Evaluation Of Uric Acid Regulation By A Synthetic Circuit

Uric Acid(UA)is the end product of purine metabolism.When it is overproduced or excreted less in the body,it will make the uric acid content in the serum too high,forming hyperuricemia and gout.At present,the main treatment for hyperuricemia and gout is to inhibit the production of uric acid and promote the excretion of uric acid by drugs.However,drug therapy has disadvantages such as ineffectiveness,hypersensitivity,drug resistance,and acute gout,a new treatment method is urgently needed to treat hyperuricemia,alleviate the pain of patients and reduce the risk of other diseases.Based on the idea of genetic circuit in synthetic biology,various functional part could be assembled to construct functional circuits from bottom to top,to achieve the goal set by researchers.In this study,we used human umbilical co’rd mesenchymal stem cells(MSCs)as vector cells to assemble uric acid sensing elements into a genetic circuit with lentiviral vectors,hoping to realize the regulation of serum uric acid at the animal level by genetic circuit.This study mainly includes the following aspects:(1)Sequences of regulatory elements mUTs,hucO8 and smUox were obtained from GenBank.Internal ribosome entry sites(IRES)were used to construct a gene circuit mUTs-IRES-huc08-smUox(MHS,about 2,700 bp).The results of digestion and sequencing showed that the pIRES2-EGFP-MHS vector was successfully constructed.The regulatory function of MHS was evaluated by the expression of uricase and the change of uric acid concentration in the environment at the cellular level.The rat hyperuricemia model was further established to evaluate the regulatory function of MHS at the animal level by cell therapy with gene circuits.(2)It requires efficient and safe lentiviral vectors to introduce a genetic circuit into MSCs.The lentivirus vector LV-MHS was successfully constructed by In-Fusion method.Using control lentivirus LV-EGFP vector,the optimum lentivirus packaging conditions including transfection reagent,ratio of liposome to DNA,composition of transfection medium,viral harvesting medium and ratio of packaging plasmid were determined.The optimal packaging conditions were as follows:Lip3000 was used as transfection reagent,and the ratio of liposome to DNA was 1.5:1(μL:μg);The molar ratio of the thr-ee packaging plasmids LV-EGFP,pGP and pVSVG was 1:1:1;Additionly,the complete medium containing 10%FBS was required to be replaced 2 h before transfection and 6 h after transfection respecclively.(3)The optimal packaging conditions were used to package the LV-FEGFP and LV-MHS respectively.The supernatant was concentrated and purified by ultracentrifugation,and the liter was detected by fluorescence method.The final packaging titer of LV-EGFP was 4×10s TU/mL,while that of LV-MHS was 2 ×106 TU/mL.(4)MSCs were isolated from human umbilical cord by the method of tissue block isolation.After amplification and culture,MSCs were identified with cell morphology,osteogenic and adipogenic differentiation characteristics and surface marker detection.The results showed that umbilical cord MSCs were successfully isolated.(5)Using the control lentivirus with titer of 2×106 TU/mL,the conditions of lentivirus-infected mesenchymal cells were optimized by adding infection enhancer,adding infectious agent Polybrene,increasing infection volume and repeating infection times.The results showed that by adding 10 μg/mL Polybrene to the complete medium,using 60μL of the control lentivirus with a titer of 2×106 TU/mL and continuous infect for 3 times could produce good infection effects for MSCs.(6)MSCs were infected with the target lentivirus LV-MHS using optimal infection conditions.Successfully infected cells were observed by the microscope and FACS,and the infection efficiency reached 10.7%.Under these conditions,after adding 1000μM uric acid for 48 hours,the uric acid in the medium could be effectively reduced to about 817 p.M,and the uric acid clearance rate reached 18.23±3.39%.This study is based on the principle of genetic circuit in synthetic biology,combined with gene therapy and stem cell therapy,in order to provide a new idea for the treatment of hyperuricemia.The expression vectors containing uric acid regulatory genetic circuit were constructed and their regulatory effects on uric acid in the environment were evaluated at the cellular and animal levels respectively.The lentiviral expression vector LV-MHS containing the genetic circuit was constructed,and the optimal lentiviral packaging conditions and optimal infection conditions for MSCs were determined.Finally,the infection e:fficiency of MSCs reached 10.7%.The concentration of uric acid in the environment was reduced from 1000 μM to 817 μM,and the uric acid clearance rate reached 18.23±3.39%.In this study,effective delivery of genetic circuit to mesenchymal stem cells was achieved,the regulation mechanism of uric acid in MSCs was preliminarily explored,which laid a foundation for the regulation of uric acid at animal level by the genetic circuit.