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Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing., Graphical Abstract, Lentiviral vectors (LVs) are powerful gene-transfer tools routinely exploited for distinct research and clinical applications. LVs produced in most research laboratories contain contaminants that can generate confounding effects in experimental studies. Soldi et al. describe a laboratory-scale workflow for purified LV production, highlighting enhanced gene-editing efficiency and diminished inflammatory responses.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

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. Transient p53 inhibition and induced cell-cycle progression increase clonal engraftment and homology-directed repair in hematopoietic stem cells.

The liver hosts an immune suppressive environment favouring metastatic seeding and proliferation of cancer cells. Pharmacological treatments, including immunotherapies, fail in the presence of liver metastases (LMS). Therefore, identifying new interventional tools and key targetable players involved in the immunosuppressive environment is of pivotal importance. We developed a lentiviral vector (LV)-based platform for selective genetic engineering of resident and tumour-associated macrophages enabling locally-sourced delivery of therapeutic molecules to LMS. Selective transgene expression is driven by a macrophage specific promoter and fine-tuned by microRNA target sequences. Upon systemic delivery of the LV, we observed enhanced transgene expression in macrophages located in areas surrounding LMS. We then equipped the LV with an IFNα-coding sequence, a cytokine with pleiotropic immune effects. Long term analysis in mice showed LV dose-dependent, sustained and well-tolerated IFNα expression. To investigate the therapeutic efficacy, we employed a colorectal cancer (CRC) organoid-based syngeneic mouse model of LMS containing molecular and histopathological hallmarks of the human disease. IFNα LV treatment significantly delayed LMS growth reaching a complete response in up to 50%. Single cell omics of LMS from IFNα LV-treated mice showed upregulation of IFNα-responsive genes, macrophage skewing to an antigen presenting (M1-like) polarization state, and expansion as well as reduced exhaustion of LMS-associated antigen specific CD8 T cells. Employing spatial transcriptomics, we found that the interface between LMS and liver parenchymal tissue was the major site of IFNα action, which was associated with enhanced immune activation and antigen presentation. Furthermore, we observed decreased angiogenesis and hypoxia in IFNα LV-treated LMS. When comparing LMS of treatment responsive to resistant mice, we found accumulation of activated CD8 T-cells in responsive whereas a high number of immunosuppressive T regulatory type 1 (TR1)-like cells in resistant mice. Molecular analyses suggest that TR1-like cell infiltration was associated with increased IL10 signaling in resistant mice. Furthermore, we found a positive correlation between IFNα and TR1-like cell signatures in human LMS and primary CRC thus supporting the link between IFNα activation and expansion of TR1-like cells in cancer. In summary, we developed an innovative gene-based platform that upon a single well-tolerated intravenous LV infusion rapidly promotes a protective therapeutic response against LMS through enabling immune activation. However, we also found that TR1-like cells might promote tumor immune evasion in presence of IFNα signaling in this setting, suggesting targeting of TR1-like cells when facing resistance to cancer immunotherapies that trigger IFNα signaling. Citation Format: Thomas Kerzel, Stefano Beretta, Eloise Scamardella, Chiara Balestrieri, Tamara Canu, Federica Pedica, Rossana Norata, Lucia Sergi Sergi, Marco Genua, Ostuni Renato, Anna Kajaste-Rudnitski, Antonio Esposito, Masanobu Oshima, Giovanni Tonon, Francesca Sanvito, Mario Leonardo Squadrito, Luigi Naldini. IFNalpha by in vivo-engineered macrophages abates liver metastases and triggers counter regulatory responses limiting efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3297.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1β production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions., Mannose is present at trace levels in blood and regulates cancer growth. Here the authors show that supraphysiological levels of mannose can also regulate macrophages, limiting their production of IL-1β and increasing resistance of mice to LPS-induced endotoxemia and DSS-induced colitis.

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit ex vivo LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow–derived CD34+ cells while better preserving their engraftment capacity in vivo. CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G0 fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels in vivo. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC ex vivo culture time.

The prominent role of dendritic cells (DC) in promoting tolerance and the development of methods to generate clinical grade products allowed the clinical application of tolerogenic DC (tolDC)-based therapies for controlling unwanted immune responses. We established an efficient method to generate tolerogenic human DC, producing supra-physiological levels of IL-10, by genetically engineering monocyte-derived DC with a bidirectional Lentiviral Vector (bdLV) encoding for IL-10 and a marker gene. DCIL−10 are mature DC, modulate T cell responses, promote T regulatory cells, and are phenotypically and functionally stable upon stimulation. Adoptive transfer of human DCIL−10 in a humanized mouse model dampens allogeneic T cell recall responses, while murine DCIL−10 delays acute graft-vs.-host disease in mice. Our report outlines an efficient method to transduce human myeloid cells with large-size LV and shows that stable over-expression of IL-10 generates an effective cell product for future clinical applications in the contest of allogeneic transplantation.

The low gene manipulation efficiency of human hematopoietic stem and progenitor cells (HSPC) remains a major hurdle for sustainable and broad clinical application of innovative therapies for a wide range of disorders. Given that all current and emerging gene transfer and editing technologies are bound to expose HSPC to exogenous nucleic acids and most often also to viral vectors, we reason that host antiviral factors and nucleic acid sensors play a pivotal role in the efficacy of HSPC genetic manipulation. Here, we review recent progress in our understanding of vector–host interactions and innate immunity in HSPC upon gene engineering and discuss how dissecting this crosstalk can guide the development of more stealth and efficient gene therapy approaches in the future.