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

Lachmann, Nico; Jagielska, Joanna; Heckl, Dirk; Brennig, Sebastian; Pfaff, Nils; Maetzig, Tobias; Modlich, Ute; Cantz, Tobias; Gentner, Bernhard; Schambach, Axel; Möritz, Thomas

doi:10.1038/gt.2011.148

Cited by 17 publications

(14 citation statements)

References 41 publications

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“…Expression of different hematopoietic and nonhematopoietic surface marker was performed as previously described (22). Antibodies used were as follows: hCD14-PE, hCD11b-PE and APC, hCD116-PE, hCD45-APC, hCD19-APC, hCD34-PE, SSEA4-FITC, isotype-controls; mouseIgG1a-PE or APC (all eBioscience) and SSEA4-Alexa555 (BD Biosciences, Heidelberg, Germany), minus one control represents the staining without the respective specific antibody.…”

Section: Flow Cytometric Analysismentioning

confidence: 99%

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Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

Lachmann

Happle²,

Ackermann³

et al. 2014

Am J Respir Crit Care Med

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These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

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Section: Flow Cytometric Analysismentioning

confidence: 99%

“…CSF2RA) was sequence-verified by DNA sequencing (SeqLab, Goettingen, Germany). Viral vector production was performed by four plasmid transfection as previously described (22). Viral titers were determined by several dilutions on SC-1 fibroblasts and flow cytometry.…”

Section: Lentiviral Vector Construction and Productionmentioning

confidence: 99%

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

Lachmann

Happle²,

Ackermann³

et al. 2014

Am J Respir Crit Care Med

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“…The Sadelain lab has explored miR‐181a as a means to segregate transgenic antigen receptor expression in developing versus post‐thymic T cells, potentially overcoming negative thymic selection . Transgene expression can be de‐targeted from mature T and B lymphocytes by miR‐150 , and the miR‐144/451 cluster shows promise in excluding expression from the erythroid lineage . Another potentially interesting expression profile can be obtained by harnessing the activity of miR‐155, an miRNA which is upregulated upon immune cell activation and which can discriminate expression between immature and mature dendritic cells .…”

Section: Post‐transcriptional Regulation Of Gene Expression By Mirnamentioning

confidence: 99%

Exploiting microRNA regulation for genetic engineering

Gentner

Naldini

2012

Tissue Antigens

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RNA interference (RNAi) has been a landmark discovery in science. A typical application is to knock down the expression of endogenous genes by delivering small interfering RNA (siRNA) into cells triggering the degradation of complementary mRNA. However, RNAi can also be exploited the other way round: making use of the huge diversity of endogenous microRNAs (miRNA), the expression of exogenously introduced genes tagged with artificial miRNA target sequences can be negatively regulated according to the activity of a given miRNA which can be tissue-, lineage-, activation- or differentiation stage specific. This has significantly expanded the regulatory potential of gene transfer vectors and will benefit both basic science and therapeutic applications. This review briefly introduces the reader to the technical basis for exploiting miRNA regulation, followed by a discussion of specific applications for miRNA-regulated vectors/viruses in basic research, gene- and virotherapy.

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“…Flow cytometric analysis was carried out as described before [33]. In brief, cells were harvested and resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) with 2% FCS and 2 mM EDTA).…”

Section: Flow Cytometrymentioning

confidence: 99%

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

et al. 2013

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Methylation‐induced gene silencing represents a major obstacle to efficient transgene expression in pluripotent cells and thereof derived tissues. As ubiquitous chromatin opening elements (UCOE) have been shown to prevent transgene silencing in cell lines and primary hematopoietic cells, we hypothesized a similar activity in pluripotent cells. This concept was investigated in the context of cytidine deaminase (CDD) gene transfer, an approach to render hematopoietic cells resistant to the chemotherapeutic agent Ara‐C. When murine induced pluripotent stem cells (iPSC)/embryonic stem cells (ESCs) were transduced with self‐inactivating lentiviral vectors using housekeeping (truncated elongation factor 1α; EFS) or viral (spleen focus‐forming virus; SFFV) promoters, incorporation of an heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 locus‐derived UCOE (A2UCOE) significantly increased transgene expression and Ara‐C resistance and effectively prevented silencing of the SFFV‐promoter. The EFS promoter showed relatively stable transgene expression in naïve iPSCs, but rapid transgene silencing was observed upon hematopoietic differentiation. When combined with the A2UCOE, however, the EFS promoter yielded stable transgene expression in 73% ± 6% of CD41+ hematopoietic progeny, markedly increased CDD expression levels, and significantly enhanced Ara‐C resistance in clonogenic cells. Bisulfite sequencing revealed protection from differentiation‐induced promoter CpG methylation to be associated with these effects. Similar transgene promoting activities of the A2UCOE were observed during murine neurogenic differentiation, in naïve human pluripotent cells, and during nondirected multilineage differentiation of these cells. Thus, our data provide strong evidence that UCOEs can efficiently prevent transgene silencing in iPS/ESCs and their differentiated progeny and thereby introduce a generalized concept to circumvent differentiation‐induced transgene silencing during the generation of advanced iPSC/ESC‐based gene and cell therapy products. STEM CELLS2013;31:488–499

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

Cited by 17 publications

References 41 publications

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

Exploiting microRNA regulation for genetic engineering

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

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