Highly efficient CRISPR/Cas9-mediated exon skipping for recessive dystrophic epidermolysis bullosa.

Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient-specific skin equivalents. Precise gene editing using homology-directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress.

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells.

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing.

Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells.

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves.

Epidermolysis Bullosa: A Review of the Tissue-Engineered Skin Substitutes Used to Treat Wounds.

Skin wound healing is a crucial process for regenerating healthy skin and avoiding the undesired consequences associated with open skin wounds. For epidermolysis bullosa (EB), a debilitating group of fragile skin disorders currently without a cure, skin blistering can often be severe and heal poorly, increasing susceptibility to life-threatening complications. To prevent these, investigational therapies have been exploring the use of tissue-engineered skin substitutes (TESSs) aimed at replacing damaged skin and promoting long-term wound closure.

Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium.

Background Aims: The ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing.

Results: We have identified a novel culture medium for the rapid growth of keratinocytes from human skin. "Kelch's medium" supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses.

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity.

The Chondrogenic Potential of First-Trimester and Term Placental Mesenchymal Stem/Stromal Cells.

Objectives: Mesenchymal stem/stromal cells (MSCs) are a well-established cell source for cartilage engineering, but challenges remain as differentiation often results in chondrocyte hypertrophy. Chondrogenic potential also varies with MSC source and donor age. We assessed the chondrogenic potential of first-trimester and term placental MSCs and compared their response to commonly used bone marrow MSCs (BM-MSCs).

Live-Cell Microscopy Reveals That Human T Cells Primarily Respond Chemokinetically Within a CCL19 Gradient That Induces Chemotaxis in Dendritic Cells.

The ability to study migratory behavior of immune cells is crucial to understanding the dynamic control of the immune system. Migration induced by chemokines is often assumed to be directional (chemotaxis), yet commonly used end-point migration assays are confounded by detecting increased cell migration that lacks directionality (chemokinesis). To distinguish between chemotaxis and chemokinesis we used the classic "under-agarose assay" in combination with video-microscopy to monitor migration of CCR7+ human monocyte-derived dendritic cells and T cells in response to a concentration gradient of CCL19.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood.

Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes.

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range of inflammatory and degenerative conditions. These lipoaspirate-derived cells are part of the abundant and accessible source of heterogeneous stromal vascular fraction (SVF). They are typically isolated and expanded from the SVF adherent cell culture for at least 2 weeks and as such represent a relatively undefined population of cells.

microRNAs in Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Undergo Rapid Culture-Induced Changes in Expression, Including miR-378 which Promotes Adipogenesis.

There is clinical interest in using human adipose tissue-derived mesenchymal stromal cells (ASC) to treat a range of inflammatory and regenerative conditions. Aspects of ASC biology, including their regenerative potential and paracrine effect, are likely to be modulated, in part, by microRNAs, small RNA molecules that are embedded as regulators of gene-expression in most biological pathways. However, the effect of standard isolation and expansion protocols on microRNA expression in ASC is not well explored.

Addendum: Precise tuning of gene expression levels in mammalian cells.

Following re-sequencing of the miSFIT constructs used in the paper, two of the construct variants inserted into the 3'UTR of PD-1, namely '12C' and '17A, 18G', have been found to contain additional insertions not present in the other construct variants. The data points corresponding to these constructs in Figs. 2c, f and Supplementary Fig.

Precise tuning of gene expression levels in mammalian cells.

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs).

AHNAK is downregulated in melanoma, predicts poor outcome, and may be required for the expression of functional cadherin-1.

The aim of this study was to further our understanding of the transformation process by identifying differentially expressed proteins in melanocytes compared with melanoma cell lines. Tandem mass spectrometry incorporating iTRAQ reagents was used as a screen to identify and comparatively quantify the expression of proteins in membrane-enriched samples isolated from primary human melanocytes or three melanoma cells lines. Real-time PCR was used to validate significant hits.

MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.

Background: microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation.

Recombinant adeno-associated virus serotype 6 efficiently transduces primary human melanocytes.

The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration.

Phosphorylation on Thr-55 by TAF1 mediates degradation of p53: a role for TAF1 in cell G1 progression.

The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, we investigated the link between TAF1 and p53. We found that TAF1 induces G1 progression in a p53-dependent manner.

Unsymmetrical DNA cross-linking agents: combination of the CBI and PBD pharmacophores.

A set of 10 compounds, each combining the seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (seco-CBI) and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores, was designed and prepared. These compounds were anticipated to cross-link between N3 of adenine and N2 of guanine in the minor groove of DNA. The compounds, which differ in the chain length separating the two alkylation subunits, and the configuration of the CBI portion, showed great variation in cellular toxicity (over 4 orders of magnitude in a cell line panel) with the most potent example exhibiting IC50s in the pM range.