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The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at short timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology at short timescales. It has been suggested that the cytoplasm of living cells can be described as a porous elastic meshwork bathed in an interstitial fluid. Microindentation tests now show that intracellular water redistribution plays a fundamental role in cellular rheology and that at physiologically relevant timescales cellular responses to mechanical stresses are consistent with such a poroelastic model.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy. In recent years, hematopoietic stem cells gene editing has emerged as a promising tool to treat blood disorders. Here the authors develop a CRISPR/Cas9-based genome editing strategy that allows the precise correction of Wiskott-Aldrich Syndrome in vitro and in vivo with high efficiency.

Mutations in genes encoding components of the immune system cause primary immunodeficiencies. Here, we study a patient with recurrent atypical mycobacterial infection and early-onset metastatic bladder carcinoma. Exome sequencing identified two homozygous missense germline mutations, P733L and P832S, in the JAK1 protein that mediates signalling from multiple cytokine receptors. Cells from this patient exhibit reduced JAK1 and STAT phosphorylation following cytokine stimulations, reduced induction of expression of interferon-regulated genes and dysregulated cytokine production; which are indicative of signalling defects in multiple immune response pathways including Interferon-γ production. Reconstitution experiments in the JAK1-deficient cells demonstrate that the impaired JAK1 function is mainly attributable to the effect of the P733L mutation. Further analyses of the mutant protein reveal a phosphorylation-independent role of JAK1 in signal transduction. These findings clarify JAK1 signalling mechanisms and demonstrate a critical function of JAK1 in protection against mycobacterial infection and possibly the immunological surveillance of cancer. JAK1 mediates intracellular signalling from multiple cytokine receptors. Here, Eletto et al . identify JAK1 mutations that disrupt multiple signalling pathways and are associated with primary immunodeficiency, atypical mycobacterial infection susceptibility and early-onset metastatic bladder carcinoma.

Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer–promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott–Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.

A form of Leber's congenital amaurosis is caused by mutant RPE65 , a critical component of the visual cycle. Two early clinical trials to assess subretinal injection of a viral vector containing RPE65 in young adults with advanced retinal degeneration show that this approach is generally safe in the short term, although one group reported an adverse event: macular hole. The authors observed improvement in some measures of visual function. Two early clinical trials to assess subretinal injection of a viral vector containing RPE65 in young adults with advanced retinal degeneration show that this approach is generally safe in the short term, although one group reported an adverse event: macular hole. The authors observed improvement in some measures of visual function. Leber's congenital amaurosis is a term used to describe a group of recessively inherited, severe, infantile-onset rod–cone dystrophies. 1 Mutation of one of several genes, including RPE65 , causes disease that involves impaired vision from birth 2 , 3 and typically progresses to blindness in the third decade of life. There is no effective treatment. RPE65 is expressed in the retinal pigment epithelium and encodes a 65-kD protein that is a key component of the visual cycle, 1 , 4 – 8 a biochemical pathway that regenerates the visual pigment after exposure to light. 9 – 14 A lack of functional RPE65 results in deficiency of 11- cis . . .

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR–Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.Haematopoietic stem and progenitor cell (HSPC) gene therapy using lentiviral or gammaretroviral vectors has now been approved for clinical use. In this Review, Ferrari, Thrasher and Aiuti discuss the history of HSPC gene therapy, the clinical promise of gene-editing HPSCs and the use of HSPC gene therapy to treat specific diseases.

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

Gene editing technologies show great promise for application to human disease as a result of rapid developments in targeting tools notably based on ZFN, TALEN, and CRISPR-Cas systems. Precise modification of a DNA sequence is now possible in mature human somatic cells including stem and progenitor cells with increasing degrees of efficiency. At the same time new technologies are required to evaluate their safety and genotoxicity before widespread clinical application can be confidently implemented. A number of methodologies have now been developed in an attempt to predict expected and unexpected modifications occurring during gene editing. This review surveys the techniques currently available as state of the art, highlighting benefits and limitations, and discusses approaches that may achieve sufficient accuracy and predictability for application in clinical settings.

The potential of gene therapy as a curative treatment for monogenetic disorders has been clearly demonstrated in a series of recent Phase I/II clinical trials. Among primary immunodeficiencies, gene transfer into hematopoietic stem (HSC)/progenitor cells has resulted in the long-term correction of immune and metabolic defects in treated patients. In most cases, successes were augmented by a recognized biological selection for successfully treated cells in vivo, perhaps even to some extent at the HSC level. In contrast, similar achievements have not turned into reality for immunodeficiencies in which gene-transduced cells lack selective advantages in vivo. This is the case for chronic granulomatous disease (CGD), a primary immunodeficiency, characterized by deficient antimicrobial activity in phagocytic cells. Several attempts to correct CGD by gene transfer in combination with bone marrow conditioning have resulted in low-level long-term engraftment and transient clinical benefits despite high levels of gene marking and high numbers of reinfused cells. This review summarizes the data from clinical trials for CGD and provides some insights into treatment options that may lead to a successful application of gene therapy for CGD.

Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10 6 transducing units/ml can be harvested from the final producer clones, which can be increased to 10 8  TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.