Summary 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.

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 upon 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 significantly shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserved 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.

To test the factorial structure of the Italian version of the Insomnia Severity Index (ISI) using a confirmatory approach and to assess its psychometric properties. ISI questionnaire was completed by 272 patients (average age 41.28, range 18-73) with insomnia diagnosis performed by a sleep medicine physician and retrospectively enrolled in the study. All patients underwent Cognitive Behavioral Treatment for Insomnia (CBT-I) and completed sleep diaries before starting the treatment. Data from sleep diaries were analyzed for assessing concurrent validity of the ISI. Confirmatory factor analysis (CFA) for ordinal Likert-type items was applied to compare four competing models proposed in the literature. 244 patients, out of the 272, completed the ISI at the end of CBT-I. A comparison of ISI score before and after treatment was performed. The CFA analysis confirmed the presence of three main factors conceptualized as severity and impact of the disease along with sleep satisfaction. Significant correlations of the first three items of the questionnaire, investigating three different subtypes of insomnia, and the subjective measures from the sleep diaries were found, thus supporting the concurrent validity of the test. Sleep efficiency (SE) had a significant inverse correlation with the severity and satisfaction factors and with ISI's total score. After CBT-I treatment, a significant reduction of ISI's scores was observed, thus confirming the effectiveness of the CBT-I treatment. The internal reliability coefficient was 0.75. The ISI questionnaire maintains good psychometric properties in the Italian version, thus confirming that this instrument is reliable for detecting insomnia severity and identifying patients' symptoms.

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.