MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy


Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono- and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220+ B and CD3+ T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220+ B and CD4+ or CD8+ T cells whereas expression in CD11b+ myeloid cells, lin and lin/Sca1+ progenitors, or lin/Sca1+/c-kit+ stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.


Due to easy accessibility, well-established in vitro manipulation procedures, longevity, and their substantial proliferation and differentiation capacities, hematopoietic stem cells (HSCs) represent a prime target for gene therapy approaches. Although the therapeutic potential of this strategy has been demonstrated by the successful treatment of several congenital immunodeficiency syndromes,1, 2, 3, 4 these studies have also highlighted several problems still associated with this approach. Thus, a number of issues need to be addressed before a more widespread use of HSC gene therapy and adequate regulation of transgene expression clearly represents one of them. So far, successful HSC gene therapy has employed enhancer/promoter elements, which are constitutively active in a wide range of target cells. However, this approach is only feasible in diseases where similar levels of transgene expression are required, or at least tolerated within the entire lymphohematopoietic system. For other diseases such as hemoglobinopathies, which require high transgene expression only in the erythroid lineage, or globoid cell leukodystrophy, in which the therapeutic transgene is required in macrophages or microglia cells but toxic to HSCs, alternative expression strategies are required.5, 6, 7 In the past, most investigators aiming for cell type-specific transgene expression have relied on transcriptional targeting by tissue- or cell-specific promoters.8, 9 This approach has its limitations, however, as transcriptional regulation usually involves a certain degree of promiscuity and strictly cell type-specific promoter/enhancer sequences are rare. In addition, many promoter/enhancer sequences have remained ill defined or exceed the technical size limits imposed by the clinically used gene transfer systems. More recently, endogenous microRNAs (miRNAs) have been exploited to direct transgene expression to the tissue, lineage or differentiation state of choice.10, 11 MiRNAs are a class of non-coding RNAs that control gene expression at the posttranscriptional level by binding to complementary sites of target mRNAs and thereby prevent translation or accelerate breakdown of these targets. As most miRNAs target multiple gene products, miRNAs constitute a network of posttranscriptional regulation involved in almost every cell function. More than 400 miRNAs have been identified in mammals so far, and most of them are well conserved between species.12, 13 Incorporation of miRNA target sequences into transgenic cDNA allows for transgene downregulation specifically in cells expressing these miRNAs.10, 11 Proof of principle for this strategy has been obtained by the incorporation of sequences targeted by the hematopoietic cell-specific miRNA-142-3p into vectors, which were systemically delivered into immunocompetent mice. This approach limited off-target transgene expression in antigen-presenting cells and allowed for stable transgene expression in the absence of an immune response.14 Applied to a murine model of hemophilia B gene therapy, the miRNA-142 regulation approach allowed achievement of stable and long-term factor IX protein expression as well as phenotypic disease correction.15 Likewise, miRNA-126 target sequences protected HSCs from the toxicity of the therapeutic transgene galactocerebrosidase in a murine gene therapy model of globoid cell leukodystrophy.16 In other settings transgene expression in the lymphatic compartment may be problematic. Substantial transgene-related lymphotoxicity has been encountered when gene transfer of the drug resistance gene cytidine deaminase as a mean to protect the hematopoietic system from the toxicity of deoxycytidine-analog type cytotoxic drugs such as cytosine arabinoside (Ara-C) or gemcitabine was investigated.17 Therefore, we here investigated targeting of miRNA-150 as a strategy to suppress transgene expression in the lymphatic compartment. MiRNA-150 is highly expressed in mature, resting T and B cells and miRNA-150 expression has been reported to be upregulated during the terminal maturation of B or T cells at the intrasplenic T1 to T2/3 or at the intrathymic double-negative to single-positive stage transition, respectively.18, 19, 20 MiRNA-150 lacks expression, however, in the stem and early progenitor compartment (including early lymphoid progenitors), or in myeloid cells.18, 21 We have investigated miRNA targeting in the context of lentivirus-mediated HSC gene transfer and demonstrate by in vitro studies as well as in a murine gene transfer model that incorporation of a quadruple repeat of a completely complementary miRNA-150 target sequence (miR-150T) into lentiviral vectors effectively suppresses transgene expression in mature T and B cells while sparing stem and progenitor cells as well as the myeloid lineage.


MiRNA-150 expression is restricted to mature T and B cells

To confirm the lymphoid-specific expression profile of miRNA-150 we performed quantitative reverse transcriptase PCR to systematically assess miRNA-150 expression levels in the various lymphohematopoietic cell compartments of adult C57Bl/6 mice. Cells were isolated from hematopoietic organs, such as the bone marrow (BM), spleen (SP) and thymus and purified to 96–98% homogeneity for the individual cell fractions using cell-type-specific surface markers and fluorescence-activated cell sorting. Results were normalized to expression levels of the small nuclear RNA U6.

Our data clearly demonstrate that the highest expression of miRNA-150 is present in terminally differentiated splenic (CD19+/B220+) B cells and thymic CD4 or CD8 single-positive T cells (Figure 1). Intermediate expression levels are observed in BM- or thymus-derived lymphoid progenitor/precursor populations such as lineage/Sca1+/cKitlow/IL7Rα+ common lymphoid progenitor cells, B220+/CD19+/IGM/IGD immature B cells or the T cell precursor fractions of CD4/CD8 double-negative cells as well as in BM-derived lineage/Sca1/cKit+/CD34/FcRγII/II megakaryocyte-erythrocyte progenitor cells. Virtually no miRNA-150 expression was detected in differentiated splenic GR1+ myeloid cells, myeloid progenitor cells in the BM such as lineage/Sca1/cKit+/CD34+/FcRγII/III common myeloid progenitor and lineage/Sca1/cKit+/CD34+/FcRγII/III+ granulocyte-macrophage progenitor, or the BM-derived lineage/Sca1+/cKit+ (LSK) stem cell enriched fraction. Thus, our data confirm the restriction of abundant miRNA-150 expression in the lymphohematopoietic system to the differentiated lymphoid lineage. Of note, miRNA-150 expression in splenic CD19+/B220+ B cells was 133- or 55-fold increased when compared with splenic GR1+ myeloid cells or the BM LSK stem cell compartment, respectively.

