpM330/sgNtni successfully disrupted the Ntnl gene in macrophages and their precursor monocytes in vivo,which reduced the expression of netrin-1(encoded by Ntnl)and subsequently improved type 2 diabetes(T2D)symptoms.Meanwhile,the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by CD68 promoter.This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/Cas9 system.2.Reconstruction of self-tolerance is becoming a powerful treatment of autoimmune diseases(AID).Thus,developing approaches to restore antigen-specific self-tolerance in vivo has always been the research focuses in recent years.We developed a novel strategy by systemically delivering CRISPR/Cas9 plasmid,three gRNAs targeting costimulatory molecules(CD80,CD86,CD40)and an autoimmune antigen peptide(2.5mi)with CLANpCa,9/gCD80,86,40/2.5mi.We demonstrated that CLAN can enter dendritic cells,leading to the knockout of costimulatory molecules and simultaneous presentation of the antigen peptide in vivo.This strategy induces a tolerogenic phenotype of dendritic cells which triggers the generation and expansion of antigen-specific regulatory CD4+ T cells in nonobese diabetic(NOD)mice,preventing diabetes development and restoring normoglycemia in NOD mice.In conclusion,our strategy provides a potential immunotherapy for treating autoimmune diabetes.