Study On Inhibition Of HIV-1with SiRNA And TRIM5Α Mutants Via Lentiviral Vectors

With global spread of the human immunodeficiency virus, HIV infection hasbecome a serious hazard to human health. Although，highly active antiretroviralretroviral theray (HAART) have markedly changed the profile of progression to AIDSin HIV-infected individuals, they are not without significant problems and drawbacks,including the lifetime treatment, cumulative toxicities and viral resistance. So far, afully effective HIV vaccine was not available. With the increasing knowledge ofmechanisms that allow control of HIV infection, more and more investigators arefocusing their attention on gene therapy, either as a stand-alone approach or as anadjuvant to HAART.RNA interference have been effectively used to inhibit the replication of HIVrecently, by targeting the structure gene gag-pol, env and the regulatory proteinsincluding tat, vpr, rev. Exogenous siRNA transfected is not stable and knocking-downeffect is transient as the siRNA is gradually degraded or diluted by cell growth andcell division. The siRNA could be produced by expressing short hairpin (shRNA)precursor, which is exported to the cytoplasm and processed by the RNAi machinery.Expressing shRNA encoding siRNAs targeting viral sequences from the backbone ofviral vectors has been used as a form of genetic therapy for HIV-1and associatedinfection, which could inhibit HIV-1replication by down-regulating the viral gene atthe HIV transcription level.In the study, we constructed several recombinant lentiviral expression plasmidscontaining the shRNA targeting vpr、tat、 rev and vif genes and evaluated inhibitoneffects of the shRNA expressed on the targeted gene. Then the inhibitory effects onHIV-1production were detected by cotransfected with the packaging plasmidspNL4-3. The recombinant lentivirus containing the shRNA was packaged and titred.MT4cell was transduced with the recombinant virus and screened by FACs. Thetransduced MT4cells were further challenged with HIV-1strains. The result showedthat shRNA targeting vpr gene could efficiently inhibited the vpr expression onmRNA, about89.2%, whereas the tatshRNA could reach to60.2%agaist tat gene.The MT4transduced the vprshRNA and tatshRNA could inhibit the HIV-1replicationeffectively, but not the revshRNA.To improve the inhibition effect on HIV, the study constructed the bispecificlentiviral vector harboring vprshRNA and tat shRNA expression cassettes from U6promotor and H1promotor respectively, which were cotransfected with recombinantplasmid expressing the vpr and tat gene. The result showed that the bispecificlentiviral vector could inhibit the vpr and tat effectively，with ratio of89.2%and62% respectively. Cotransfected with pNL4-3in293T cell, the bispecific plasmids showedhigher efficacy in down regulating the HIV NL4-3strains packaging production thanthe recombinant plasmids expressing the single shRNA. MT4cell clone transducedwith recombinant lentiviral vectors were screened and challenged with HIV NL4-3.P24ELISA test showed that MT4transduced with the combinational lentiviral vectorcould inhibit virus replication efficiently in vitro.However, as HIV-1is prone to generating escape mutants, using a singleanti-HIV construct would not be adequate to afford long range viral protection.Combination of therapeutic genes targeting different viral products and steps in theviral life cycle was consider as an alternative effectively solution. Here, we reported anovel expression construct into the backbone of a replication defective lentiviralvector, encoding the Trim5ahumutant and short hairpin RNA targeting HIV-1vpr gene,which were expressed under control of the CMV promoter and U6promoter,respectively. Both of the two anti-HIV genes could block the HIV infection andinhibit replication by a distinct mechanism. Co-transfection assays in293T cellsshowed that the vprshRNA in the combination vector could knock down vprexpression with ratio of84.05%and inhibit the HIV-1NL4-3strain productioneffectively with ratio of98.45%. The western blotting showed that the TRIM5ahumutant could be expressed highly in transduced cells. Luciferase assay in Tzm-bl cellsstably expressing the TRIM5ahu showed that the ectopic protein could inhibitpseudotype HIV effectively with ratio of63.30%. MT4cell tranduced with thecombination lentiviral vector were challenged with HIV-1NL4-3. P24ELISA testshowed that MT-4transduced the combination vector could inhibit virus replicationefficiently for long time. These results suggested that combination of TRIM5ahumutant and shRNA targeting HIV-1vpr gene could lead to an effective gene therapyfor the HIV-AIDS control.Microvesicles as vesicles are released from cells with donor cells’ ligand andlipids which could serve as delivery vehicle transporting proteins, mRNA and miRNA.Use of microvesicles as siRNA carrier may be a key step toward clinical applicationof siRNA. Human umbilical cord is an alternative source of adult stem cells with lowimmunogenicity, which do not trigger an immense immune reaction in unrelateddonor transplantation. However, hMSCs have a limited life, gradually lose theirdifferentiation potential after several passages and will be senescent and die, whichrestricts the scientific and clinical application. Ectopic expression of humantelomerase reverse transcriptase (hTERT) extended life-span in certain cell types andpotentially immortalize them. In this study, we constructed a recombinant lentiviralvector including htert gene to infect the hucMSCs at PD2. Then hucMSCs transducedwith htert was screened by drug resistance and underwent a total of approximately105population doublings so far and continued to proliferate further. PCR amplified from genomic DNA and quantitative PCR manifested the ectopic hTERT has beenintegrated into the genomic DNA and expressed. TRAP PCR was used to determinewhether the exogenous hTERT gene affected the telomerase activity of the hucMSCs,which showed that the telomerase activity recovered in hucMSCs-hTERT cells.Moreover, our study showed that the established hucMSC-TERT were stronglypositive for the crucial markers of hMSCs, such as CD29, CD44and CD105andnegative for CD106, CD45, CD19and HLA-DR. When the hucMSCs-htert werecultured in osteogenic stimulatory medium, the cell could form the osteoblast,demonstrating the hucMSC-htert still maintain the differentiation potential. We alsoassessed the capacity of the immortalized hucMSCs-hTERT to produce heterologousproteins in vitro. The hucMSCs-htert maintained normal cell characteristics inmorphology, phenotype, karyotype and differentiation potential without malignanttransformation. The hucMSCs-htert cell line has the potential as a cell source for genetherapy as well as transplantation.The established hucMSCs-htert cell was transducedwith recombinant lentiviral vectors containing shRNA targeting the HIV-1and cellswere screened. The microvesicles were isolated and detected with quatitative PCR.The inhibition of the target gene with the microvesicles was studied. Themicrovesicles derived from hucMSCs containing the siRNA targeting HIV-1withoutimmunogenicity would be a novel therapy for AIDS-HIV.In summary, we constructed recombinant lentiviral vetors containing singleshRNA targeting the regutory protein of HIV-1, bispecific lentiviral vector containingvpr and tatshRNA and combinational lentiviral vector containing the human TRIM5amutant and shRNA targeting vpr gene and evaluated their inhibitory effect on HIV-1.The recombinant lentivirus would be used to infect hemopoietic stem cell for genetherapy on HIV. Combination of therapeutic genes targeting different viral productsand steps was considered as an effective gene therapy for the HIV-AIDS control. Then,we have successfully immortalized human umbilical cord derived MSCs, whichdemonstrated ectopic expression of hTERT in hucMSCs could reconstitute theirtelomerase activity and promote their proliferative ability. The establishedhucMSCs-htert cell maintained stem cell markers, normal karyotype anddifferentiation potential without any tumorigenesis in SCID mice. The establishedhucMSCs-htert could be a potential cell source for transplantation and gene therapy.The cells will be used in producing the micorvesicles containing siRNA targeting theHIV-1for their low immunogenicity, which could be used in gene therapy on HIV.