Figure 1

Expression profile of miRNA-150 in defined murine lymphohematopoietic cell compartments. Relative expression levels of miRNA-150 were assessed by quantitative reverse transcriptase PCR analysis (mean±s.e.m., n=3). Data are relative to the LSK compartment and normalized to levels of the small nuclear RNA U6. Cells were isolated from BM, SP or thymus (Th). Compartments analyzed include the following: LSK (lineage/Sca1+/cKit+) stem cells (BM), LK (lineage/cKit+) progenitor cells (BM), CMP (lineage/Sca1/cKit+/CD34+/FcRγII/III) common myeloid progenitors (BM), GMP (lineage/Sca1/cKit+/CD34+/FcRγII/III+) granulocyte-macrophage progenitors (BM), MEP (lineage/Sca1/cKit+/CD34/FcRγII/II) megakaryocyte-erythroid progenitors (BM), CLP (lineage/Sca1+/cKitlow/IL7Rα+) common lymphoid progenitor (BM), immature (CD19+ B220+ IgM IgD) B cells (BM), DP double-positive (CD4+ CD8+) T cells (Th), myeloid (GR1+/CD11b+) cells (SP), single-positive CD4+ T cells (Th), single-positive CD8+ T cells (Th), mature (CD19+ B220+ IgM+ IgD+) B cells (SP). Significance was calculated by Student's t-test (**P0.01).

MiRNA-150 targeting allows for cell-type-specific gene expression in-vitro

Next, miRNA-150-regulated gene expression was investigated in vitro, utilizing third generation self-inactivating lentiviral vectors that express the green fluorescence protein (GFP) marker gene from the spleen focus forming virus promoter/enhancer and coupled to a totally complementary, tetrameric binding cassette for miRNA-150 (LV.SFFV. GFP.miR150T). The same vector without the miR-150T tetrameric binding cassette (LV.SFFV.GFP) served as a control (Figure 2). These experiments were performed with immunomagnetically separated splenic B220+ B and CD3+ T cells (90–96% purity) as well as BM-derived lineage-negative (lin) progenitor cells utilizing transduction protocols specifically suited for these cell types. In addition, following transduction of lin cells, mature myeloid GR1+ cells were obtained by G-CSF-induced terminal myeloid differentiation.

Figure 2

Self-inactivating lentiviral (SIN-LV) miRNA-150-targeting vectors. Monodirectional SIN-LV vector-expressing green fluorescence protein (GFP) unregulated (LV.SFFV.GFP) (a) or in combination with a target sequence for miRNA-150 (LV.SFFV.GFP.miR150T) (b) from the spleen focus-forming virus (SFFV) promoter/enhancer. Bidirectional SIN-LV vector expressing a destabilized GFP version (d2GFP) unregulated (LV.Bd.PGK.GFP) (c) or in combination with a target sequence for miRNA-150 (LV.Bd.PGK.GFP.miR150T) (d) from the phosphoglycerate kinase (PGK) promoter. Bidirectional constructs also express the mCherry reporter gene from a minimal cytomegalovirus (mCMV) promoter-driven cassette carrying the SV40 polyadenylation signal (pA) in antisense orientation. The miRNA-150 target sequence (miR150T) contains four repeats of 22 basepairs (bp) completely complementary to the sequence of miRNA-150 including its seed sequence. All SIN-LV vectors contain 5′ and 3′ long terminal repeats with SIN deletion in the U3 region (LTRs, ΔU3, R, U5), splice donor (SD) and splice acceptor (SA) sites, the posttranscriptional regulatory element of woodchuck hepatitis virus (wPRE), a central polypurine tract (cPPT), the Rev responsive element (RRE) and an extended encapsidation signal (ψ) including the 5′ region of gag (ΔGA).

Control transductions performed with the LV.SFFV.GFP vector yielded gene transfer rates of 6 to 35% for the individual cell types. Successfully transduced cells were easily identified in all four lineages by an 102- to 103-fold increased mean green fluorescence intensity as compared with non-transduced cells (Figures 3a–d, upper panels). In comparison, almost no GFP expression was observed in CD19+ B or CD3+ T cells following transduction with the LV.SFFV.GFP.miR150T vector containing a quadruple repeat of a specific miRNA-150 targeting sequence (Figures 3a and b, middle panels). This was in contrast to mature GR1+ myeloid or lin progenitor cells, for which clear-cut expression of GFP at levels similar to the LV.SFFV.GFP construct was observed also upon LV.SFFV.GFP.miR150T transduction (Figures 3c and d, middle panels). Semiquantitative PCR analysis for the vector-specific posttranscriptional regulatory element sequence of the woodchuck hepatitis virus (wPRE) was applied and confirmed similar transduction levels by LV.SFFV.GFP and LV.SFFV.GFP.miR150T in all cell types (Figures 3a–d, lower panels). These data were recapitulated in two further experiments (Supplementary Figure 1). In one of these experiments similar transduction-efficiency in B and T cells was confirmed by copy number analysis yielding 2.7 or 2.8 copies for LV.SFFV.GFP and LV.SFFV.GFP.miR150T transduced B cells as well as 2.7 or 1.7 copies for LV.SFFV.GFP and LV.SFFV.GFP.miR150T transduced T cells, respectively.

Figure 3

Cell type-specific in vitro transgene expression by miRNA-150 targeting. GFP expression in CD19+ B cells (a), CD3+ T cells (b), GR1+ myeloid cells differentiated in vitro from lin (CD3ɛ, CD45R/B220, CD11b, TER119, GR1) cells (c), and lin cells from bone marrow (d) transduced with LV.SFFV.GFP (upper panel) and LV.SFFV.GFP.miR150T (middle panel) are shown. Transduction efficiency was assessed by semiquantitative PCR of the vector-specific posttranscriptional regulatory element (PRE) utilizing the housekeeping gene FLK1 as an internal control (lower panel). Negative controls were non-transduced murine CD19+ B cells, CD3+ T cells, GR1+ myeloid cells or Lin cells as well as water controls.

As downregulation of miRNA-150 has been described upon in vitro activation of terminally differentiated CD4+ as well as CD8+ T cells,18 we investigated miRNA-150 expression in lymphocytes during in vitro culture of T and B cells. Our data clearly demonstrate that in vitro cultivation of murine CD19+/B220+ splenic B cells for up to 72 h as utilized in our transduction experiments had almost no effect on miRNA-150 expression level when compared with freshly isolated cells. Cultivation of freshly isolated CD8+ thymic T cells, however, in accordance with the literature resulted in a substantial decrease of miRNA-150 expression to 50 and 20% of starting levels after 48 and 72 h, respectively. Even this reduction, however, did not interfere with robust transgene suppression in our in vitro system as evident from Figure 3 and Supplementary Figure 1.

‘Lymphocyte-sparing’ transgene expression by miRNA-150 targeting in a murine in vivo model

To further confirm the feasibility of miRNA-150 targeting to avoid transgene expression in the lymphoid compartment during HSC gene therapy, a murine in vivo BM transplantation/gene transfer model was utilized. In these studies, the LV.Bd.PGK.GFP and the LV.Bd.PGK.GFP.miR150T constructs were employed to allow for the easy identification of gene-transduced cells and their progeny. In these bidirectional constructs a destabilized d2GFP marker gene (either miRNA-150 regulated or unregulated) is transcribed from the phosphoglycerate kinase (PGK) promoter, which transactivates an antisense-oriented minimal cytomegalovirus (CMV) promoter driving the expression of the mCherry fluorescent protein. Transduced cells and their progeny can thus be identified via constitutive mCherry expression, and miRNA-150-mediated regulation can be detected by d2GFP fluorescence intensity (Figures 2c and d). Figure 4 gives expression data for peripheral blood-derived CD11b+/GR1+ myeloid cells and CD19+/B220+ B cells in recipient mice 16 weeks after transplantation. Strategies for gating of transduced myeloid (mCherry+/GR1+/CD11b+) or B cells (mCherry+/CD19+/B220+) and assessment of GFP expression in transduced versus untransduced (mCherry) populations are depicted in Figures 4a and b, respectively.

Figure 4

Cell-type-specific transgene expression by miRNA-150 targeting in murine B and myeloid cells in vivo. Data of peripheral blood-derived myeloid and B-lymphoid cells 16 weeks after transplantation of LV.Bd.PGK.GFP- or LV.Bd.PGK.GFP.miR150T-transduced cells are given. Gating strategies for myeloid (a) and B lymphoid (b) cells included pre-gating for viable cells according to side scatter and forward scatter (FSC) determination (data not shown) followed by analysis of the CD11b and GR1 myeloid-specific or the B220 and CD19 B cell-specific surface markers. Cells positive for both markers were gated for mCherry expression and histograms of green fluorescence protein (GFP) expression were generated for the mCherry-positive and the mCherry-negative fraction as indicated by arrows and finally presented as a histogram overlay of GFP expression within the mCherry-positive (red) and the mCherry-negative population (blue). Histogram overlays of GFP expression in peripheral blood CD11b+/GR1+ myeloid (c) and B220+/CD19+ B (d) cells from a total of four recipient mice transplanted with LV.Bd.PGK.GFP- (M1.1 to M1.4) or LV.Bd.PGK.GFP.miR150T- (M2.1 to M2.4) transduced cells are depicted. Histogram overlays were used to calculate a corrected mean fluorescence activity for GFP (mean fluorescence index (MFI) corrected (GFP) or MFIcorr; also see Figures 5 and 6) by subtracting the MFI (GFP) from mCherry-negative cells from the MFI (GFP) of mCherry-positive cells. (MFIcorr=MFIGFP(mCherry+)−MFIGFP(mCherry−)).

As evident from Figure 4c (upper panel) in the myeloid GR1+/CD11b+ compartment LV.Bd.PGK.GFP-transduced control cells exhibited an 10-fold increased mean green fluorescence intensity as compared with non-transduced cells reflecting the expression of the d2GFP marker protein from the PGK promoter. The considerably lower fluorescence intensity of transduced cells in comparison to the data obtained in vitro with the monocistronic LV.SFFV.GFP construct (Figure 3a) reflects the lower activity of the PGK as compared with the SFFV promoter in hematopoietic cells as well as the shorter half-life of the d2GFP protein. The same pattern was observed when the LV.Bd.PGK.GFP.miR150T construct was used in GR1+/CD11b+ myeloid cells indicating clear-cut GFP expression from the LV.Bd.PGK.GFP.miR150T vector construct in mature myeloid progenies of transduced cells (Figure 4c, lower panel). A completely different pattern was detected in mature lymphoid cells such as CD19+/B220+ B cells (Figure 4d). Here, in contrast to the marked GFP expression in control LV.Bd.PGK.GFP-transduced cells (Figure 4d, upper panel) markedly reduced GFP expression was detected in the mCherry+ fraction of LV.Bd.PGK.GFP.miR150T-transduced B cells, thus demonstrating efficient transgene silencing by miRNA-150 (Figure 4d, lower panel).

GFP expression data for various lymphohematopoietic cell compartments are summarized in Figure 5. Again, animals were analyzed 16 weeks after transplantation. MiRNA-150 targeting sequences had few if any effects on GFP expression in mature peripheral blood CD11b+/GR1+ myeloid cells (Figure 5d) and BM-derived lin or lin/cKit+ progenitor populations (Figures 5e and f). In contrast, GFP expression in mature peripheral blood B220+/CD19+ B cells as well as single CD4+ or CD8+ T cells was significantly impaired (Figures 5a–c), and expression levels in these cells were virtually indistinguishable from untransduced cells (data not shown). Of note, mean GFP-expression levels were markedly higher in the progenitor than in mature myeloid or lymphoid cells, reflecting the differential properties of the PGK promoter in these compartments.22 Efficacy of miRNA-150 targeting was confirmed in secondary transplant experiment and demonstrated stable long-term regulation of the GFP transgene by the miR150T sequence. As evident from Figure 6, again miRNA-150-targeting sequences had little effect on GFP expression in the myeloid lineage, that is, splenic GR1+/CD11b+ cells (Figure 6d) and in the progenitor compartment, such as lin or lin/cKit+ populations (Figures 6e and f). A similar tendency was noted in the LSK compartment (Figure 6g), although the low numbers of animals may make it difficult to draw definite conclusions. In contrast no transgenic GFP expression was detectable in the differentiated lymphoid compartments, such as peripheral blood CD19+/B220+ B cells (Figure 6a), CD4+ and CD8+ T cells (Figures 6b and c).

Figure 5

MiRNA-150 targeting directs cell type-specific transgene expression in murine primary recipients. Corrected mean fluorescence index (MFI) of green fluorescence protein (GFP) (see Figure 4) from animals (mean values, n=4) transplanted with LV.Bd.PGK.GFP- or LV.Bd.PGK.GFP.miR150T-transduced cells are given for peripheral blood CD19+/B220+ B cells (a), CD4+ T cells (b), CD8+ T cells (c), CD11b+/GR1+ myeloid cells (d) as well as for bone marrow lin (e) and lin/cKit+ (LK) (f) progenitor cells 16 weeks following transplantation. Significance was calculated by Student's t-test (**P0.01).

Figure 6

MiRNA-150 targeting directs cell type-specific transgene expression in murine secondary recipients. Corrected mean fluorescence index (MFI) of green fluorescence protein (GFP) (see Figure 4) from animals (mean values, n=4) secondarily transplanted with LV.Bd.PGK.GFP- or LV.Bd.PGK.GFP.miR150T-transduced cells are given for peripheral blood CD19+/B220+ B cells (a), CD4+ T cells (b), CD8+ T cells (c), splenic CD11b+/GR1+ myeloid cells (d) as well as for bone marrow lin (e) and lin/cKit+ (f) progenitor and lin/cKit+/Sca1+ stem (g) cells 12 weeks following secondary transplantation. Significance was calculated by Student's t-test (**P0.01).

No evidence for in vivo toxicity of miRNA-150 targeting

So far, our analysis of primary as well as secondary recipient mice has failed to demonstrate any adverse effects of miRNA-150 targeting and peripheral blood counts within normal ranges were observed in all animals transplanted with the LV.Bd.PGK.GFP.miR150T vector (Tables 1 and 2, Supplementary Table 2). Even more importantly, also the contribution of transduced versus untransduced cells to the different lymphohematopoietic compartments remained fairly stable during the entire 28 weeks observation period (16 weeks primary, 12 weeks secondary recipients). To rule out consumption of endogenous miRNA-150, which in turn could lead to disrupted regulation of miRNA-150 targets by our transgene targeting approach, we examined the expression of c-myb in differentiated B cells. C-myb represents a major miRNA-150 target gene in B lymphopoiesis as an increase of miRNA-150 levels during B cell differentiation ensures the suppression of c-myb activity following the pro-B to pre-B cell transition.20 To screen for potential miR150T-induced offsite target effects caused by LV.Bd.PGK.GFP.miR150T expression, endogenous expression level of c-myb, a major target gene of miRNA-150 in B lymphopoiesis, was analyzed by quantitative reverse transcriptase PCR in peripheral blood CD19+ B cells from primary recipients of LV.Bd.PGK.GFP.miR150T-transduced cells and non-transplanted C57Bl/6 control mice. No significant changes from the low c-myb background expression levels in B cells of control wild-type mice were observed in B cells of the LV.Bd.PGK.GFP.miR150T experimental group. These background levels were 30-fold decreased when compared with lin cells from C57Bl/6 control mice (Figure 7).

Table 1 Cellularity and contribution of gene-modified cells to defined lymphohematopoietic compartments in primary recipients
Table 2 Cellularity and contribution of gene-modified cells to defined cell compartments of the peripheral blood in secondary recipients
Figure 7

Expression of c-Myb in murine primary recipients. Expression levels of c-myb are given for bone marrow lin cells and CD19+ peripheral blood B cells of wild-type (WT) mice and peripheral blood CD19+ B cells of mice transplanted with LV.Bd.PGK.GFP.miR150T-transduced cells (mean±s.e.m., n=4). Expression levels are normalized to WT B cells.


