The New Way Of Gene Therapy Through Recombinant Adeno-Associated Virus 2 Transfer To Mesenchymal Stem Cell Which Derived From Bone Marrow

Background and ObjectiveRecombinant Adeno-Asociated Viral Vector(rAAV) is the safest and most effective viral vector in gene therapy attempt till now.Due to different Cap protein it has,There are 8 different serotypes of rAAV, Each permissive to certain cell and tissue.But one of the mostly used is the serotype 2. Bone marrow mesenchymal stem cell (BMSC) is one of adult stem cell which derived from bone marrow, is an essential part of bone marrow hematopoietic microenvironment which have effect of hematopoietic support,immunomodulation and accelerating hematopoietic stem cell engraftment. It can be isolated through bone marrow aspirate,after culture and proliferation in vitro, home to certain hematopoietic organ such as bone marrow,liver,spleen through intravenous transfusion, and maintain long time survival in vivo after transfusion. A variety of studies using different viral vector systems have attempted to transduce BMSCs. BMSCs have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. So it may be an ideal vehicle for systemic gene delivery, especially in treatment of inherited and malignant hematological diseases. The documented reports of rAAV transduction to BMSCs haven't appeared yet. and granulocyte macrophage colony stimulating factor(GMCSF) have been used for years in clinical therapeutics of hematological diseases. Could the advantages of the rAAV and BMSCs be combined? To verify this proposal, We constructed rAAV-2 vector which include green fluorescent protein(GFP), human granulocyte macrophage colony stimulating factor(hGMCSF), mouse granulocyte macrophagecolony stimulating factor(mGMCSF) respectively. To explore whether rAAV-2-eGFP, rAAV-2-hGMCSF,rAAV-2-mGMCSF could be efficiently transduced to BMSCs. and to find the expression profile in vitro and in vivo. Furthermore to get some evidence for further use of BMSC and rAAV-2 for cytotherapy and gene therapy in malignant hematological diseases.MethodsTo construct rAAV-2 vector which include reporter gene green fluoresent protein and granulocyte macrophage colony stimulating factor gene respectively. To detect whether the reporter gene and target gene have been successfully cloned to the multiple cloning site of rAAV-2 vector, Then massively produce rAAV-2-eGFP, rAAV-2-hGMCSF and rAAV-2-mGMCSF. The rAAV-2 vector purity,viability and biological titer was detected for the usage of next step study. Isolate human bone marrow mesenchymal stem cell from normal volunteers and leukemia patients in vitro. To comparatively study the biological characteristics of these two sources of BMSCs, proliferation characteristics,cell surface marker,multiple differentiation potential etc. and whether the leukemia patients derived BMSCs have the malignant clonal abnormal gene changes which always maintain in leukemia cells. To evaluate the possibility of using BMSC as a vehicle for cytotherapy and gene therapy, and at the same time collect the BMSC,proliferation invitro and frozen in liquid nitrogen for the further usage of research. Firstly, we used rAAV-2-eGFP to infect BMSC derived from normal volunteers and acute myelogenous leukemia patients at different multiplicity of infection (MOI) to find a condition which could acquire a higher transduction efficiency in vitro. At the condition which could acquire a relatively high transduction efficiency, We transduced rAAV-2-hGMCSF, rAAV-2-mGMCSF to BMSC derived from acute myelogenous leukemia patients in vitro, and observed the expression profile as the transduced BMSCs were cultured by passaging at certain interval period. After we have known the in vitro gene expression profile, We transduced the BMSC derived from acute myelogenous leukemia patients by rAAV-2-hGMCSF, rAAV-2-mGMCSF respectively, Then transfuse the in vitro gene modified BMSC after 2 weeks proliferation in vitro to 6 weeks old nude mice through tail vein. 2,4,6,8weeks after transfusion, detected