Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown ...impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly ...characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs. However, functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs.
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•DNA DSBs induced by programmable nucleases transiently activate the DDR in HSPCs•Single-cell transcriptomics show that induced DSBs activate the p53 pathway•AAV6-mediated genome editing aggravates p53 activation and delays HSPC proliferation•Transient p53 inhibition alleviates clonogenic and repopulation defects in edited HSPCs
Precise gene editing has the potential to treat immune and hematological diseases. Genovese, Naldini, Di Micco, and colleagues now show that gene-editing procedures are well tolerated by hematopoietic stem cells and provide molecular evidence of the feasibility of seamless gene editing, strengthening translation of such approaches to humans.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Undoubtedly, a better understanding and the further development of approaches for damage tolerant component design of AM parts are among the most significant challenges currently facing the use of ...these new technologies.
This article presents a thorough overview of the discussion at an international workshop on the topic. It aims to provide a review of the parameters affecting the damage tolerance of parts produced by additive manufacturing (shortly, AM parts) with special emphasis on the process parameters intrinsic to the AM technologies, the resulting defects and the residual stresses. Based on these aspects, basic concepts are reviewed and critically discussed specifically for AM materials:-Criteria for damage tolerant component design;-Criteria for the determination of fatigue and fracture properties;-Strategies for the determination of the fatigue life in dependence of different manufacturing conditions;-Methods for the quantitative characterization of microstructure and defects;-Methods for the determination of residual stresses;-Effect of the defects and the residual stresses on the fatigue life and behaviour.We see that many of the classic concepts need to be expanded in order to fit with the particular microstructure (grain size and shape, crystal texture) and defect distribution (spatial arrangement, size, shape, amount) present in AM (in particular laser powder bed fusion). For instance, 3D characterization of defects becomes essential, since the defect shapes in AM are diverse and impact the fatigue life in a different way than in the case of conventionally produced components. Such new concepts have immediate consequence on the way one should tackle the determination of the fatigue life of AM parts; for instance, since a classification of defects and a quantification of the tolerable shapes and sizes is still missing, a new strategy must be defined, whereby theoretical calculations (e.g. finite element modeling) allow determining the maximum tolerable defect size, and non-destructive testing (NDT) techniques are required to detect whether such defects are indeed present in the component. Such examples show how component design, damage and failure criteria, and characterization (and/or NDT) become for AM parts fully interlinked. We conclude that the homogenization of these fields represents the current challenge for the engineer and the materials scientist.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ABSTRACT
Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid ...cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (
BRAF
V600E
). All mice transplanted with
BRAF
V600E
-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms.
Space and aerospace industries has been starting in the recent years the replacement process of parts and components obtained by traditional manufacturing processes with those produced by Additive ...Manufacturing (AM). The complexity of the obtainable parts makes, in general, challenging the superficial post processing of some zones, making a stringent requirement the investigation of the fatigue performances of components with rough superficial state or machined. The aim of this work is then to analyse and compare the fatigue performances of an additively manufactured (AMed) AlSi10Mg material considering both the effects of the manufacturing defects and residual stresses related to three different superficial states, namely machined, net-shape and sandblasted. The residual stress profiles of the three superficial states were found to play a key role in determining the fatigue properties of the analysed material, while the manufacturing defects at the failure origin were found to be comparable among the three series. To take into account the combined effect of residual stresses and manufacturing defects a fracture mechanics approach was considered for the estimation of the fatigue performances in both infinite and finite life regimes. It was found that by considering the nominal measured residual stress profiles in the fracture mechanics model the estimations were satisfactory compared to the experimental data-point. To increase the accuracy of the fatigue life estimations a series of numerical analyses were performed aimed to investigate the residual stresses relaxation during the cyclic loading. The adoption of the relaxed residual stress profiles in the fracture mechanics model resulted in good estimations respect to the experimental data-points, highlighting the necessity in adopting such developed approaches during the design phase of AM parts and components.
Gene editing by engineered nucleases has revolutionized the field of gene therapy by enabling targeted and precise modification of the genome. However, the limited availability of methods for clonal ...tracking of edited cells has resulted in a paucity of information on the diversity, abundance and behavior of engineered clones. Here we detail the wet laboratory and bioinformatic BAR-Seq pipeline, a strategy for clonal tracking of cells harboring homology-directed targeted integration of a barcoding cassette. We present the BAR-Seq web application, an online, freely available and easy-to-use software that allows performing clonal tracking analyses on raw sequencing data without any computational resources or advanced bioinformatic skills. BAR-Seq can be applied to most editing strategies, and we describe its use to investigate the clonal dynamics of human edited hematopoietic stem/progenitor cells in xenotransplanted hosts. Notably, BAR-Seq may be applied in both basic and translational research contexts to investigate the biology of edited cells and stringently compare editing protocols at a clonal level. Our BAR-Seq pipeline allows library preparation and validation in a few days and clonal analyses of edited cell populations in 1 week.
