Gene Specificity of Suppression of Transgene-Mediated Insertional Transcriptional Activation by the Chicken HS4 Insulator

Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation between an enhancer and a promoter. This has important implications for our theoretical understanding of the possible effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 decreased the level of expression of the globin transgene. Therefore, the benefits of partially preventing insertional gene activation are in part negated by the lower expression level of the transgene. A cost/benefit analysis of the utility of incorporation of insulators in gene therapy vectors will require further studies in which the effects of insulators on both the therapeutic gene and the flanking genes are determined at a large number of integration sites. Identification of insulators with minimal promoter specificity would also be of great value

Differentiation of human induced pluripotent stem cells towards notochordal-like cells: the role of tissue source

INTRODUCTION: Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and they are considered an exciting target for cell-based therapy. However, NCs are scarcely available as they are lost early in life, and attempts at in vivoexpansion have failed because NCs lose their specific phenotype. The production of Notochordal-like cells (NLCs) from human induced pluripotent stem cells (iPSCs) is a viable alternative. However, current attempts have been challenged by the low differentiation efficiency into the NC lineage. Therefore, the aim of this study was to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD progenitor cells (TIE2+-cells) to improve hiPSC differentiation towards mature, matrix-producing NLCs. METHODS: hiPSCs were generated from TIE2⁺ cells of three adult donors. As a comparison, donormatched minimally invasive peripheral blood mononuclear (PBM) cell-derived iPSCs were used. Firstly, the iPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)¹. Thereafter, the cells were further driven towards the NClineage by transfection with synthetic NOTO mRNA¹ and further matured using a 3D pellet culture in discogenic medium containing 10ng/mL TGF-β1. Read-out parameters included cell morphology, gene and protein expression and matrix deposition. RESULTS: Both TIE2⁺ and PBM cell-derived hiPSC showed successful differentiation towards mesendodermal progenitor cells following Wnt activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt-target genes (LEF1, NODAL) and mesendodermal markers (TBXT, FOXA2, TBX6) was observed compared to mTESR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. At day 3, we confirmed a 9-fold increase in NOTO mRNA levels after transfection in all donor lines. At day 28, the appearance of vacuolated NLCs was observed in both TIE2⁺ and PBM cell-derived pellet cultures confirming successful commitment towards the NC-lineage. Interestingly, while DMMB-assay detected GAG deposition in both lines, a significant increase in GAG content was seen in the TIE2⁺ cell-derived pellets. DISCUSSION & CONCLUSIONS: Tissue-specific TIE2⁺ cell-derived iPSCs may allow for an improved iPSNLC differentiation efficiency, indicated by the increased potency for deposition of GAG-rich matrix. Detailed analysis of the phenotypic markers and matrix deposited at the end of the 28 day maturation is ongoing to further document the phenotype of these iPS-NLCs. Delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the differences in their differentiation capacity. REFERENCES: ¹Colombier et al., 202

DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation

Production of Embryonic and Fetal-Like Red Blood Cells from Human Induced Pluripotent Stem Cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications

The Molecular Machinery of Somatic Cell Reprogramming

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primer used for RT-PCR.

<p>primer used for RT-PCR.</p

DNA segment inserted in the various cassettes used in this study.

<p>The cHS4 insulator used in this study contained 2 tandem duplicated copies of U78775.</p

The cHS4 insulator block activation of Map17 but not Tal1.

<p>A: PCR analysis demonstrating insertion of 5 cassettes at RL5 in each orientation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#s4" target="_blank">methods</a>). At least 2 clones in each orientation are shown. B and C: Schematic of the structure of RL5 loci in the presence of the various tested cassettes in each orientation, and histograms illustrating the average activation (±standard deviation) of the flanking genes (relative to the control cDNA cassette). D: FACS Analysis: Whisker plots of the mean linear fluorescence of 5 to 15 clones containing cassette 234-β-EGFP flanked on either sides or on both sides by the 2.4 kb cHS4 insulator. Presence of one insulator decreases EGFP expression by more than 2-fold. Presence of 2 insulators has an even more pronounced effect. E: Histograms illustrating a Q-PCR analysis of EGFP expression of the clones analyzed by FACS in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#pone-0005956-g003" target="_blank">Figure 3A</a>. The RT-PCR results are similar to the FACS results. EGFP expression was normalized to expression of the β-2-microglobulin gene. The effect of the insulator was independent of its location within the cassette and of the orientation of the cassette in the locus.</p

Insertion of hHS4, hHS5 and hG8 repeat do not block activation of Tal1 and Map17.

<p>A: PCR analysis demonstrating insertion of the various cassettes. B and C: Diagram illustrating the structure of the RL5 region after insertion of the various cassettes and histograms summarizing Q-RT-PCR determinations of the average fold increases (±standard deviation) of the flanking genes relative to the cDNA control cassette. The three cassettes tested had minimal effects on expression of Tal1 and Map17. The black bars represent the fold increase of the 234-β-EGFP cassette with and without cHS4 which was used as a control in this experiment. D and E: FACS and Q-RT-PCR analyses of EGFP expression (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#pone-0005956-g003" target="_blank">Figure 3D and 3E</a>) when cassettes 234-β-EGFP plus hHS4, hHS5 or G8 were inserted at RL5. Levels of expression in the presence of HS5, HS4 and G8 are respectively lower or higher than the controls both at the protein and mRNA levels.</p