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The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the ''Genome Editing to treat Human Diseases'' (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi’s primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing’s potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Fanconi Anemia (FA) is caused by deficiencies in DNA repair, making it hard to correct. Here the authors report a therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells.

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34+ cells/μL required to initiate apheresis. A median of 21.8 CD34+ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34+ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34+ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34+ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34+ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.

Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage–associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D–NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D–NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.

Abstract: Hematopoietic stem cell (HSC)–based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein–displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)–LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc−/− (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.

Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41+, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

Abstract 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.

Despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use Illumina to sequence small (~400 bp) hypervariable regions of the 16S rRNA SSU for phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With Nanopore sequencing now providing raw simplex reads with quality scores above Q20 using the kit 12 chemistry, the ease, cost, and portability of Nanopore play a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate the sequencing of bacterial rrn operons and to support import into phyloseq. Benchmarking RESCUE showed that fully processed reads are now parallel or exceed the quality of Sanger, with median quality scores of approximately Q20+, using the R10.4 and Guppy SUP basecalling. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual Illumina data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using short reads such as Illumina.

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene, which encodes for the CD18 subunit of β2-integrins. Deficient expression of β2-integrins results in impaired neutrophil migration in response to bacterial and fungal infections. Using a lentiviral vector (LV) that mediates a preferential myeloid expression of human CD18 (Chim.hCD18-LV), we first demonstrated that gene therapy efficiently corrected the phenotype of mice with severe LAD-I. Next, we investigated if the ectopic hCD18 expression modified the phenotypic characteristics of human healthy donor hematopoietic stem cells and their progeny. Significantly, transduction of healthy CD34+ cells with the Chim.hCD18-LV did not modify the membrane expression of CD18 nor the adhesion of physiological ligands to transduced cells. Additionally, we observed that the repopulating properties of healthy CD34+ cells were preserved following transduction with the Chim.hCD18-LV, and that a safe polyclonal repopulation pattern was observed in transplanted immunodeficient NOD scid gamma (NSG) mice. In a final set of experiments, we demonstrated that transduction of CD34+ cells from a severe LAD-I patient with the Chim.hCD18-LV restores the expression of β2-integrins in these cells. These results offer additional preclinical safety and efficacy evidence supporting the gene therapy of patients with severe LAD-I.

Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-β and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.

Abstract CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA− T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

Despite the impressive results of autologous CAR-T cell therapy in refractory B lymphoproliferative diseases, CAR-NK immunotherapy emerges as a safer, faster, and cost-effective approach with no signs of severe toxicities as described for CAR-T cells. Permanently scrutinized for its efficacy, recent promising data in CAR-NK clinical trials point out the achievement of deep, high-quality responses, thus confirming its potential clinical use. Although CAR-NK cell therapy is not significantly affected by the loss or downregulation of its CAR tumor target, as in the case of CAR-T cell, a plethora of common additional tumor intrinsic or extrinsic mechanisms that could also disable NK cell function have been described. Therefore, considering lessons learned from CAR-T cell therapy, the emergence of CAR-NK cell therapy resistance can also be envisioned. In this review we highlight the processes that could be involved in its development, focusing on cytokine addiction and potential fratricide during manufacturing, poor tumor trafficking, exhaustion within the tumor microenvironment (TME), and NK cell short in vivo persistence on account of the limited expansion, replicative senescence, and rejection by patient’s immune system after lymphodepletion recovery. Finally, we outline new actively explored alternatives to overcome these resistance mechanisms, with a special emphasis on CRISPR/Cas9 mediated genetic engineering approaches, a promising platform to optimize CAR-NK cell function to eradicate refractory cancers.

Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders.

Background: Cardiovascular diseases are a public health concern worldwide, with high rates of morbidity and mortality. Depression is a frequent comorbidity in coronary heart disease (CHD). It can be caused by the experience of suffering from heart disease, but it can also influence the prognosis of the CHD. The prevalence of depression in patients with cardiovascular disease is twice as high as that in the general population. Aim: Assess the influence of depression in the prognosis at 5 years in patients with CHD. Methods: 145 patients diagnosed with CHD were recruited between September 2013 and June 2015. Depression was assessed based on the PHQ-9 results at the time of hospitalization and 3 months after discharged. Sociodemographic and clinical variables were collected. A 5-year follow-up was carried out to verify death, reinfarction or any adverse outcome. Results: 20% of the study population had depression at hospital admission compared with 11% at 3 months. Depression at 3 months after discharged was a differentiating factor to present complications (42.6 months, CI 95% 27.3–57.9) compared with patients without depression (55 months, CI 95%, 50.9–59.1) (Log-Rank p = 0.034). In the unadjusted model, the risk of heart complications increased with patients that have comorbidities, such as diabetes (HR 2.78, 95% CI 1.21–6.3) or hypothyroidism (HR 2.5 95% CI 1.09–5.7). Also, patients with post-hospitalization depression at 3 months were 3 times (95% CI 1.023–8.8) more likely to have complications during the follow-up period than nondepressed patients. After risk factor adjustment, the HR for depression was 2.01 (95% CI 0.57–6.9). Findings: Patients with depression at 3 months following the coronary event, presented complications sooner than those without depression.

With the current advancements in DNA sequencing technology, the limiting factor in long-read metagenomic assemblies is now the quantity and quality of input DNA. Although these requirements can be met through the use of axenic bacterial cultures or large amounts of biological material, insect systems that contain unculturable bacteria or that contain a low amount of available DNA cannot fully utilize the benefits of third-generation sequencing. The citrus greening disease insect vector Diaphorina citri is an example that exhibits both of these limitations. Although endosymbiont genomes have mostly been closed after the short-read sequencing of amplified template DNA, creating de novo long-read genomes from the unamplified DNA of an insect population may benefit communities using bioinformatics to study insect pathosystems. Here all four genomes of the infected D. citri microbiome were sequenced to closure using unamplified template DNA and two long-read sequencing technologies. Avoiding amplification bias and using long reads to assemble the bacterial genomes allowed for the circularization of the Wolbachia endosymbiont of Diaphorina citri for the first time and paralleled the annotation context of all four reference genomes without utilizing a traditional hybrid assembly. The strategies detailed here are suitable for the sequencing of other insect systems for which the input DNA, time, and cost are an issue.

As a result of a recently completed research, the aim of this work is to investigate, from the point of view of teachers of Children’s Education, how gender marks are present in the interaction among children of a Rio de Janeiro public school. Given the need to destabilize the heterocentric regime that seeks, unceasingly, to normalize bodies, genders and sexualities, this research is anchored in a proposal that encourages other ways of looking/(re)thinking the world in favor of the need to create strategies to contest heteronormativity. In order to do this, debates held with teachers from this public school encouraged us to reflect on how the games played by children destabilize the regulatory norms of gender, since it favors the (re)creation of other (im)possible worlds.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

This study examined the putative link between the entrainment to the slow rhythmic structure of speech, speech intelligibility and reading by means of a behavioral paradigm. Two groups of 20 children (Grades 2 and 5) were asked to recall a pseudoword embedded in sentences presented either in quiet or noisy listening conditions. Half of the sentences were primed with their syllabic and prosodic amplitude envelope to determine whether a boost in auditory entrainment to these speech features enhanced pseudoword intelligibility. Priming improved pseudoword recall performance only for the older children both in a quiet and a noisy listening environment, and such benefit from the prime correlated with reading skills and pseudoword recall. Our results support the role of syllabic and prosodic tracking of speech in reading development.

Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.