| Background and objection: Recombinant adeno-associated virus(rAAV)vector has become one of the main therapeutic vectors in the field of gene therapy because of its long-term expression and high safety.However,in clinical application,rAAV vectors have a series of problems such as low production efficiency,poor stability,and low transduction efficiency.The inverted terminal repeat(ITR)existing at both ends of the rAAV vector genome is the only reserved wild-type AAV gene sequence,and researchers generally believe that the D sequence is important for the replication,rescue,and packaging of the AAV and rAAV vector genomes.Nevertheless,due to its complex configuration,how this structure participates in functional regulation remains to be clarified Therefore,this study mainly investigates how the D sequence structure regulates the packaging and transduction efficiency of rAAV vectors and lays a theoretical foundation for the further optimization of rAAV vectors.Methods: This research is mainly divided into two parts.The first part is to study the effect of D sequence structural modification on the packaging efficiency of rAAV vector.Three different D sequence libraries were firstly constructed,and the D sequence and related sequence in the ITR of the rAAV packaging plasmid were replaced by the libraries individually.Sequences that can be packaged and transduced were send for sequencing,and alternative sequences were screened from the sequencing results for D sequence modification.Next,the effect of D sequence structural modification in rAAV vector on the replication and packaging efficiency was investigated by Southern blot and real-time quantitative PCR.The second part is to study the effect of D sequence and its mutants on the expression efficiency of rAAV vector.The transduction efficiency to different cell lines of rAAV vector with structural modification of D sequence was evaluated by the detection of firefly luciferase reporter gene.Results:(1)Alternative sequences with better packaging efficiency than wild-type D sequence could be obtained through D sequence library screening.The packaging efficiency in self-complementary rAAV vectors were increased by 39.13% and 34.24%in D08127 and D24081,respectively.D24054 and S sequence in single-stranded rAAV vectors were increased by 259.49% and 202.82%,respectively;(2)the encapsidation efficiency of rAAV was positively correlated with the replication efficiency;(3)the pseudo-D sequence of the plasmid backbone was one of the reasons for the contamination of the plasmid sequence in rAAV packaging.The proportion of plasmid DNA packaged into the genome of the rAAV vector capsid is about 10%;(4)The transduction efficiency of the rAAV vector can be improved by D sequence modification.Among self-complementary rAAV vectors,D24058 increased 83.74% in h CMEC/D3 cells,D08010 increased 76.25% in NCI-H446 cells,and D18119 increased 41.35% and33.49% in LO2 and He La cells,respectively.In single-stranded rAAV,D08121 increased by 147.05% in LO2 cells,while D08009 increased by 91.20% in NCI-H446 cells.Conclusion: The D sequence in the ITR structure,which is indispensable in the genome of the rAAV vector,plays a critical role in the packaging and transduction of the rAAV vector.The modification of the D sequence can effectively improve the packaging and transduction efficiency of rAAV.The presence of the pseudo-D sequence is responsible for the contamination of the plasmid backbone in the rAAV vector.This study lays a foundation for further optimizing the design,detection,and application of rAAV vectors. |
