While this has been accomplished in earlier studies 1,2  the new twist is that they cleverly incorporate the cellular microRNA machinery to inhibit promiscuous lentiviral-mediated transgene expression in hematopoietically derived immune-participatory cells that become inadvertently transduced. As a result, these vectors dampen the cell-mediated immune response directed against human factor IX in vector-transduced cells, prolonging human factor IX expression in immunocompetent animals.

Various flavors of the immune response can limit the efficacy of any gene therapy approach, many of which have been experimentally demonstrated in animals and/or humans. The adaptive immune response can be directed against the vector, vector remnants in transduced cells, and the transgene product, resulting in a humoral or cell-mediated response, the loss of the transgene product, and a limited period of efficacy. While lentiviral and other vectors show promise in preclinical gene transfer studies, there is no known way to completely restrict the dissemination of a vector when delivered by systemic (eg, intravascular) routes. In addition, even tissue-specific promoters designed to confine transgene expression to a specific cell type can be leaky when placed into some vectors. Taken together, even small amounts of inadvertent transduction can result in transgene expression in nontargeted professional antigen-presenting cells, resulting in an unwanted immune response.

MicroRNAs are a class of short (20-22 nt long) regulatory RNAs that represent up to 4% of the mammalian genome, encoding more than 400 transcripts that are believed to regulate as many as 30% of all genes. Some of these microRNAs are tissue specific and fine-tune genetic circuits, which are critical for normal development, cellular differentiation, and normal cellular homeostasis. In mammals, most of the currently known microRNA targets are localized to the 3′ untranslated region of mRNAs and contain sequence mismatches with their corresponding microRNA. In the absence of perfect complementarity, the primary mode of gene control is at the level of translational inhibition of the corresponding mRNA, by mechanism(s) that are still not well understood. If the target and microRNA have perfect complementarity, the mRNA is eliminated by a RNA degradation pathway. MicroRNA expression profiles are being harnessed as diagnostic tools and as means to predict possible reactions to different treatment options being considered for complex diseases such as cancer, while individual micro RNAs are being targeted to treat diseases such as hepatitis C.

Naldini and colleagues suggest a new therapeutic endeavor for microRNAs. The group previously inserted a modified microRNA 142–3p target that contained several copies of a perfect complement to the corresponding microRNA into the 3′ untranslated region of a test reporter gene3 ; they have now done so with the human factor IX expression cassette. After intravenous infusion, most of the lentiviral vector is taken up by the intended target, the liver, although some hematopoietic-derived cells become transduced. Since the 142–3p microRNA is only expressed in hematopoietic cells, the reporter gene or factor IX mRNAs are degraded, thwarting antigen production and the potential immune response against the transgene product. Moreover, because the microRNA is not produced in liver cells, transgene expression is unabated, resulting in a sustained therapeutic level of the coagulation factor.

The current approach has its limitations. It will not ameliorate cell-mediated responses directed against vector particles taken up by antigen-presenting cells or transgene product synthesized in the target cells but then taken up by other cells that may participate in evoking an immune response. The former process was believed to limit factor IX gene expression from the liver when the transgene was delivered in a recombinant AAV-2 vector in a clinical trial,4  although the mechanisms for such responses have not been fully characterized. In addition, the Naldini study used a cross-species transgene, human factor IX in a mouse, a scenario that is not likely to be tried in clinical trials. Nonetheless, this study shows the proof of concept that the strategic inclusion of microRNA targets in a transgene expression cassette can help thwart at least one of the robust processes that can result in an immune-mediated elimination of the therapeutic. The utility of this approach for use in humans will need to be further evaluated.

This work was supported by National Institues of Health grant HL 64 274.

Conflict-of-interest disclosure: The author declares no competing financial interests. ■

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