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Chimeric Antigen Receptor (CAR) Cytokine-Induced Killer (CIK) cell therapy is an emerging treatment for acute myeloid leukemia (AML) although some implementations are required. Specifically, it appears crucial to improve CAR-CIK infiltration ability into the bone marrow (BM) niche to eradicate leukemia stem cells (LSCs) at their location. CAR-CIK ex vivo manipulation influences the expression of several chemokine receptors and may dampen the capacity of infused cells to migrate to the BM. The chemokine ligand 12 (CXCL12), produced by mesenchymal stromal cells (MSCs) within the niche, and its chemokine receptor 4 (CXCR4) modulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 overexpressed on blasts, enhancing their homing in the niche. CXCR4 expression is consistently downregulated during the culture of CIKs. Therefore, combining the expression of CD33.CAR and CXCR4 might promote CAR-CIK homing to the BM and subsequent leukemia eradication, specifically of LSCs. Two bicistronic Sleeping Beauty transposon vectors were designed: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. Phenotype and CAR-related in vitro effector functions were compared. Migration in vitro toward rhCXCL12 or MSC supernatants was tested using a transwell migration assay. CAR-CIKs in vivo BM homing ability was evaluated in sub-lethally irradiated NSG mice. CAR-CIKs engraftment was assessed in BM, blood, spleen, and lungs. We noticed that both CD33.CAR(2A)CXCR4-CIKs (n=22, P

Chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has been quite elusive due to tumor heterogeneity, lack of ideal target antigens and the role of the tumor microenvironment in leukemia protection and leukemia stem cells (LSCs) maintenance. Therefore, CAR design must be improved for an effective anti-AML activity. Besides selecting a good target antigen, it is crucial to improve CAR T-cells infiltration, to eradicate LSCs at their location within the bone marrow (BM) niche. The chemokine receptor 4 (CXCR4) and its ligand, CXC motif ligand 12 (CXCL12) are two fundamental factors orchestrating leukocytes trafficking to the niche. In AML, the CXCL12-CXCR4 axis is co-opted by leukemic cells. CXCL12, released by mesenchymal stromal cells (MSCs), interacts with CXCR4 overexpressed on blasts, boosting their homing in the BM, where they can find a protecting environment. We hypothesize that CXCR4 overexpression by Cytokine-Induced Killer cells (CIKs) combined with a CD33.CAR may improve their homing to the BM and subsequent leukemia eradication. We designed two bicistronic vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. CIKs were non-virally engineered with the Sleeping Beauty transposon system to express our constructs or the monocistronic CD33.CAR, used as control. We assessed the chemotactic activity of the bicistronic constructs employing the transwell migration assay, toward rhCXCL12 and human MSCs supernatants. Additionally, in vitro effector functions were compared. Considering that CXCR4 expression on CAR-CIKs is downregulated over culture, we induced it artificially. CXCR4(IRES)CD33.CAR-CIKs (n=8) expressed higher levels of CXCR4, but lower CD33.CAR expression compared with CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=9) showed a significant co-expression of both proteins, as compared to control (P≤ 0.05). The analysis of T-cell markers and memory phenotype uncovered that CXCR4(IRES)CD33.CAR-CIKs (n=7) are enriched in CD8+ compartment and include a higher fraction of TEMRA contrary to CD33-CAR CIKs (P≤0.05), while CD33.CAR(2A)CXCR4-CIKs (n=8) phenotype is comparable to control. Chemotaxis assays toward rhCXCL12 confirmed that CXCR4(IRES)CD33.CAR-CIKs (n=7, P≤0.05) and CD33.CAR(2A)CXCR4-CIKs (n=7, P≤0.05) achieved a migration advantage over CD33.CAR-CIKs alone (n=11), with a mean percentage of migration of 58.5% and 68.8% respectively, compared to 40.5%. We were also able to observe a specific chemotactic response toward MSCs supernatants, as proved by the use of Plerixafor. Moreover, correlation analysis strengthened our observation, as higher CXCR4 expression resulted in increased migratory activity. Concerning CAR-related effector functions, CXCR4(IRES)CD33.CAR-CIKs and CD33.CAR(2A)CXCR4-CIKs displayed similar killing of the CD33+ KG1 cell line, compared to CD33.CAR-CIKs alone, with a mean lysis of 56.6% (E:T ratio 5:1, n=7) for CXCR4(IRES)CD33.CAR-CIKs, and of 66.9% (n=9) for CD33.CAR(2A)CXCR4-CIKs, compared to a mean lysis of 62.5% (n=12) of CD33.CAR-CIKs. The bicistronic constructs also maintained their capacity to produce IL-2 and IFN-y, and to proliferate after CD33 antigen exposure. CXCR4(IRES)CD33.CAR-CIKs displayed not significant but inferior effector responses as compared to control, due to lower CAR expression; CD33.CAR(2A)CXCR4-CIKs performed similarly to CD33.CAR CIKs alone. Compared to conventional CD33.CAR-CIKs, CXCR4-overexpressing CD33.CAR-CIKs exhibit enhanced migration capacity while retaining functional activity against CD33+ target cells. Considering these results, we believe that CD33.CAR(2A)CXCR4 is the best candidate for further in vivo homing and anti-leukemic tests.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency in lysosomal enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs). The current therapeutic strategies of enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation have been reported to reduce patient morbidity and to improve their quality of life, but they are associated with persistence of residual disease burden, in particular at the neurocognitive and musculoskeletal levels. This indicates the need for more efficacious treatments capable of effective and rapid enzyme delivery to the affected organs, especially the brain and the skeleton. Gene therapy (GT) strategies aimed at correcting the genetic defect in patient cells could represent a significant improvement for the treatment of MPS when compared with conventional approaches. While in-vivo GT strategies foresee the administration of viral vector particles directly to patients with the aim of providing normal complementary DNA to the affected cells, ex-vivo GT approaches are based on the ex-vivo transduction of patient cells that are subsequently infused back. This review provides insights into the state-of-art accomplishments made with in vivo and ex vivo GT-based approaches in MPS and provide a vision for the future in the medical community.

Purpose: To investigate the role of the overall antioxidant activity of diet, we estimated the relation between three dietary indices of total antioxidant capacity (TAC) and endometrial cancer risk Methods: We analyzed data from an Italian case–control study including 454 women with incident, histologically confirmed endometrial cancer, and 908 frequency-matched controls admitted to the same hospitals as cases for acute non-neoplastic conditions. A reproducible and valid food frequency questionnaire was used to assess subjects’ habitual diet. TAC was measured using Italian food composition tables in terms of Ferric-reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), and total radical-trapping antioxidant parameter (TRAP). We computed odds ratios (OR) and corresponding 95 % confidence intervals (CIs) using conditional multiple logistic regression models, including terms for recognized endometrial cancer risk factors and total energy intake. Results: TAC was inversely related to endometrial cancer risk with ORs for the highest versus the lowest quartile of 0.69 (95 % CI 0.47–1.00) for FRAP, 0.68 (95 % CI 0.46–0.99) for TEAC, and 0.68 (95 % CI 0.47–0.98) for TRAP. The relations appeared consistent in strata of selected risk factors and decreased when considering TAC without the contribution of coffee. Conclusions: Our findings suggest a favorable role of a diet high in TAC on endometrial cancer risk, which can be partially driven by coffee consumption. © 2016, Springer International Publishing Switzerland.