Within the regulatory network of the cell, miRNAs constitute an important element of posttranscriptional control, which can be exploited for regulated, cell type-specific transgene expression. This also holds true for the lymphohematopoietic system, where miRNA-based gene regulation is observed at almost every stage of differentiation.23, 24 Thus, in the context of HSC gene therapy, miRNAs represent an attractive approach to detarget gene expression from unwanted cells, thereby preventing transgene toxicity. Based on our previous experience with transgenic expression of the drug resistance gene cytidine deaminase in lymphohematopoietic cells,17 we have specifically investigated transgene detargeting in the lymphoid compartment. We here demonstrate effective shut-down of transgene expression in differentiated B and T cells by a complementary, tetrameric binding cassette for miRNA-150, while expression in primitive stem and progenitor cells or in the myeloid lineage remains unaltered. These results were obtained by in vitro studies with primary lymphohematopoietic cells and subsequently confirmed in murine BM chimeras. Both of these models demonstrated robust miR150T-regulated, cell type-specific transgene expression as well as an absence of unwanted offsite or toxic effects in the targeted lymphohematopoietic cell populations.

These observations further support the concept of miRNA targeting to achieve a desired cell type-specificity of transgene expression in gene therapy approaches. This strategy was introduced by L Naldini's group, who employed miRNA-142-3p to detarget transgene expression in lymphohematopoietic versus liver cells.15 Meanwhile, it has been extended to avoid transgene expression in HSCs versus myeloid lineage cells,16 undifferentiated versus mature dendritic cells, myeloid versus lymphoid cells, pluripotent embryonic stem cells versus differentiated progeny thereof,10 or to avoid transgene expression in hepatic or muscle cells during AAV- or Coxsackievirus-mediated in vivo gene therapy.25, 26, 27 Our work now adds miRNA-150 targeting as another component to this regulatory armamentarium, which appears particularly suited to avoid transgene-mediated lymphotoxicity in HSC gene therapy approaches.

Though certain differences in the potential of individual miRNAs to mediate transgene knock-down exist,10 absolute expression levels currently still represent the most reliable method to select candidate miRNAs for transgene regulation in specific cell types,11 and 50–100 copies per pg small RNA correlating to 100 copies per cell appear to represent the threshold level for repression of target-containing constructs.10, 11 Thus, miRNA-150 as one of the most abundant miRNAs in mature B as well as T cells represents a natural choice of target-miRNA to achieve transgene suppression in these cells. This concept also should be applicable to the human situation as miRNA-150 profiling in human hematopoietic cells has demonstrated high miRNA-150 expression in B cells, and even more in CD4+ and CD8+ T cell populations, whereas only low levels were detectable in the CD11b+ myeloid and CD34+ progenitor/stem cell compartment (own unpublished data). The suitability of miRNA-150 is further underlined by the fact that even a 50–80% reduction of miRNA expression levels, as observed in CD8+ T cells during our in vitro transduction protocol, still had no major effect on stable transgene knock-down, further supporting the concept that miRNA activity is dependent on a threshold level rather than directly proportional to their expression level.10 Although this work mainly demonstrates the efficacy of miR150T sequences in terminally differentiated, SP- or peripheral blood-derived B and T cells, miRNA-150 expression data would suggest that this strategy may also be efficient in late developmental stages of T or B lymphopoiesis. In murine B cell development, miRNA-150 has been described to be upregulated in B2 cells during the progression from T1 to T1/2 stage, whereas in T lymphopoiesis, upregulation is observed during the intrathymic transition from the double-negative to the single-positive stage.18, 19, 21 These studies are confirmed by our own quantitative reverse transcriptase-PCR data. Thus, at least to some extent, also late stages of intra-BM B and intrathymic T lymphopoiesis may be amenable to miR150T-mediated transgene suppression. To target earlier developmental stages of B or T lymphopoiesis, alternative miRNAs, which are specifically expressed at these differentiation stages,18, 21, 23 may be employed. In this context, miRNA-181, which has major regulatory functions in T as well as B cell development,28 appears particularly attractive. MiRNA-181 targeting already has been successfully employed to direct transgene expression to early stage intra-thymic versus late stage post-thymic T cells in tumor immunotherapy approaches.29 Targeting of such ‘early acting’ miRNAs may also be combined with miRNA-150-targeting to prevent gene expression over a broader range of lymphoid differentiation stages. Such combinatorial targeting strategies have already been successfully applied to restrict lentiviral-mediated transgene expression in both hepatic and hematopoietic target cell populations.10, 29

For potential clinical applications of miRNA-based cell-targeting approaches safety considerations clearly constitute a major issue. It has been shown that an abundance of miRNA substrates may result in competitive inhibition of endogenous miRNA targets and/or decreased intracellular levels of the corresponding miRNA, resulting in ‘miRNA insufficiency’.10 Given the major regulatory role of miRNA-150 in B-lymphopoiesis, this could prove to be problematic. MiRNA-150 has been defined as the crucial factor to mediate c-myb downregulation at the pre-B cell stage, and its downregulation has been associated with an increase in intrasplenic B1 cells as well as an enhanced humoral immune response most likely due to insufficient c-myb repression.20 In addition, ectopic expression of miRNA-150 in hematopoietic stem cells reduces mature B cell levels in the circulation, SP and lymph nodes.30 Though both of these studies experienced little effects of miRNA-150 modulations on the T cell compartment, more recently miRNA-150 also has been described as a tumor suppressor in NK/T cell lymphomas.31 Furthermore miRNA-150 controls cell fate decisions in megakaryocyte-erythrocyte progenitors.32, 33 Given this background it is reassuring that in our studies no adverse effects of miRNA targeting were observed. Total B and T cell numbers as well as the contribution of transduced cells and their various differentiated progeny to overall hematopoiesis remained fairly constant throughout the 28 weeks observation period of our in vivo model. Likewise, no effect of miRNA-150 targeting on c-myb levels in mature B cells was noted. Thus, with respect to a potential clinical relevance of miRNA-150 targeting for cell type-specific transgene suppression in HSC gene therapy approaches, our data clearly support further evaluation of these strategies for specific applications or disease entities, in which transgene expression in lymphoid cells is not desired.

