Study On The Mechanism And Gene Delivery Of Novel Composite Vectors

An often overlooked problem in adenovirus(Ad)-mediated gene therapy is an efficient gene delivery system.Currently,electrostatic complexation of cationic liposomes and Ad is an effective means to overcome these limitations and enhance gene transduction,but this Complexation produces cytotoxicity,and at high doses of Ad,the large dose of Ad also causes cell death,thus limiting the application of this method.Based on the above background,we have constructed a novel adenovirus/polyaminoglycoside composite vector(Ad-GFP/SS-HPT).We have carried out the following two works on the Ad-GFP/SS-HPT composite vector:(1)To investigate the transfection effect of Ad-GFP/SS-HPT complex at the cellular level and the complexation mechanism of Ad-GFP and SS-HPT.We first explored the effective delivery capacity and low cytotoxicity of SS-HPT on H9C2 cells,and then compared the percentageof GFP-positive cells transfected with H9C2 by naked Ad-GFP and Ad-GFP/SS-HPT at different MOI values.The percentage of GFP-positive cells at MOI=10,20,and 40 was 5.65%±0.68%?10.23%±1.26%?22.53%±1.03%,respectively,transfected with naked Ad-GFP.However,in the presence of SS-HPT,the Ad-GFP transfection efficiency of MOI=10,20,40 increased by 3.8,4.2,and 3.1 times,respectively(p<0.001),indicating that SS-HPT can effectively enhance naked Ad--Transfection of GFP in H9C2 cells.Next,we further explored the complexation mechanism of Ad and SS-HPT,and found that SS-HPT and Ad particles form a complex by electrostatic interaction,the particle size is about 250 nm,and the complexation of SS-HPT makes the surface of Ad particles.The charge changes from a negative value to a high positive value,which facilitates the binding of Ad to the cell membrane.At the same time,the endocytosis inhibitor is used to inhibit the transfection of Ad-GFP/SS-HPT.We have discovered a new mechanism of entry into the cell,ie cell membrane.The acupoint-like invagination-dependent endocytosis,and we also found that serum does not affect the enhanced transfection of SS-HPT,and does not require additional pre-incubation time.(2)In order to explore the gene delivery ability of Ad-GFP/SS-HPT complex in vivo,we first explored the in vivo potentiation of Ad-GFP/SS-HPT at the protein and RNA levels of liver and heart;The injected mouse serum is then incubated with Ad-GFP/SS-HPT and transfected into cells.It was found to reduce the innate immune response of Ad;The mRNA expression of various inflammatory factors such as CXCL1 and CXCL9 also demonstrated that the electrostatic complexation of SS-HPT with Ad after 8 h affected the pre-existing antibody recognition in vivo and reduced the inflammatory response;Finally,HE staining of liver,kidney and spleen and detection of serum biochemical factors such as ALT and AST further showed that Ad-GFP/SS-HPT has low biological toxicity in vivo.In summary,we studied the gene delivery ability of Ad-GFP/SS-HPT composite vector at the cellular and animal levels,and we further explored the complexation mechanism of Ad and SS-HPT.Ad-GFP/SS-HPT is expected to be a new generation of safe and effective cardiovascular gene delivery systems.