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Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.

ABSTRACT: Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 "safe harbor" locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line and faithfully recapitulates endogenous CD43 expression. In addition, using the same gene targeting strategy we demonstrate that constitutive expression of short hairpin RNAs within a microRNA backbone can suppress expression of PU.1, an important regulator of myeloid cell development. We show that PU.1 knockdown cell lines display an inhibition in myeloid cell formation and skewing towards erythroid development. Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown.

SUBMITTER: Tiyaboonchai A

PROVIDER: S-EPMC4048956 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.

Tiyaboonchai Amita A Mac Helen H Shamsedeen Razveen R Mills Jason A JA Kishore Siddarth S French Deborah L DL Gadue Paul P

Stem cell research 20140221 3

Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 "safe harbor" locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line ...[more]

PMID: 24631742

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Project description:Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral long terminal repeat (LTR)-driven vectors, which semirandomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSCs) and can impede transgene expression during differentiation. In this study, we aimed to correct p47phox deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47phox transgene into the AAVS1 locus of human induced PSCs (iPSCs) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes, and myeloid-related protein 8 [MRP8]). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoters achieved therapeutically relevant levels of p47phox expression and nicotinamide adenine dinucleotide phosphate oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells.

Project description:Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca 2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

Dataset Information

Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.

Publications

Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.

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