Materials and methods

Vector design

The murine miRNA-150-specific target sequence (miR-150T) was ordered as a 121-bp oligo-strand representing a tetrameric repeat of 22 bp of miRNA-150 completely complementary interrupted by three 5 bp spacer sequences from GenScript (Piscataway, NJ, USA). Mono- or bicistronic third generation self-inactivating lentiviral vectors were utilized for miRNA-150-targeted transgene expression (Figure 2). Monocistronic LV.SFFV.GFP.miR150T expressing the GFP from a SP focus-forming virus (SFFV) promoter/enhancer element, was generated by cloning the miRNA-150 tetrameric binding site cassette into the 3′ untranslated region of LV.SFFV.GFP.34 To construct the bidirectional LV.Bd.PGK.GFP.miR150T construct, the miRNA-150 tetrameric binding site cassette was excised from the parental plasmid with BsrGI/NotI, filled with Klenow fragment and ligated into the SmaI-digested control vector LV.Bd.PGK.GFP.35 In these bidirectional vectors the first transcription unit expresses the mCherry reporter protein in antisense orientation from a minimal cytomegalovirus promoter (mCMV) transactivated by the PGK promoter. The second sense transcription unit expressed a destabilized d2GFP from a human PGK.

Production of viral supernatants and titration

Lentiviral supernatant preparations were generated by transient transfection of 293T cells as described previously.9, 34 In brief, 293T cells were cultured in Dulbecco's-modified Eagle's Medium (PAA, Pasching, Austria) supplemented with 10% fetal calf serum, 100 U ml−1 penicillin/streptomycin, 20 mmol l−1 HEPES (all PAA), 2 mmol l−1 glutamine (GIBCO, Darmstadt, Germany) and 25 μmol l−1 chloroquine (Sigma-Aldrich, Steinheim, Germany). Cells were transfected using calcium phosphate precipitation in the presence of 8 μg ml−1 gag/pol, 5 μg ml−1 pRSV-Rev, 5 μg ml−1 lentiviral vector plasmid and either 2 μg ml−1 pMD.G9, 36 (encoding for VSVg) or 5 μg ml−1 plasmid encoding for the ecotropic envelope.37, 38 Viral supernatants were harvested 36 and 48 h post transfection, filtered and concentrated by ultracentrifugation (Becton Dickinson, Krefeld, Germany) for 3 h at 30.000 g and 4 °C for VSVg or 12h at 8.000 g and 4°C for ecotrophic. To increase viral titers of bidirectional vectors an additional suppressor of the RNA interference Nodamuravirus B2 protein-encoding plasmid (NovB2) was used as described previously.35, 39 Titration was performed on murine (SC-1) fibroblasts using serial dilution transduction and flow cytometric GFP-analysis as read-out.

Isolation and transduction of murine T and B cells

T and B cells of BALB/c (Central Animal Facility of Hannover Medical School, Germany) were isolated from SPs using Pan T Cell Isolation Kit or CD45R (B220) Microbeads, respectively, (both Miltenyi, Bergisch Gladbach, Germany). Purified T- and B cells were cultured in RPMI1640 medium (PAA) enriched with 10% heat-inactivated fetal calf serum. Prestimulation was performed with 100 IU ml−1 IL-2, 2 ng ml−1 rmIL-7 and mouse T Cell Activation/Expansion Kit (Miltenyi) for T cells or 10 μg ml−1 bacterial lipopolysaccharide (Sigma-Aldrich) for B cells, respectively. Following 24 h prestimulation T and B cells were transduced on Retronectin (10 μg cm−2; Takara, Otsu, Japan) coated plates using ecotropic infectious particles.

TaqMan real-time PCR miRNA analysis

Quantitative PCR was performed on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, ABI, Darmstadt, Germany). For miRNA-150 profiling, RNA was isolated from defined lymphohematopoietic cell compartments of C57Bl/6N mice (Charles River Laboratories, Wilmington, MA, USA) using directly conjugated antibodies and fluorescence-activated cell sorting (see Supplementary Table 1). Samples for miRNA-150 expression analysis were also collected from in vitro stimulated T and B cells, cultured for 72 h in respective culture conditions in 24 h intervals. Subsequently, RNA was purified with RNeasy micro kit and RNase-Free DNase (both Qiagen, Hilden, Germany). Reverse transcription of RNA samples was performed using Taqman MicroRNA Reverse Transcription kit and mature Taqman MicroRNA assay-, 150 and U6, respectively (both ABI). Real-Time analysis was performed using Taqman Universal Master Mix II with UNG and Taqman MicroRNA assay-, 150 and U6, respectively (both ABI). U6 was used for normalization (ΔCt=ΔCtmiRNA150−ΔCtU6). Endogenous c-Myb expression levels were analyzed from C57Bl/6N mice transplanted with LV.Bd.PGK.GFP or LV.Bd.PGK.GFP.miR150T transduced cells after fluorescence-activated cell sorting of peripheral blood CD19+ B cells using QuantiTect Primer Assay for c-Myb and GAPDH as endogenous control (both Qiagen).

