Optimization Of Efficient Transduction Of Hematopoietic Stem Cells By Recombinant Adeno-Associated Virus

Objective:Recombinant adeno-associated virus (rAAV) is considered to be the most promising gene therapy virus carrier.application of rAAV for beta Mediterranean anemia gene therapy is a great and valuable research.however,the low infection efficiency of rAAV has been a major problem of the researchers. Especially for hematopoietic stem cells which is difficult to be infect, the infection efficiency is more than satisfactory. This study improve rAAV from the packaging to each infection process and optimize the materials and methods of packaging and infection process of the rAAV,that to get high infection efficiency of hematopoietic stem cells and the best rAAV infection condition.Methods and Results:This study focuses on the optimization and improvement of the virus packaging process and infection process. In the packaging process of rAAV,we employ PEI transfection and Calcium phosphate respectly to package rAAV.Then the speeding centrifugal chromatography purification and heparin-agarose affinity purification were use to purificate virus.Lastly, we determined the titer of virus by qPCR analysis and quantitative DNA slot blot analysis.The influence and difference of the rAAV that package with various methods were determined.The steps which cause the reduction of the rAAV activity in packaging process were improved and optimized.Finally we get a set of optimum packaging prosess of rAAV.In the infection process of rAAV, scAAV combined with tyrosine-mutant AAV were employed, that have improved the infection efficieny of HSC greatly. At the same time, we found that each infection factor of the rAAV infection system has a big impaction on the infection efficieny of rAAV. These factors include length of infecrtion, concentration of serum, multiplicities of infection and post-infection processing.These factors were studied to obtain a optimum rAAV infection system to HSC which improves the rAAV infection efficieny greatly compared to previous system.We aslo explored the treatment of cells before infection and for the first found that pretreatment with ZY can improve the infection efficiency. ZY combinant with urea hydroxyl can increase the infection efficiency largely. This is an improtant finding.Conclusion:Through a series of optimization and improvment, this study obtain a set of optimum packaging prosess of rAAV and an optimum rAAV infection system to HSC which improve the rAAV infection efficieny greatly. What is more, we found for the first time that pretreatment with ZY can improve the infection efficiency.