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microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389., (© 2015 AlphaMed Press.)

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

We previously showed that incorporating target sequences for the hematopoietic-specific microRNA miR-142 into an antigen-encoding transgene prevents antigen expression in antigen-presenting cells (APCs). To determine whether this approach induces immunologic tolerance, we treated mice with a miR-142-regulated lentiviral vector encoding green fluorescent protein (GFP), and subsequently vaccinated the mice against GFP. In contrast to control mice, no anti-GFP response was observed, indicating that robust tolerance to the transgene-encoded antigen was achieved. Furthermore, injection of the miR-142-regulated vector induced a population of GFP-specific regulatory T cells. Interestingly, an anti-GFP response was observed when microRNA miR-122a was inserted into the vector and antigen expression was detargeted from hepatocytes as well as APCs. This demonstrates that, in the context of lentiviral vector-mediated gene transfer, detargeting antigen expression from professional APCs, coupled with expression in hepatocytes, can induce antigen-specific immunologic tolerance.

A longstanding goal for the treatment of hemophilia B is the development of a gene transfer strategy that can maintain sustained production of clotting factor IX (F.IX) in the absence of an immune response. To this end, we have sought to use lentiviral vectors (LVs) as a means for systemic gene transfer. Unfortunately, initial evaluation of LVs expressing F.IX from hepatocyte-specific promoters failed to achieve sustained F.IX expression in hemophilia B mice due to the induction of an anti-F.IX cellular immune response. Further analysis suggested that this may be a result of off-target transgene expression in hematopoietic-lineage cells of the spleen. In order to overcome this problem, we modified our vector to contain a target sequence for the hematopoietic-specific microRNA, miR-142-3p. This eliminated off-target expression in hematopoietic cells, and enabled sustained gene transfer in hemophilia B mice for more than 280 days after injection. Treated mice had more than 10% normal F.IX activity, no detectable anti-F.IX antibodies, and were unresponsive to F.IX immunization. Importantly, the mice survived tail-clip challenge, thus demonstrating phenotypic correction of their bleeding diathesis. This work, which is among the first applications to exploit the microRNA regulatory pathway, provides the basis for a promising new therapy for the treatment of hemophilia B.