thehGMCSF, mGMCSF concentration in nude mice serum and count the total white blood cell at that time point.Results1. BMSC derived from normal volunteers and leukemia patients have no significant differences in aspect of morphology, proliferation characteristics, cell surface marker and adipogenesis,oesteogenesis differentiation ability. The malignant clonal marker of leukemia cell couldn't be found in the BMSC derived from leukemia patients. So BMSC derived from leukemia patients might have a promising future for cytotherapy and gene therapy.2. To detect the target gene DNA sequence after vector was successfully constructed, and to compare the detected sequence to the sequence in genebank. and find they overlapped completely, So it proved the gene cDNA sequenc we cloned to the vector is correct. The rAAV-2 vector package cell line BHK-21 and HEK-293 are permissive to rAAV-2 vector. Therefore, BHK-21 and HEK-293 cell line are ideal tool to detect whether the constructed rAAV-2 be biologically active. After transduced the rAAV-2-eGFP, rAAV-2-hGMCSF, rAAV-2-mGMCSF to BHK-21 and HEK-293 cell line, We find high level expression of the GFP,hGMCSF and mGMCSF. All above proved rAAV-2-eGFP, AAV-2-hGMCSF and rAAV-2-mGMCSF construction are successful, and the effective biological titer of rAAV-2-eGFP, rAAV-2-hGMCSF and rAAV-2-mGMCSF is 10n-1012v.g/mL.3. The transduction picture of rAAV-2-eGFP to BMSC derived from normal volunteers and acute myelogenous leukemia patients have no significant differences(p>0.05). GFP began to express 10 tol4 days after transfection, The transduction efficiency is from 0.3 to 1.4%. By changing infection condition, We couldn't make a higher transduction efficiency. The in vitro GFP expression profile of the BMSC derived from acute myelogenous leukemia patients tranduced by rAAV-2-eGFP at MOI=1 X 105 is, The percentage of GFP expression cell gradually decreased from 1.14% at day 12 after transduction to 0.6% as cell passaged 2 to 3 passages, and maintained at a level of 0.5 to 0.6% later on, till 61 days after transduction. In 60 days of observation period, The in vitro gene expression profile of rAAV-2-hGMCSF, rAAV-2-mGMCSF transduced BMSC is, 3 days aftertransduction ,we can find a hGMCSF, mGMCSF expression at level of 167pg/mL,175 pg/mL . It reaches peak level of 849pg/mL, 339pg/mL at 24 days after transduction. The expression level goes down gradually until it attained 83.75 pg/mL, 42pg/mL 60 days after transduction.4. We used rAAV-2-hGMCSF, rAAV-2-mGMCSF gene modified BMSCs which derived from acute myelogenous leukemia patients , proliferated 2 weeks in vitro, Then tansfused to 6 weeks old nude mice 8X 106cell per mouse, and hGMCSF, mGMCSF expressed in nude mice serum at a stable level in 8 weeks's observation period.Conclusion1. BMSC derived from leukemia patients have no biological differences compared with normal volunteers? and it has no malignant clonal abnormal marker of leukemia cell either. So it is possible to use BMSC derived from leukemia patients as a vehicle for cell therapy and gene therapy.2. The transduction efficiency of rAAV to BMSC is low, After transduced interest gene to BMSC, The transduced gene could maintain a long time expression in vitro, As the gene modified BMSC passaged on, The intensity of the expressed gene gradually decayed. The target gene had a long term expression after the gene modified BMSC transfused to nude mice body. So the BMSC and rAAV-2 could be used together for cytotherapy and gene therapy. But the main obstacle for further usage of this technology is the low level transduction efficiency of rAAV-2 to BMSC.3. The rAAV-2 transduced BMSC derived from acute myelogenous patients maintains multiple differentiation potential.and it can maintain a stable level of target gene expression without significant decay within the 8 weeks observation period of our study, no immunological injury to host.Therefore,The combination of rAAV-2 and BMSCs may be a new alternative for cytotherapy and gene therapy in hematological diseases.