Inhibition Of Transgene Product Antigen Presentation In Mature Dendritic Cells Via MicroRNA Technology

A longstanding goal for the gene therapy of genetic diseases is the development ofa gene transfer vector that can maintain sustained gene expression in the absence ofan immune response. Since1990, the number of clinical trials in gene therapy haveresearched1,996, with a major breakthrough being made by the sucessful treatmentof Leber’s congenital amaurosis, hemophilia and other diseases. However, due tothe immune response occurred in gene therapy clinical trials, especially the cellularimmune toxicity, wider range application of gene therapy in clinical trials bas beenlargely limited.The cause of cellular immune toxicity is yet not determined, neither is anappropriate strategy to cope with it. Dendritic cells (DC) are professional antigenpresenting cells (APC) pivotal for regulating immune response, they have a role in theactivation and function of both innate and adaptive immune responses. DC residesin an immature state in peripheral tissues, where they scan their environment for‘danger signals’. In this resting state they express various receptors for pathogenrecognition. Upon pathogen encounter, signaling through these receptors activates theDCs, initiating their migration to the draining lymph nodes. During this process, DCdifferentiate into the mature dendritic cells (mDC), which allows them to presentantigens captured in the periphery and to stimulate proliferation of antigen-specific Tcells, and producing cellular immune toxicity. Therefore, this study proposed thatreducing the APC of immune system, especially the transgene expression in mDC,without affecting the expression of trasngene expression in normal cells may be aeffective methods to circumvent the cellular immune toxicity induced by transgeneproducts.MicroRNA (miRNA) is an evolutionarily ancient class of endogenous smallnoncoding RNA, posttranscriptionally regulate gene expression by binding to3’untranslated regions (3’ UTR) of target mRNAs, thereby inhibiting their translation.MicroRNA-155(miR-155) is not only the characteristic of DC maturation, but alsothe necessity for DC maturation, which provides us with a favorable condition to construct novel vectors that specifically silence transgene expression in mDC. Thus,reverse complementary sequence corresponding to miR155was inserted into the3’UTRs of gene expression cassettes to construct the novel recombinant vectors, then,gene expression in different cells was evaluated, and whether endogenous miR155could reduce the transgene expression of miR155T vectors in DCs was tested. Then,our academic hypothesis was evaluated using non-viral vector and lentiviral vector.Our results identified that the miR155could inhibit the transgene expression of thenovel recombinant vector containing miR155T via miR155mimic and novel genetherapy vector cotransfection experiments. The expression of miR155was graduallyincreased during the conversion process of immature to mature DC,the geneexpression of novel recombinant vector was also decreased. Subsequently, weconstructed a strong antigen protein ovalbumin (OVA) transgene lentiviral vectorcontaining miR155T, transduced the iDC and mDC, and investigated whether theOVA antigen presentation and activation of T cells could be effectively inhibited inmDC.In conclusion, miR155is specifically and highly expressed in mDC, andendogenous miR155can inhibit the transgene expression of the novel recombinantvector containing miR155T, besides, the novel vector can effectively inhibit theeffective antigen presentation and activation of T cells in mDC. Neverhteless, Innormal cells, the transgenic expression of the novel vector is substantially unaffected.The development of this novel vector provides a new insight for the future study ofgene medicine, especially the cellular immune response in clinical trials.