| Background: About 50 years ago,the concept of gene therapy to treat human genetic diseases through genetic modification by exogenous DNA was first proposed.This gene therapy strategy provides a theoretical advantage that long-lasting and possibly curative clinical benefits could be derived from a single treatment.Although it has experienced a long and tortuous journey from concept proposal to clinical application,gene therapy is currently bringing new treatment options to many medical fields.Its treatment methods mainly include direct in vivo administration of viral vectors,adoptive transfer of hematopoietic stem cells or genetically engineered T cells,and genome editing technologies.Adeno-associated virus(AAV)is a non-pathogenic,non-enveloped parvovirus,which has natural replication defects and requires the presence of helper viruses such as adenovirus or herpes virus to complete its life cycle.Since AAV can mediate long-term stable transgene expression in vivo without causing any known human diseases,it has become a promising gene therapy viral vector.From 2012 to 2019,three AAV-based gene therapy drugs were approved by the European Medicines Agency or the U.S.Food and Drug Administration for the treatment of lipoprotein deficiency(Glybera),retinal diseases caused by RPE65mutations(Luxturna)and spinal muscular atrophy(Zolgensma).Currently,more than200 interventional clinical trials involving AAV have been registered on Clinical Trials.gov,including gene therapy for hemophilia,Parkinson’s disease,Duchenne muscular dystrophy,giant axonal neuropathy,heart disease and other diseases.Because vascular endothelial cells are in direct contact with the bloodstream and are closely related to major human diseases such as chronic heart failure,hypertension,atherosclerosis,diabetes,and stroke,they have become an ideal target for gene therapy of cardiovascular diseases,and the stable and effective delivery of therapeutic genes to the cardiovascular endothelium has important clinical significance.In the early research,adenovirus vector could mediate effective transgene expression in vascular endothelium,but because of its strong immunogenicity,the long-term expression of transgene could not be achieved,so it is impossible to achieve truly effective gene therapy in vivo.For the natural AAV serotypes,although it can achieve effective transduction of some tissues such as liver and muscle,it has a lower transduction efficiency to vascular endothelial cells in vivo,which limits its application to cardiovascular disease gene therapy.In recent years,with the rapid progress of gene therapy vector technology,it has been possible to improve the transduction efficiency of AAV to specific tissues and organs through rational design or directed evolution genetic modification methods.Among them,DNA shuffling and random peptide display are two important directed evolution methods,which have been effectively used in the targeting of some organs such as the heart and nervous system.DNA shuffling through homologous recombination of the capsid gene of the AAV serotypes to obtain a more functional progeny library,while the random peptide display library changes its tissue tropism by inserting random peptides at specific sites of the AAV2 capsid gene.These two library construction methods combined with in vitro or in vivo screening platforms have produced many improved AAV vectors,however,they have not been used to optimize the transduction of AAV to cardiovascular endothelial cells in vivo.Objective: The purpose of this study is to improve the transduction efficiency of AAV to cardiovascular endothelial cells in vivo,provide more effective viral vectors for gene therapy of cardiovascular endothelial cell-related diseases,and broaden the application range of AAV vectors.Methods: In order to achieve the above objective,we used directed evolution method,combined with DNA shuffling library and AAV2 random peptide display library,and performed two consecutive rounds of screening in Tie2-GFP transgenic mice expressing GFP specifically in endothelial cells.Flow cytometry was used to isolate cardiac endothelial cells and the capsid gene was amplified by polymerase chain reaction(PCR).Luciferase activity and Real-time PCR experiment were used to identify AAV variants with a higher degree of enrichment after two rounds of screening to characterize their transgene expression efficiency and biodistribution in endothelial cells of various tissues and organs.Combining immunofluorescence experiments and flow cytometry techniques to identify the co-localization of variants and endothelial cells at the cellular level.The β-galactosidase assay and X-gal staining were used to characterize the ability of the variants to infect primary endothelial cells in vitro.In order to determine whether the variants could achieve long-term stable transgene expression in vivo,we conducted continuous monitoring of the transgene expression in four vital organs for up to four months.To further prove the gene therapy potential of the variants,the AAV vector carrying the endothelial nitric oxide synthase(eNOS)gene was used for gene therapy in the mouse model of myocardial infarction.After two months of modeling,the cardiac function was evaluated by echocardiography and ventricular pressure catheter,the expression and activity of eNOS in mouse organs were detected by Western-blot and enzyme activity experiments,respectively.Finally,through the heparin affinity and the AAVR combination experiment,the mechanism of changing the tissue tropism of the variants was studied.Results: First,the constructed DNA shuffling library and the AAV2 random peptide display library were mixed in equal proportions.After two rounds of in vivo screening in transgenic mice,the AAV capsid gene was isolated from cardiac vascular endothelial cells,and 30 clones were randomly selected for reverse sequencing with CAP-3` primer,of which two clones showed higher gene frequency and enrichment score,named EC71(gene frequency of 10%,enrichment score of 0.98)and EC73(gene frequency of 63%,enrichment score of 0.92).Sequencing results showed that the two mutants were derived from the AAV2 random peptide display library.The peptide sequence inserted after the R588 site