Bioinformatics tools designed to identify lentiviral or retroviral vector insertion sites in the genome of host cells are used to address the safety and long-term efficacy of hematopoietic stem cell ...gene therapy applications and to study the clonal dynamics of hematopoietic reconstitution. The increasing number of gene therapy clinical trials combined with the increasing amount of Next Generation Sequencing data, aimed at identifying integration sites, require both highly accurate and efficient computational software able to correctly process "big data" in a reasonable computational time.
Here we present VISPA2 (Vector Integration Site Parallel Analysis, version 2), the latest optimized computational pipeline for integration site identification and analysis with the following features: (1) the sequence analysis for the integration site processing is fully compliant with paired-end reads and includes a sequence quality filter before and after the alignment on the target genome; (2) an heuristic algorithm to reduce false positive integration sites at nucleotide level to reduce the impact of Polymerase Chain Reaction or trimming/alignment artifacts; (3) a classification and annotation module for integration sites; (4) a user friendly web interface as researcher front-end to perform integration site analyses without computational skills; (5) the time speedup of all steps through parallelization (Hadoop free).
We tested VISPA2 performances using simulated and real datasets of lentiviral vector integration sites, previously obtained from patients enrolled in a hematopoietic stem cell gene therapy clinical trial and compared the results with other preexisting tools for integration site analysis. On the computational side, VISPA2 showed a > 6-fold speedup and improved precision and recall metrics (1 and 0.97 respectively) compared to previously developed computational pipelines. These performances indicate that VISPA2 is a fast, reliable and user-friendly tool for integration site analysis, which allows gene therapy integration data to be handled in a cost and time effective fashion. Moreover, the web access of VISPA2 ( http://openserver.itb.cnr.it/vispa/ ) ensures accessibility and ease of usage to researches of a complex analytical tool. We released the source code of VISPA2 in a public repository ( https://bitbucket.org/andreacalabria/vispa2 ).
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
While the reconstruction of transcripts from a sample of RNA-Seq data is a computationally expensive and complicated task, the detection of splicing events from RNA-Seq data and a gene annotation is ...computationally feasible. This latter task, which is adequate for many transcriptome analyses, is usually achieved by aligning the reads to a reference genome, followed by comparing the alignments with a gene annotation, often implicitly represented by a graph: the splicing graph.
We present ASGAL (Alternative Splicing Graph ALigner): a tool for mapping RNA-Seq data to the splicing graph, with the specific goal of detecting novel splicing events, involving either annotated or unannotated splice sites. ASGAL takes as input the annotated transcripts of a gene and a RNA-Seq sample, and computes (1) the spliced alignments of each read in input, and (2) a list of novel events with respect to the gene annotation.
An experimental analysis shows that ASGAL allows to enrich the annotation with novel alternative splicing events even when genes in an experiment express at most one isoform. Compared with other tools which use the spliced alignment of reads against a reference genome for differential analysis, ASGAL better predicts events that use splice sites which are novel with respect to a splicing graph, showing a higher accuracy. To the best of our knowledge, ASGAL is the first tool that detects novel alternative splicing events by directly aligning reads to a splicing graph.
Source code, documentation, and data are available for download at http://asgal.algolab.eu .
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Alloys used for turbine blades have to safely sustain severe thermomechanical loadings during service such as, for example, centrifugal loadings, creep and high temperature gradients. For these ...applications, cast Ni-based superalloys characterized by a coarse-grained microstructure are widely adopted. This microstructure dictates a strong anisotropic mechanical behaviour and, concurrently, a large scatter in the fatigue properties is observed. In this work, Crystal Plasticity Finite Element (CPFE) simulations and strain measurements performed by means of Digital Image Correlations (DIC) were adopted to study the variability introduced by the coarse-grained microstructure. In particular, the CPFE simulations were calibrated and used to simulate the effect of the grain cluster orientations in proximity to notches, which reproduce the cooling air ducts of the turbine blades. The numerical simulations were experimentally validated by the DIC measurements. This study aims to predict the statistical variability of the strain concentration factors and support component design.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these ...insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy.HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.
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