Study On The Therapeutic Effect Of AAV2 Mediated Stable Expression Of FGF21 In T2DM Mice In Vivo

Background Type 2 diabetes(T2DM)is the result of genetic and lifestyle factors,and its prevalence is on the rise worldwide.At present,although there are relatively mature drug treatment methods,some drugs such as insulin may experience side effects such as hypoglycemia and subcutaneous sclerosis during treatment.Therefore,exploring new treatment methods for T2 DM has become an important way to improve related clinical treatment deficiencies.Fibroblast growth factor 21(FGF21)is considered a promising therapeutic agent for T2 DM.Due to the poor pharmacokinetics of natural FGF21,the strategy of gene therapy to achieve sustained protein cycling levels has become attractive.Adeno associated virus(AAV)is currently the main delivery vector for gene therapy.The albumin binding domain(ABD)is currently a suitable scaffold for extending the half-life of peptides and proteins.The research on the gene delivery system targeting ABD-FGF21 is not yet sufficient,and the development of AAV vectors with T2 DM targeting is one of the research focuses of T2 DM gene therapy.Objective This study explores the gene therapy effect of FGF21 on T2 DM and constructs an AAV-FGF21 and AAV-ABD-FGF21 system targeting mouse liver by inserting FGF21 and ABD-FGF21 into the reverse terminal repeat(ITR)of AAV2,providing a theoretical basis for clinical trials.Methods(1)Insert the coding sequences of FGF21 and ABD-FGF21 into the reverse terminal repeat sequence(ITR)of AAV2 using homologous recombination technology,and construct pAAV-FGF21 Luciferase and pAAV-ABD-FGF21 Luciferase plasmids;Then,it was identified through enzyme digestion.(2)Transfect the plasmid pAAV-FGF21-Luciferase(or pAAV-Null Luciferase,pAAV-ABD-FGF21-Luciferase),pHelper,and capsid plasmids into the HEK 293 T cell line to prepare rAAV virus.Through virus titer detection and Western blot verification.(3)The T2 DM mouse model was established,and rAAV was injected into the mice by tail vein injection.On the 7th day,the distribution of Luciferase signal in the mice was observed.(4)Detect the effects of rAAV injection on different indicators in mice using a reagent kit.(5)Hematoxylin eosin staining was used to observe the toxic and side effects of rAAV virus complex on the main organs,liver,and fat of T2 DM mice.Results(1)Successfully constructed pAAV-FGF21 Luciferase and pAAV-ABD-FGF21 Luciferase plasmids inserted into FGF21 and ABD-FGF21 coding sequences,and prepared rAAV viruses.(2)The prepared rAAV complex and its control AAV-null were successfully expressed in mouse liver.(3)When the complex entered T2 DM mice,it was found that compared with the model group,overexpression of FGF21 in the liver not only reduced the fasting blood glucose and GSP levels of T2 DM mice,but also improved their insulin resistance and sensitivity;At the same time,the level of FGF21 in circulation was significantly increased,and blood glucose metabolism was significantly improved;Overexpression of FGF21 in the liver can reverse liver injury in T2 DM mice and significantly intervene in liver lipid deposition;And it has a significant intervention effect on fat accumulation in T2 DM mice.(4)The rAAV virus complex has almost no toxic side effects on major organs in mice.Conclusion This study prepared rAAV virus complexes targeting mouse liver by modifying the AAV2 core protein,and the AAV-ABD-FGF21 complex significantly increased the level of FGF21 in serum;Targeted gene hypoglycemic effect in T2 DM mice;In addition,the virus complex does not cause significant toxic side effects.The above research has established a novel recombinant adeno-associated virus vector complex with universal potential,providing a promising new strategy for further development of T2 DM gene therapy.