of the AAV2 capsid gene of the EC71 variant is AEGDVAR,and the peptide sequence inserted in the EC73 variant is NHPPGGV.The results of the luciferase experiment showed that after systemic administration,the EC71 and EC73 variants had transgene expression in the endothelial cells of the heart,liver,brain,and lung,but mainly concentrated in the heart.Among them,EC71 and EC73 variants mediated transgene expression in cardiac endothelial cells were 8.8-fold and 6.3-fold higher than the natural optimal serotype AAV1,and 16.5-fold and 11.8-fold higher than the parent serotype AAV2,respectively.At the same time,the transgene expression mediated by the EC71 and EC73 variants in liver endothelial cells was 8.3-fold and 8.9-fold lower than that of AAV1,and 4.3-folds and 4.6-fold lower than that of AAV2,respectively.The transgene expression ratio of the two variants to the heart and liver increased to 2:1,while the heart-to-liver transduction ratios of AAV1 and AAV2 were 1:31 and 1:30,respectively,which increased the tissue specificity by approximately 60-fold.The results of Real-time PCR were basically the same as those of luciferase,AAV1 and AAV2 were mainly enriched in mouse liver after systemic administration,while EC71 and EC73 variants significantly reduced liver metastasis.Compared with AAV1,the genome copy numbers of EC71 and EC73 in the liver were reduced by 23.3-fold and 28.5-fold,respectively;compared with AAV2,the genome copy numbers of EC71 and EC73 in the liver were reduced by 15.7-fold and 19.3-fold,respectively.In contrast,the vector genomic distribution of EC71 and EC73 in mouse heart was very similar to that of AAV1,with slightly increasing of 1.5-fold and 1.4-fold,respectively.Therefore,the ratio of the copy number of the vector genome in the heart and liver also showed a trend similar to luciferase activity.Both the luciferase and the Real-time PCR assay showed that the EC71 and EC73 variants have significantly improved transduction ability and specificity to cardiac endothelial cells in mice compared to the natural serotypes.The immunofluorescence results showed that after systemic administration,AAV1 preferentially transduce cardiomyocytes in the heart and both AAV1 and AAV2 had strong transduction to hepatocytes in the liver,while EC71 and EC73 significantly reduce the transduction of cardiomyocytes or hepatocytes in the heart and liver.In the brain and lung,no obvious GFP expression was observed in the four viruses.The results of flow cytometry showed that about 2.0% of endothelial cells in the heart were transduced by AAV1,1.0% of endothelial cells were transduced by AAV2,and EC71 and EC73 respectively infected 2.2% and 1.2% of cardiac endothelial cells.In the liver,about 3.2% of endothelial cells were transduced by AAV1 and 1.1% of endothelial cells were transduced by AAV2.In contrast,the transduction of liver endothelial cells by EC71 and EC73 were decreased to 0.7% and1.0%,respectively.Therefore,the transduction efficiency of EC71 was slightly higher than that of AAV1,and was significantly higher than that of parent serotype AAV2(P<0.05).In addition,EC71 also showed significant off-target from liver endothelial cells,compared with AAV1 and AAV2,there were extremely significant differences(P<0.01)and significant differences(P<0.05),respectively.The long-term monitoring of the transgene expression of AAV1,EC71 and EC73 in vivo showed that in the heart,the transgene expression of the three vectors could be maintained for at least four months,and the expression levels was gradually increased,and always maintain EC71>EC73>AAV1.In the liver,the transgene expression of AAV1 reached a peak at one month and then gradually decreased,while the expression of EC71 and EC73 in the liver was always maintained at a very low level.In the brain,all three viruses were stable for four months.In the lung,it gradually increased two months ago and then showed a downward trend.The X-gal staining results of primary endothelial cells infected in vitro showed that when MOI=30000,the percentage of HUVECs infected with EC71 and EC73 were less than 1%,while the percentages of infected AAV1 and AAV2 were about 2%and 9%,respectively.The quantitative analysis showed that β-galactosidase activities of AAV1-,AAV2-and EC73-infected HUVECs were 68-fold,297-fold and 2-fold,respectively,of that of EC71.For RMMVECs,< 1% of EC71-and EC73-infected endothelial cells expressed the Lac Z gene,but approximately 3% and 5% of the AAV1 and AAV2 cells respectively expressed the Lac Z gene in same infection multiplicity.Quantitative analysis showed that β-gal enzyme activities of AAV1-,AAV2-and EC73-infected endothelial cells were 185-fold,250-fold and 1-fold,respectively,of that of EC71.EC71 vector carrying eNOS gene was used to treat myocardial infarction mouse model.After two months,a significant increase in eNOS activity was detected in the heart and lung,and to a certain extent,the heart function of the mice with myocardial infarction was improved,specifically in the systolic(%FS)and diastolic(LVEDP and-d P/ dt)partial improvement of heart function.Finally,the heparin affinity experiment of EC71 and EC73 combined with AAVR binding experiment showed that the heptapeptide insertion caused the loss of or greatly reduced the dependence on the primary receptor HSPG,but retained the use of the protein receptor AAVR.These results inferred the possible mechanism of the change of tissue tropism of variants is discussed.Conclusions: Two variants EC71 and EC73 were screened in mice by the method of directed evolution.After identification,the two variants have significantly improved the transduction efficiency and specificity of mouse heart endothelial cells in vivo,and could maintain long-term stable transgene expression in cardiac endothelial cells.At the same time,the infection efficiency of the two variants on primary endothelial cells in vitro was significantly reduced.In addition,we have verified that the EC71 vector can be used for gene therapy of cardiovascular-related diseases and has certain clinical value.Further,we clarified the possible mechanism of the tissue tropism change of the variants through the receptor binding experiments. |
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