Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery

Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells

The Use of Artificial Intelligence for the Classification of Craniofacial Deformities

Positional cranial deformities are a common finding in toddlers, yet differentiation from craniosynostosis can be challenging. The aim of this study was to train convolutional neural networks (CNNs) to classify craniofacial deformities based on 2D images generated using photogrammetry as a radiation-free imaging technique. A total of 487 patients with photogrammetry scans were included in this retrospective cohort study: children with craniosynostosis (n = 227), positional deformities (n = 206), and healthy children (n = 54). Three two-dimensional images were extracted from each photogrammetry scan. The datasets were divided into training, validation, and test sets. During the training, fine-tuned ResNet-152s were utilized. The performance was quantified using tenfold cross-validation. For the detection of craniosynostosis, sensitivity was at 0.94 with a specificity of 0.85. Regarding the differentiation of the five existing classes (trigonocephaly, scaphocephaly, positional plagiocephaly left, positional plagiocephaly right, and healthy), sensitivity ranged from 0.45 (positional plagiocephaly left) to 0.95 (scaphocephaly) and specificity ranged from 0.87 (positional plagiocephaly right) to 0.97 (scaphocephaly). We present a CNN-based approach to classify craniofacial deformities on two-dimensional images with promising results. A larger dataset would be required to identify rarer forms of craniosynostosis as well. The chosen 2D approach enables future applications for digital cameras or smartphones

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

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

Energietransfer und Relaxation an N2-Aggregaten in Edelgasmatrizen

SIGLECopy held by FIZ Karlsruhe; available from UB/TIB Hannover / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman