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Presented is an experimental analysis of the stability of transgene expression, the perturbation of endogenous expression and the perturbation of epigenetic organization upon site-directed delivery of transgenes to the CCR5 and AAVS1 loci in human cells. It provides guidelines for optimal cassette design for stable and nonperturbative gene transfer. Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13–39%) of editing at the IL-2 receptor common γ-chain gene ( IL2RG ) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.

Impairments of inhibitory circuits are at the basis of most, if not all, cognitive deficits. The impact of OPHN1, a gene associate with intellectual disability (ID), on inhibitory neurons remains elusive. We addressed this issue by analyzing the postnatal migration of inhibitory interneurons derived from the subventricular zone in a validated mouse model of ID (OPHN1−/y mice). We found that the speed and directionality of migrating neuroblasts were deeply perturbed in OPHN1−/y mice. The significant reduction in speed was due to altered chloride (Cl⁻) homeostasis, while the overactivation of the OPHN1 downstream signaling pathway, RhoA kinase (ROCK), caused abnormalities in the directionality of the neuroblast progression in mutants. Blocking the cation–Cl⁻ cotransporter KCC2 almost completely rescued the migration speed while proper directionality was restored upon ROCK inhibition. Our data unveil a strong impact of OPHN1 on GABAergic inhibitory interneurons and identify putative targets for successful therapeutic approaches.

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large‐scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell‐derived polymorphic class‐I major histocompatibility complexes (MHC‐I) are incorporated into the LV surface and trigger allogeneic T‐cell responses. By disrupting the beta‐2 microglobulin gene in producer cells, we obtained MHC‐free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single‐copy inducible vector components, which can be reproducibly converted into high‐yield LV producers upon site‐specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement‐mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen‐free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy. Synopsis Lentiviral vectors (LV) lacking major histocompatibility complexes (MHC) and with low vesicular stomatitis virus glycoprotein G (VSV.G) content escape immune recognition by human T cells and complement. Cell line‐packaged LV offer scalable and consistent LV production for in vivo gene therapy. Site‐specific integration of the LV genome into an inducible packaging cell line allows consistent generation of high‐yield producers of the LV of interest. Cell line‐produced LV achieve equivalent gene transfer in the mouse liver as LV produced by conventional transfection but are more stable in human sera, due to a lower VSV.G content. Genetic inactivation of beta‐2 microglobulin (B2M) in LV producer cells allows production of MHC‐free LV with preserved infectivity, devoid of immunogenic alloantigens in human recipients. Graphical Abstract Lentiviral vectors (LV) lacking major histocompatibility complexes (MHC) and with low vesicular stomatitis virus glycoprotein G (VSV.G) content escape immune recognition by human T cells and complement. Cell line packaged LV offer scalable and consistent LV production for in vivo gene therapy.

Galectin-3 (Gal-3) is an extracellular matrix glycan-binding protein with several immunosuppressive and pro-tumor functions. The role of Galectin-3 in cancer stem-like cells (CSCs) is poorly investigated. Here, we show that prostate CSCs also colonizing prostate-draining lymph nodes of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice overexpress Gal-3. Gal-3 contributes to prostate CSC-mediated immune suppression because either Gal-3 silencing in CSCs, or co-culture of CSCs and T cells in the presence of the Gal-3 inhibitor N-Acetyl-D-lactosamine rescued T cell proliferation. N-Acetyl-D-lactosamine also rescued the proliferation of T cells in prostate-draining lymph nodes of TRAMP mice affected by prostate intraepithelial neoplasia. Additionally, Gal-3 impacted prostate CSC tumorigenic and metastatic potential , as Gal-3 silencing in prostate CSCs reduced both primary tumor growth and secondary invasion. Gal-3 was also found expressed in more differentiated prostate cancer cells, but with different intracellular distribution as compared to CSCs, which suggests different functions of Gal-3 in the two cell populations. In fact, the prevalent nuclear and cytoplasmic distribution of Gal-3 in prostate CSCs made them less susceptible to apoptosis, when compared to more differentiated prostate cancer cells, in which Gal-3 was predominantly intra-cytoplasmic. Finally, we found Gal-3 expressed in human and mouse prostate intraepithelial neoplasia lesions and in metastatic lymph nodes. All together, these findings identify Gal-3 as a key molecule and a potential therapeutic target already in the early phases of prostate cancer progression and metastasis.

