Functional Study And Application Of Truncated Inverted Terminal Repeats Of RAAV Vector

Due to many advantages such as infecting dividing and non-dividing cells,high level and persistent of transgene expression,high safety and high stability,adeno-associated virus(AAV)has been widely used in the development of gene therapy for many diseases,especially monogenic diseases.The presence of inverted terminal repeats(ITRs)is the sole cis requirement for the rescue,replication,packaging,and integration of the AAV genomes.Wild-type(wt)ITRs,at two ends of AAV genome,contain 145 nucleotides(nt).The terminal 125 nt of each ITR self-anneals to form a palindromic double-stranded T-shaped hairpin(HP)structure,in which the small palindromic B-B' and C-C' regions form the cross arm and the large palindromic A-A' region forms the stem.Each HP is followed by a unique 20 nt D(or D')region.ITRs are very important for AAV,but various deletions have been identified in recombinant AAV(r AAV)vector plasmids.The diversity of ITR deletions has suggested that some regions may be dispensable.Therefore,it is of great scientific significance and potential application value to explore the function of ITR regions and its effects on r AAV.In this thesis,a series of truncated ITRs have been designed systematically and rAAV vector plasmids with one or two truncated ITRs were constructed for the first time.After the r AAV vectors were packaged by using these plasmids,we analyzed the effects of truncated ITRs on r AAV packaging and expression.According to the findings,a novel r AAV vector harboring truncated ITRs with high expression was used in gene therapy for hemophilia B.To study the effects of truncated ITRs on rAAV packaging and expression,we designed a series of truncated ITRs including ITR?B(deletion of the B-B' region),ITR?BC(deletion of the B-B' and C-C' regions),ITR?BC1/2A(deletion of the B-B',C-C',and half of the A-A' regions),ITR?BCA(deletion of the B-B',C-C',and A-A'(excluding trs)regions),and ITR?BCAtrs(deletion of the B-B',C-C',and A-A'(including trs)regions).Then they were used to replace the one wt ITR of r AAV vector plasmid p AAV2 wt containing two wt ITRs to generate the p AAV2?B,p AAV2?BC,p AAV2?BC1/2A,p AAV2?BCA,and p AAV2?BCAtrs.We also constructed a r AAV vector plasmid p AAV2?ITR,in which one wt ITR was completely absent.This series of r AAV vector plasmids harboring one truncated ITR and their control p AAV2wt were used in r AAV packaging based on serotype DJ,three times under identical conditions.Depending on the regions of deletion,these vectors were named as r AAVDJ?B,r AAVDJ?BC,r AAVDJ?BC1/2A,r AAVDJ?BCA,r AAVDJ?BCAtrs and r AAVDJ?ITR,while their control was named as r AAVDJwt.The purity determination of these r AAV demonstrated that the capsid proteins of them were obtained with high purity.Only VP1,VP2,and VP3 were present in purified viral vectors.And we found no significant differences between the concentration of all viral capsid proteins by BCA detection.Southern blotting analysis of the purified viral DNA from all types of r AAVDJ showed that single bands corresponding to genomic DNA of r AAVDJ?B,r AAVDJ?BC,r AAVDJ?BC1/2A,and r AAVDJ?BCA were apparent,indicating that the majority of their genomes were homogeneous and intact.While the strips of r AAVDJ?BCAtrs and r AAVDJ?ITR were smear seriously,which suggested that their genomes were heterogeneous.Subsequently,the titers of 4 types of r AAVDJ harboring one truncated ITR with intact genomes and their control r AAVDJwt were detected by DNA dot blotting and q PCR,while another two with incomplete genomes were abandoned.The result revealed that the titers of r AAVDJ?B,r AAVDJ?BC,r AAVDJ?BC1/2A,and r AAVDJ?BCA were similar to that of r AAVDJwt.After quality testing,the transgene expression levels of these 4 types of r AAVDJ with one truncated ITR were further compared with the control,we found that the expression of r AAVDJ?B,r AAVDJ?BC,and r AAVDJ?BC1/2A were similar to that of the control,while the r AAVDJ?BCA was slightly lower.These results indicated that when one wt ITR of r AAV vector plasmid mutated into ITR?B,ITR?BC,or ITR?BC1/2A,the packaging and expression of r AAV were not affected.And when one wt ITR mutated into ITR?BCA,the packaging of r AAV was not affected,but the expression decreased slightly.However,when one wt ITR mutated into ITR?BCAs or it was deleted,the packaging of r AAV was damaged.However,there is an efficient mechanism for correcting deletions with AAV termini.To avoid the interference of this correcting and further study the effects of deletion mutations in ITR regions on r AAV packaging and expression,we constructed the p AAV2bi?BC,p AAV2bi?BC1/2A,and p AAV2 bi RTD by replacing the two wt ITRs of p AAV2 wt with two ITR?BCs,ITR?BC1/2As or ITR-RTDs(only containing Rep binding element,terminal resolution site,and D region).Then this series of r AAV vector plasmids harboring two truncated ITRs and their control p AAV2 wt were used in r AAV packaging based on serotype DJ,five times under identical conditions.Depending on the regions of deletion,these vectors were named as r AAVDJbi?BC,r AAVDJb?BC1/2A,and r AAVDJbi RTD,while their control was still r AAVDJwt.The purity determination of these r AAV demonstrated that the capsid proteins of them