Transduction of lineage-negative (lin) cells and granulocytic differentiation in vitro

Lin cells of untreated 10–12-weeks-old male C57Bl/6N mice were isolated from femurs and tibias using lineage specific antibodies (Lineage Cell depletion kit; Miltenyi), prestimulated for 24 h in StemSpan (StemCell Technologies, Grenoble, France) supplemented with (10 ng ml−1 rmSCF, 20 ng ml−1 rmTPO, 20 ng ml−1 rmIGF2, 10 ng ml−1 rhFGF1; all PeproTech, Hamburg, Germany)40 and subsequently transduced on Retronectin (10 μg cm−2; Takara) for 24 h using VSVg pseudotyped lentiviral particles as recommended by the manufacturer. At 48 h after transduction, cells were either subjected to quantitative reverse transcriptase PCR and integration analysis or transferred to Iscove's-modified Dulbeccos medium supplemented with 10% FSC, 100 U ml−1 penicillin/streptomycin (all PAA), 2 mmol l−1 glutamine (GIBCO), 100 ng ml−1 rhG-SCF and 20 ng ml−1 rmIL-3 (both Peprotech) for granulocytic differentiation for another 72 h.

Integration analysis

Genomic DNA from transduced or non-transduced cells was isolated using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). Quantitative real-time PCR was performed on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems) using primers detecting a 94-bp specific sequence for wPRE and SYBR Green (Applied Biosystems). To normalize wPRE signal, primers for the housekeeping gene murine flk1 were used. Primer and PCR conditions were used as previously described.34

Murine BM transplant transfer model

Treatment of animals

Lin BM cells of 10–12-weeks-old male C57Bl/6N mice were isolated, prestimulated and transduced as described above. Transduced cells were transplanted intravenously (i.v.) into lethally irradiated (10Gy) 10–12-weeks-old female C57Bl/6N recipients. All mice were kept in IVC racks (Allentown Deutschland, Moembris, Germany) under pathogen-free conditions in the animal facility of the Hannover Medical School, Germany. All animal experiments were approved by the local animal wellfare committee and performed according to their guidelines. Peripheral blood samples were taken retro-orbitally and analyzed using scil Vet ABC (scil animal care company, Viernheim, Germany).

Flow cytometric analysis

Expression of the fluorescent marker proteins d2GFP and mCherry was measured at different time points in defined hematopoietic cell compartments using LSRII or FACSCalibur (both Becton Dickinson). In brief, cells were isolated from different hematopoietic organs using a cell strainer (Becton Dickinson), washed with PBS supplemented with 2 mM EDTA and 2% fetal calf serum and stained for 45 min with conjugated antibodies. All antibodies were used as recommended by the manufacturer. Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Statistical analysis

Statistical analysis was performed using Prism 4 software (GraphPad, La Jolla, CA, USA). Unless otherwise noted statistical analysis was performed using Student's t-test (unpaired, one-tailed).


  1. 1

    Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296: 2410–2413.

    CAS  Article  Google Scholar 

  2. 2

    Boztug K, Schmidt M, Schwarzer A, Banerjee PP, Diez IA, Dewey RA et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med 2010; 363: 1918–1927.

    CAS  Article  Google Scholar 

  3. 3

    Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M . Gene therapy of X-linked severe combined immunodeficiency. Int J Hematol 2002; 76: 295–298.

    CAS  Article  Google Scholar 

  4. 4

    Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–409.

    CAS  Article  Google Scholar 

  5. 5

    Negre O, Fusil F, Colomb C, Roth S, Gillet-Legrand B, Henri A et al. Correction of murine {beta}-thalassemia after minimal lentiviral gene transfer and homeostatic in vivo erythroid expansion. Blood 2011; 117: 5321–5331.

    CAS  Article  Google Scholar 

  6. 6

    Perumbeti A, Higashimoto T, Urbinati F, Franco R, Meiselman HJ, Witte D et al. A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction. Blood 2009; 114: 1174–1185.

    CAS  Article  Google Scholar 

  7. 7

    Sakai N . Pathogenesis of leukodystrophy for Krabbe disease: molecular mechanism and clinical treatment. Brain Dev 2009; 31: 485–487.

    Article  Google Scholar 

  8. 8

    Goverdhana S, Puntel M, Xiong W, Zirger JM, Barcia C, Curtin JF et al. Regulatable gene expression systems for gene therapy applications: progress and future challenges. Mol Ther 2005; 12: 189–211.

    CAS  Article  Google Scholar 

  9. 9

    Heckl D, Wicke DC, Brugman MH, Meyer J, Schambach A, Busche G et al. Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia. Blood 2011; 117: 3737–3747.

    CAS  Article  Google Scholar 

  10. 10

    Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, Zingale A et al. Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 2007; 25: 1457–1467.

    CAS  Article  Google Scholar 

  11. 11

    Brown BD, Naldini L . Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications. Nat Rev Genet 2009; 10: 578–585.

    CAS  Article  Google Scholar 

  12. 12

    Bissels U, Wild S, Tomiuk S, Holste A, Hafner M, Tuschl T et al. Absolute quantification of microRNAs by using a universal reference. RNA 2009; 15: 2375–2384.

    CAS  Article  Google Scholar 

  13. 13

    Petriv OI, Kuchenbauer F, Delaney AD, Lecault V, White A, Kent D et al. Comprehensive microRNA expression profiling of the hematopoietic hierarchy. Proc Natl Acad Sci USA 2010; 107: 15443–15448.

    CAS  Article  Google Scholar 

  14. 14

    Brown BD, Venneri MA, Zingale A, Sergi Sergi L, Naldini L . Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med 2006; 12: 585–591.

    CAS  Article  Google Scholar 

  15. 15

    Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, Della Valle P et al. A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice. Blood 2007; 110: 4144–4152.

