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Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis., Graphical Abstract, In a GLP study, Jofra Hernández and colleagues demonstrate that lentiviral vector-mediated HSPC gene therapy effectively corrects long-term X-linked chronic granulomatous disease in a mouse model of the disease. A small proportion of mice develops hematopoietic tumors originating from rare dominant clones harboring vector insertions near oncogenes.

Cell-selective drug release in the central nervous system (CNS) holds great promise for the treatment of many CNS disorders but it is still challenging. We previously demonstrated that polymeric nanoparticles (NPs) injected intra-parenchyma in the CNS can be internalized specifically in microglia/macrophages surrounding the injection site. Here, we explored NPs administration in the cerebrospinal fluid (CSF) to achieve a wider spreading and increased cell targeting throughout the CNS; we generated new NPs variants and studied the effect of modifying size and surface charge on NPs biodistribution and cellular uptake. Intra-cerebroventricular administration resulted in prevalent localization of the NPs in proximity to stem-cell niches, such as around the lateral ventricles, the subventricular zone and the rostral migratory stream. NPs internalization occurred preferentially in brain myeloid cells/microglia. We demonstrated that brain biodistribution and extent of internalization in microglia are influenced by NPs dimensions and can be improved by applying a transient disruption of the blood-brain barrier with mannitol, leading to NPs internalization in up to 25% of brain myeloid/microglia cells. A fraction of the targeted cells was positive for markers of proliferation or stained positive for stemness/progenitor-cell markers such as Nestin, c-kit, or NG2. Interestingly, through these newly formulated NPs we obtained controlled and selective release of drugs otherwise difficult to formulate (such as busulfan and etoposide) to the target cells, preventing unwanted side effects and the toxicity obtained by direct brain delivery of the not encapsulated drugs. Overall, these data provide proof of concept of the applicability of these novel NP-based drug formulations for achieving internalization not only in mature microglia but also possibly in more immature myeloid cells in the brain and pave the way for brain-restricted microglia-targeted drug delivery regimens.

Natural killer (NK) cells are immune innate effectors playing a pivotal role in the immunosurveillance of multiple myeloma (MM) since they are able to directly recognize and kill MM cells. In this regard, among activating receptors expressed by NK cells, NKG2D represents an important receptor for the recognition of MM cells, being its ligands expressed by tumor cells, and being able to trigger NK cell cytotoxicity. The MHC class I-related molecule A (MICA) is one of the NKG2D ligands; it is encoded by highly polymorphic genes and exists as membrane-bound and soluble isoforms. Soluble MICA (sMICA) is overexpressed in the serum of MM patients, and its levels correlate with tumor progression. Interestingly, a methionine (Met) to valine (Val) substitution at position 129 of the α2 heavy chain domain classifies the MICA alleles into strong (MICA-129Met) and weak (MICA-129Val) binders to NKG2D receptor. We addressed whether the genetic polymorphisms in the MICA-129 alleles could affect MICA release during MM progression. The frequencies of Val/Val, Val/Met, and Met/Met MICA-129 genotypes in a cohort of 137 MM patients were 36, 43, and 22%, respectively. Interestingly, patients characterized by a Val/Val genotype exhibited the highest levels of sMICA in the sera. In addition, analysis of the frequencies of MICA-129 genotypes among different MM disease states revealed that Val/Val patients had a significant higher frequency of relapse. Interestingly, NKG2D was downmodulated in NK cells derived from MICA-129Met/Met MM patients. Results obtained by structural modeling analysis suggested that the Met to Val dimorphism could affect the capacity of MICA to form an optimal template for NKG2D recognition. In conclusion, our findings indicate that the MICA-129Val/Val variant is associated with significantly higher levels of sMICA and the progression of MM, strongly suggesting that the usage of soluble MICA as prognostic marker has to be definitely combined with the patient MICA genotype.

Exosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNγ production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-κB pathway in a TLR2/HSP70-dependent manner. Interestingly, HSP70+ exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNγ production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors.

In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for mucopolysaccharidosis I (MPS I), biosafety studies were conducted to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted, employing Good Laboratory Practice principles. Vector integration site (IS) studies were applied in order to predict adverse consequences of vector gene transfer and to obtain HSC-related information. Overall, the results obtained in these studies provided robust evidence to support the safety and tolerability of high-efficiency LV-mediated gene transfer and above-normal IDUA enzyme expression in both murine and human HSPCs and their in vivo progeny. Taken together, these investigations provide essential safety data to support clinical testing of HSC gene therapy in MPS I patients. These studies also underline criticisms associated with the use of currently available models, and highlight the value of surrogate markers of tumorigenicity that may be further explored in the future. Notably, biological evidence supporting the efficacy of gene therapy on MPS I disease and its feasibility on patients' HSCs were also generated, employing clinical-grade LVs. Finally, the clonal contribution of LV-transduced HSPCs to hematopoiesis along serial transplantation was quantified in a minimum of 200-300 clones, with the different level of repopulating cells in primary recipients being reflected in the secondary.

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine–threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell–mediated cytotoxicity. We could exclude that increased expression of β-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr705 phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.