were obtained with high purit,and no other protein was found.In addition,there were no significant differences between the concentration of all viral capsid proteins.However,compared with the r AAVDJwt,the titer of r AAVDJbi?BC decreased by 75.7±1.6% while r AAVDJb?BC1/2A and r AAVDJbi RTD decreased sharply.The extremely low productivity of r AAVDJb?BC1/2A and r AAVDJbi RTD indicated that when both of two wt ITRs mutated into ITR?BC1/2As or ITR-RTDs,the packaging of r AAV was destroyed.The result of Southern blotting analysis showed that the genome of r AAVDJbi?BC was intact and not destroyed by low productivity.To investigate the reason for the decline in r AAVbi?BC productivity,we compared the rescue and replication efficiencies of the genomes of r AAVbi?BC and r AAVwt.The result showed that compared to r AAVwt,the rescue and replication efficiency of r AAVbi?BC decreased 8 fold,which may be the main reason for the decrease in productivity.Then we detected the expression of r AAVbi?BC based on several types of serotypes and reporter genes.To our surprise,although the productivity of r AAVbi?BC was decreased,its expression was significantly higher than that of the control in vitro and in vivo.After this phenomenon was discovered,we further deleted the B-B' and C-C' regions in the two ITRs of self-complementary AAV(sc AAV)vector plasmid to explored the impact of the deletion on the packaging and expression of sc AAV.The results revealed that although the genome encapsulating of rsc AAVbi?BC was not be affected by the deletion,its productivity decreased by 76.6±3.0%,compared to the control rsc AAVwt.And the expression of rsc AAVbi?BC also increased significantly.Deletion of the B-B' and C-C' regions in the two ITRs reduced the rescue and replication efficiency of the r AAV genome,which resulted in a decline in viral productivity.However,the reason why the deletion caused increased expression of r AAV was unclear.Therefore,we preliminarily explored the molecular mechanisms.To eliminate the interference by sc AAV,r AAVDJbi?BC and its control r AAVDJwt which accommodated a large genome(about 4.4 k-nt)were packaged.Because of the large genome,none of the rsc AAVDJbi?BC and rsc AAVDJwt can be packaged.However,the expression of r AAVDJbi?BC was still significantly higher than that of r AAVDJwt at this time,which suggested that high expression of r AAVbi?BC may not be attributable to the presence of sc AAV.In the following research,Ataxia telangiectasia mutated(ATM)and Mre11/Rad50/Nbs1 complex(MRN)which inhibited the expression of r AAV with two wt ITRs were locked as targets.Our findings showed that although inhibition of ATM enhanced the expression of r AAVbi?BC and r AAVwt,the increase of r AAVbi?BC was far less than its control.Similarly,inhibition or degradation of MRN also improved the expression of r AAVbi?BC and r AAVwt,but the increase of the control was higher than r AAVbi?BC.In particular,the degradation of MRN dramatically improved the expression of r AAVwt,and even made it higher than that of r AAVbi?BC.These results suggested that deletion of the B-B' and C-C' regions in the two ITRs may weaken the inhibitory effects of ATM and/or MRN on AAV expression,which provided further clues to elucidate the molecular mechanisms of increased expression of r AAVbi?BC.It is remarkable that the expression of r AAVbi?BC was not higher than r AAVwt when it was transduced into cells as plasmid or genomic DNA extracted from virions.In genomes within viral particles ITRs from HP conformation,while they form linear double-stranded and open duplex structure,respectively,in r AAV vector plasmid and genomic DNA extracted from virions.The results suggested that in addition to the difference of primary structures,the specific secondary HP structure also contributed to the differences in expression between r AAVwt and r AAVbi?BC as a result of infection.After we found that deletion of the B-B' and C-C' regions in the two ITRs increased the expression of r AAV in vivo,we tried to use r AAVbi?BC in gene therapy research for hemophilia B.Gene drug r AAV8bi?BC-h FIX and its control r AAV8wt-h FIX were prepared and injected into the model mice,respectively.For the next 12 weeks,we continually detected the human blood clotting factor IX(h FIX)levels in mice plasma and found that r AAV8bi?BC-h FIX can not only express h FIX efficiently in model mice,but also its expression level was higher than that of r AAV8wt-h FIX.These results suggested that r AAVbi?BC can be used in the efficient expression of non-reporter genes,which provided a new option for the development of gene therapy drugs for hemophilia B.In conclusion,we established a systematic research method for deletion mutations in ITR regions and investigated the effects of a series of truncated ITRs on r AAV packaging and expression.We also analyzed the deletion of which regions in the one or two ITRs did not affect the packaging and expression of r AAV.In the study of deletion mutations of ITR regions,a novel r AAVbi?BC with high expression was obtained and preliminarily used in gene therapy for hemophilia B.Our findings suggested that deletion in ITR can affect the packaging and expression of r AAV,which provided a new way to improve the r AAV expression through ITRs modification.