TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity

Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications

Targeted genome editing restores T cell differentiation in a humanized X-SCID pluripotent stem cell disease model

Abstract The generation of T cells from pluripotent stem cells (PSCs) is attractive for investigating T cell development and validating genome editing strategies in vitro. X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the IL2RG gene and characterised by the absence of T and NK cells in patients. IL2RG encodes the common gamma chain, which is part of several interleukin receptors, including IL-2 and IL-7 receptors. To model X-SCID in vitro, we generated a mouse embryonic stem cell (ESC) line in which a disease-causing human IL2RG gene variant replaces the endogenous Il2rg locus. We developed a stage-specific T cell differentiation protocol to validate genetic correction of the common G691A mutation with transcription activator-like effector nucleases. While all ESC clones could be differentiated to hematopoietic precursor cells, stage-specific analysis of T cell maturation confirmed early arrest of T cell differentiation at the T cell progenitor stage in X-SCID cells. In contrast, genetically corrected ESCs differentiated to CD4 + or CD8 + single-positive T cells, confirming correction of the cellular X-SCID phenotype. This study emphasises the value of PSCs for disease modelling and underlines the significance of in vitro models as tools to validate genome editing strategies before clinical application

<i>In vitro</i> differentiation of iPSCs to proT-cells and T-cells.

<p>(A) Schematic of <i>in vitro</i> T-cell differentiation from iPSCs. Differentiation of iPSCs starts with formation of embryoid bodies that are dissociated to give rise to hematopoietic stem and progenitor cells (HPC). DL-1 mediated Notch signaling coaxes HPC development towards early proT-cells (DN2), which undergo DNA-PK dependent V(D)J recombination. After passing through DN3 and DN4 stages, preT-cells mature into double-positive (DP) T-cells that express the beta chain of the T-cell receptor (TCRß). Dashed lines indicate to what stage iPSC clones are expected to differentiate. (B) Assessment of T-cell differentiation. <i>In vitro</i> T-cell differentiation was analyzed by flow cytometry after two weeks of co-cultivation on OP9-DL1. Gating (indicated on top of each column) was applied in the following order: FSC/SSC and CD45⁺/DAPI^—to assess CD4/CD8 expression; CD8^–/CD4^—to gate for DN1-DN4 stage cells; CD8^–/CD4^– (DN) or CD8⁺/CD4⁺ (DP) to assess TCRß expression. Numbers indicate percentage of cells in each quadrant. HPC, lineage-negative bone marrow cells; iPS.WTX, wild-type iPSC; iPS.S6X, SCID iPSC clone; iPS.T25, gene targeted SCID iPSC clone.</p

Polyclonal T-cell receptor recombination.

<p><i>In vitro</i> generated T-cells were analyzed by spectratyping, i.e. quantitative RT-PCR expression analysis of the variable beta chains. Exemplarily shown are results for Vß1, Vß8.3 and Vß10 (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005239#pgen.1005239.s008" target="_blank">S5 Fig</a>). X-axis depicts detected PCR fragment size in bp, Y-axis depicts counts of obtained PCR fragments. Thymus, T-cells isolated from thymus as a positive control; HSC, <i>in vitro</i> generated T-cells from lineage-negative bone marrow cells; iPS.WTX, wild-type iPSCs; iPS.S6X, SCID iPSC clone; iPS.T25X, gene targeted SCID iPSC clone.</p

Evaluation of pluripotency of generated iPSCs.

<p>(<b>A</b>) Pluripotent stem cell marker gene expression. Oct3/4, Nanog, Zfp42, Esg1, Eras, and fgf4 mRNA expression was determined by qualitative RT-PCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005239#pgen.1005239.s002" target="_blank">S2 Table</a>). Housekeeping gene 36B4 (+/- reverse transcriptase) were included as controls. ES.CCE, murine embryonic stem cell line; Fib.S, SCID fibroblasts; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, gene targeted SCID iPSC clones; iPS.WT, wild-type iPSC clone. (<b>B</b>) <i>In vivo</i> differentiation analysis. Teratoma formation was induced by subcutaneous injection of iPSCs into mice. <b>Hematoxylin/eosin</b>-stained sections of teratoma-derived from clones iPS.S6 and iPS.T25 are shown. (<b>C</b>) Karyotype analysis. Spectral karyotyping (SKY) was performed to detect microscopic genomic abnormalities, translocations and aneuploidies in untreated or genetically corrected SCID iPSC clones. SKY analysis of clone iPSC.T25 is shown (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005239#pgen.1005239.s005" target="_blank">S2 Fig</a>).</p

Targeted genome editing in SCID-derived iPSCs.

<p>(<b>A</b>) Verification of gene targeting. Inside-out PCR strategies (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005239#pgen.1005239.g001" target="_blank">Fig 1A</a>) were used to verify correct 5´ (J5) and 3´-junctions (J3) of the integrated donor. Allelic discrimination (AD) PCR was used to assess mono- vs. bi-allelic integration. Targeted allele runs at 2.99 kb. Sizes of all expected PCR amplicons are indicated on the right. iPS.WT, wild-type iPSC; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, iPS.T45 and iPS.T60, targeted SCID iPSC clones. (<b>B</b>) Expression of corrected <i>prkdc</i> mRNA. Successful splicing from exon 83 to cDNA encompassing exons 84/85 was detected by an inside-out RT-PCR strategy using primers RT-F/RT-R.</p

Targeted genome editing in RS-SCID fibroblasts.

<p>(<b>A</b>) Schematic of genome editing strategy. Homology-directed repair (HDR) between the <i>prkdc</i> locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding <i>prkdc</i> exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (<i>NeoR</i>). The SCID underlying mutation in exon 85 (mut*), and primer binding sites for PCR analysis (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/RT-R) are indicated. (<b>B</b>) Genome editing. After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (<b>C</b>) Expression of corrected <i>prkdc</i> mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with an inside-out RT-PCR strategy using primers RT-F/RT-R.</p

Biodegradable nanocarriers resembling extracellular vesicles deliver genetic material with the highest efficiency to various cell types

Abstract Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co‐transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10–4 pmol of siRNA, and 1 × 10–3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo