Preclinical Study Of RAAV8-hFⅧ In The Treatment Of Hemophilia A

Objective:To learn the treatment of a novel AAV recombinant virus (rAAV8-hFVIII) for hemophilia A, and the introduction of site-specific integration concept virus designed and built, to provide direction for gene therapy of hemophilia A.Methods:In this experiment, we used AAV mice and FVIII KO mice as observation to watch the effect of the rAAV8-hFVIII to the treatment of hemophilia A. Group will be divided into high-pressure injection, injection group and normal control group.Gene expression of the target, we used ELISA detecting the expression of hFVIII protein antigen-antibody days1,7,14,21,35,60,90;100days, the mice were sacrificed whichever heart, liver, spleen, lung, kidney, using RT-PCR detection of viral transcription situation. Physiological coagulation indicators, we used the activated partial thromboplastin time(APTT) assay within30days to observe changes in endogenous coagulation FVIII KO mice, while mice tails assay was used to observe physiological changes in bleeding time. AAVS1site-specific integration, we add on some AAV mice high-pressure injection with Rep, using nested PCR to detect the linker sequence, and100days for mice liver resection surgery, using Real-time PCR to observe the change.Results:ELISA detection of hFVIII expression results in the same injection, the approximate expression of the two animals, the expression of the virus in about60days is relatively low, near the base90days. High pressure injection and normal injection is to bring a different expression trends, high-pressure injection of the virus on the seven day produced expression, then the expression of relatively stable; whereas injection of the virus in a normal day there is a very high expression, but declined rapidly. We used the APTT assay to physiological observation, viral expression in mice found that higher physiological coagulation performance has also been a corresponding increase of1%of antigen expression can bring about5%of the activity. Nested-PCR detected rAAV8-hFVIII and Rep plasmids were injected, and it show group consolidation occurs. To test whether the ratio of the integration would expression longer, we did liver resection after100days of injection, approximately2/3/of the liver was removed, and then allowed the growth of the mice one month, liver has been re-grown, and then killed the mices. Using Real-time PCR detection, we compared before and after the liver resection, and we found the copies of FVIII decreased in the liver.Conclusion:The final results showed that, rAAV8-hFVIII achieve a better therapeutic effect in early, a very good improvement in the pathological condition of hemophilia A, but the expression is short,it was near the base on90days. We found that compared to pressure injection, normal injection is more conducive to expression of the viral vector. rAAV8-hFVIII can be integrated into the human AAVS1sites with the Rep-mediated site-specific integration system, which greatly increasing the safety of viral vector applications.