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)‐mediated insertion of a non‐therapeutic EGFP‐reporter donor in the AAVS1 “safe harbor” locus of FA‐A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA‐A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA‐A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. Synopsis Zinc finger nuclease‐mediated gene editing facilitates insertion of a therapeutic gene in the AAVS1 “safe harbor” locus in human hematopoietic cell lines and primary HSPCs from healthy donors and patients with Fanconi anemia‐A, leading to their phenotypic correction. FANCA is not essential for zinc finger nuclease (ZFN)‐mediated gene editing in human hematopoietic cells. ZFN‐mediated gene editing facilitates the targeting of FANCA in a “safe harbor” locus of lymphoblastic cell lines (LCLs) from Fanconi anemia A (FA‐A) patients. ZFN‐mediated gene editing facilitates the targeting of donor constructs in long term hematopoietic stem and progenitor cells (HSPCs) from healthy donors. Gene targeting of therapeutic constructs in HSPCs from FA‐A patients is thus feasible and mediates the phenotypic correction of these cells. Graphical Abstract Zinc finger nuclease‐mediated gene editing facilitates insertion of a therapeutic gene in the AAVS1 “safe harbor” locus in human hematopoietic cell lines and primary HSPCs from healthy donors and patients with Fanconi anemia‐A, leading to their phenotypic correction.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T‐cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene‐modified postnatal murine TECs into three‐dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline‐induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4‐expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107–1122 Transient Oct4 expression promoted thymic epithelial cells expansion without drastically changing the cell lineage identity of adult thymic epithelial cells. iOCT4 thymic epithelial cells were able to grow into three‐dimensional collagen type I scaffolds both in vitro and in vivo demonstrating that the collagen structure reproduced a three‐dimensional environment similar to the thymic extracellular matrix, perfectly recognized by thymic epithelial cells. in vivo results showed that thymic organoids transplanted subcutaneously in athymis nude mice were vascularized but failed to support thymopoiesis because of the limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of three‐dimensional biomimetic scaffolds, represents a novel approach allowing the use of postnatal thymic epithelial cells for thymic regeneration.

Background: Pseudomonas aeruginosa infection is a major driver of morbidity and mortality in cystic fibrosis (CF), yet disease severity varies widely among people with CF (pwCF). This clinical heterogeneity suggests the involvement of host genetic modifiers beyond CFTR. We previously identified sphingosine 1-phosphate receptor 1 (S1PR1) as a candidate gene associated with susceptibility to P. aeruginosa. Here, we investigated its role in modulating airway epithelial responses to infection. Methods: Using CRISPR/Cas9, we generated S1PR1-knockout bronchial epithelial cells with (IB3-1) and without (C38) CFTR mutations. We assessed cell viability, cytotoxicity, and interleukin-8 secretion following exposure to P. aeruginosa exoproducts. S1PR1 protein expression was evaluated in lung tissue from pwCF and non-CF individuals using immunohistochemistry. Results: S1PR1-mutant cells produced truncated, non-functional peptides. In CFTR-mutant cells, S1PR1 loss reduced viability, increased cytotoxicity, and significantly enhanced interleukin-8 production in response to P. aeruginosa exoproducts. These effects were not observed in CFTR-competent cells. Notably, S1PR1 protein levels were markedly lower in lung tissue from pwCF compared to non-CF individuals. Conclusions: S1PR1 deficiency exacerbates epithelial damage and inflammatory responses to P. aeruginosa in CF models. These findings highlight S1PR1 as a potential contributor to infection severity and a promising target for therapeutic strategies in pwCF.

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology‐directed DNA repair. In this study, we used zinc finger nucleases and integrase‐defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA‐A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene‐edited FA fibroblasts were then reprogrammed and re‐differentiated toward the hematopoietic lineage. Analyses of gene‐edited FA‐iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease‐free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene‐targeting and cell reprogramming strategies. Synopsis This study shows for the first time the possibility of performing targeted gene therapy in a DNA‐repair deficiency syndrome, known as Fanconi anemia. By reprogramming targeted cells, asymptomatic gene‐edited iPSCs and hematopoietic progenitor cells are generated. Fanconi anemia cells of the FA‐A subtype have been efficiently targeted with zinc finger nucleases and a donor construct harboring the therapeutic FANCA gene. The specific insertion of FANCA in the AAVS1 “safe harbor locus” of FA‐A fibroblasts efficiently corrected the genetic instability characteristic of these cells. The reprogramming and re‐differentiation of gene‐edited FA‐A fibroblasts generated disease‐free hematopoietic progenitors. Graphical Abstract This study shows for the first time the possibility of performing targeted gene therapy in a z‐repair deficiency syndrome, known as Fanconi anemia. By reprogramming targeted cells, asymptomatic gene‐edited iPSCs and hematopoietic progenitor cells are generated.