    CAS  Article  Google Scholar 

  16. 16

    Gentner B, Visigalli I, Hiramatsu H, Lechman E, Ungari S, Giustacchini A et al. Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy. Sci Transl Med 2010; 2: 58ra84.

    CAS  Article  Google Scholar 

  17. 17

    Rattmann I, Kleff V, Sorg UR, Bardenheuer W, Brueckner A, Hilger RA et al. Gene transfer of cytidine deaminase protects myelopoiesis from cytidine analogs in an in vivo murine transplant model. Blood 2006; 108: 2965–2971.

    CAS  Article  Google Scholar 

  18. 18

    Monticelli S, Ansel KM, Xiao C, Socci ND, Krichevsky AM, Thai TH et al. MicroRNA profiling of the murine hematopoietic system. Genome Biol 2005; 6: R71.

    Article  Google Scholar 

  19. 19

    Spierings DC, McGoldrick D, Hamilton-Easton AM, Neale G, Murchison EP, Hannon GJ et al. Ordered progression of stage specific miRNA profiles in the mouse B2 B cell lineage. Blood 2011; 117: 5340–5349.

    CAS  Article  Google Scholar 

  20. 20

    Xiao C, Calado DP, Galler G, Thai TH, Patterson HC, Wang J et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 2007; 131: 146–159.

    CAS  Article  Google Scholar 

  21. 21

    Neilson JR, Zheng GX, Burge CB, Sharp PA . Dynamic regulation of miRNA expression in ordered stages of cellular development. Genes Dev 2007; 21: 578–589.

    CAS  Article  Google Scholar 

  22. 22

    Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler RH, Trono D . High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 2000; 96: 3392–3398.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Chen CZ, Li L, Lodish HF, Bartel DP . MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303: 83–86.

    CAS  Article  Google Scholar 

  24. 24

    Ramkissoon SH, Mainwaring LA, Ogasawara Y, Keyvanfar K, McCoy Jr JP, Sloand EM et al. Hematopoietic-specific microRNA expression in human cells. Leuk Res 2006; 30: 643–647.

    CAS  Article  Google Scholar 

  25. 25

    Geisler A, Jungmann A, Kurreck J, Poller W, Katus HA, Vetter R et al. microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors. Gene Therapy 2011; 18: 199–209.

    CAS  Article  Google Scholar 

  26. 26

    Kelly ME, Zhuo J, Bharadwaj AS, Chao H . Induction of immune tolerance to FIX following muscular AAV gene transfer is AAV-dose/FIX-level dependent. Mol Ther 2009; 17: 857–863.

    CAS  Article  Google Scholar 

  27. 27

    Qiao C, Yuan Z, Li J, He B, Zheng H, Mayer C et al. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Therapy 2011; 18: 403–410.

    CAS  Article  Google Scholar 

  28. 28

    Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 2007; 129: 147–161.

    CAS  Article  Google Scholar 

  29. 29

    Papapetrou EP, Kovalovsky D, Beloeil L, Sant’angelo D, Sadelain M . Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras. J Clin Invest 2009; 119: 157–168.

    CAS  PubMed  Google Scholar 

  30. 30

    Zhou B, Wang S, Mayr C, Bartel DP, Lodish HF . miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc Natl Acad Sci USA 2007; 104: 7080–7085.

    CAS  Article  Google Scholar 

  31. 31

    Watanabe A, Tagawa H, Yamashita J, Teshima K, Nara M, Iwamoto K et al. The role of microRNA-150 as a tumor suppressor in malignant lymphoma. Leukemia 2011; 25: 1324–1334.

    CAS  Article  Google Scholar 

  32. 32

    Lu J, Guo S, Ebert BL, Zhang H, Peng X, Bosco J et al. MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors. Dev Cell 2008; 14: 843–853.

    CAS  Article  Google Scholar 

  33. 33

    Nagalla S, Shaw C, Kong X, Kondkar AA, Edelstein LC, Ma L et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011; 117: 5189–5197.

    CAS  Article  Google Scholar 

  34. 34

    Modlich U, Navarro S, Zychlinski D, Maetzig T, Knoess S, Brugman MH et al. Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther 2009; 17: 1919–1928.

    CAS  Article  Google Scholar 

  35. 35

    Maetzig T, Galla M, Brugman MH, Loew R, Baum C, Schambach A . Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors. Gene Therapy 2010; 17: 400–411.

    CAS  Article  Google Scholar 

  36. 36

    Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK . Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 1993; 90: 8033–8037.

    CAS  Article  Google Scholar 

  37. 37

    Morita S, Kojima T, Kitamura T . Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Therapy 2000; 7: 1063–1066.

    CAS  Article  Google Scholar 

  38. 38

    Schambach A, Galla M, Modlich U, Will E, Chandra S, Reeves L et al. Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research. Exp Hematol 2006; 34: 588–592.

    CAS  Article  Google Scholar 

  39. 39

    Sullivan CS, Ganem D . A virus-encoded inhibitor that blocks RNA interference in mammalian cells. J Virol 2005; 79: 7371–7379.

    CAS  Article  Google Scholar 

  40. 40

    Zhang CC, Lodish HF . Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 2005; 105: 4314–4320.

    CAS  Article  Google Scholar 

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We thank Matthias Ballmaier and his team from the Core-Facility Cell-Sorting of Hannover Medical School for cell sorting and Doreen Lüttge (Hannover) for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft: Cluster of Excellence REBIRTH (Exc 62/1), SPP1230 Grant MO 886/3-1 (UM and TM).

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Correspondence to T Moritz.

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Lachmann, N., Jagielska, J., Heckl, D. et al. MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy. Gene Ther 19, 915–924 (2012). https://doi.org/10.1038/gt.2011.148

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  • microRNA
  • hematopoiesis
  • transgene regulation

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