Observational Study On The Infection Efficiency Of Mesenchymal Stem Cells Derived From Amniotic Fluid Of A Duchenne Muscular Dystrophy Fetus By Three Viruses

BackgroundDuchenne muscular dystrophy(DMD)is a common X-linked recessive genetic disease.At present,there is no effective treatment for DMD in clinical practice.DMD has been mainly dealt with physical therapy,glucocorticoid therapy,orthopedic treatment and so on.However,these therapies can not prolong the lifespan of patients.The gene therapy of DMD is considered one of the most promising therapy,which always relies on efficient viral vector systems.Such viral vectors as AAV,AdV and LV are most widely used in the pathogenesis and gene therapy of DMD.However,virus packaging technology is still at its early stage,the virus titer can not meet the experimental requirements,and its price is extremely high.Therefore,the establishment of the viral packaging platform,efficient and economical,can better promote the clinical application of DMD gene therapy study and research.Recently,patient-derived induced pluripotent stem cells(iPSCs)have been the common practice in the gene therapy research of DMD.However,iPSCs are less efficient to acquire and can not avoid the potential risks in viral vectors.Mesenchymal stem cells derived from amniotic fluid(AFMSC)is a kind of amniotic fluid-derived stem cells(AFSC).AFMSC is obtained from prenatal diagnosis specimens.What's more,AFMSC does not have ethical restrictions and tumorigenicity.AFMSC provides a new source of stem cells for the gene therapy research of DMD.This study established a packaging system of AAV6,AdV5 and LV viruses in vitro.AFMSC was isolated and identified from amniotic fluid of a DMD fetus.The AAV6,AdV5 and LV viruses infected the AFMSC,and the infection efficiency of the three viruses was observed.An efficient and economical viral packaging platform was intended to set up to provide a certain research basis for the gene therapy research of DMD.Objectives1.Establishment of a packaging system of AAV6,AdV5 and LV viruses.2.Isolation and identification of AFMSC from a DMD fetus.3.Observation the infection efficiency of AFMSC by the three viruses.Methods1.The packaging system of AdV5,AAV6 and LV viruses was accomplished in vitro,and the virus titer was determined by Real-time Quantitative PCR Detection System(qPCR).2.A amniotic fluid sample was collected from a DMD fetus containing a deletion of exon 10-15 of the DMD gene.AFSMC was isolated by the direct adherence method from the sample.AFMSC was identified by the cells morphology,karyotyping and flow cytometry(CD34,CD45,CD73,CD105).3.The AAV6,AdV5 and LV viruses infected the AFMSC,and the infection efficiency of the three viruses was observed.Results1.The packaging of AAV6,AdV5 and LV viruses in vitro was successfully completed,and the virus titer was successfully detected by qPCR.Among the three viruses,the virus titer of AAV6,AdV5,and LV were 1.890×1013 vg/mL,1.280×108 vg/mL,and 1.252×1011 vg/mL,respectively.2.AFMSC from a DMD fetus was successfully isolated.The AFMSC had a long fusiform shape with a single layer of growth and a spiral arrangement.Karyotype analysis indicated that the isolated AFMSC had a normal karyotype.Flow cytometry showed that the isolated AFMSC surface markers CD34 and CD45 were negative,and CD73 and CD105 were positive.3.All three viruses successfully infected the AFMSC.In terms of infection efficiency,AdV5 virus had the highest infection efficiency,followed by LV virus.And AAV6 virus had the lowest infection efficiency.ConclusionThis study successfully established an efficient and economical packaging system of AdV5,AAV6 and LV viruses.And AFMSC from a DMD fetus was successfully isolated.The three viruses could successfully infect AFMSC.In terms of infection efficiency,AdV5 virus had the highest infection efficiency,followed by LV virus.And AAV6 virus had the lowest infection efficiency.