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Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

Vasoactive intestinal peptide (VIP) is a potent antiinflammatory agent with immunoregulatory properties, skewing the immune response to a Th2 pattern of cytokine production. Here, we studied the effect of treatment with VIP in the development of diabetes in nonobese diabetic (NOD) mice, an annual model of type 1 diabetes. Mice treated with VIP from 4 weeks of age did not develop diabetes and showed milder insulitis than nontreated mice. The protective mechanism of VIP was associated with a reduction in the circulating levels of Th1 cytokines. In the pancreas of VIP-treated animals, regulatory T cell markers predominate, as indicated by the upregulation of FoxP3 and transforming growth factor-β (TGF-β), and the downregulation of the transcription factor, T-bet. These findings indicate that VIP restores tolerance to pancreatic islets by promoting the local differentiation and function of regulatory T cells. Fil: Rosignoli, F.. Universidad Complutense de Madrid; España Fil: Torroba, M.. Universidad Complutense de Madrid; España Fil: Juarranz, Y.. Universidad Complutense de Madrid; España Fil: García Gómez, M.. Universidad Complutense de Madrid; España Fil: Martinez, C.. Universidad Complutense de Madrid; España Fil: Gomariz, R. P.. Universidad Complutense de Madrid; España Fil: Perez Leiros, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Leceta, J.. Universidad Complutense de Madrid; España

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD is the most common erythroid inherited enzymatic defect causing chronic non-spherocytic hemolytic anemia, high reticulocytosis, acute splenomegaly and intense iron overload in the liver, being life-threatening in severe patients. Up to date allogeneic bone marrow transplant represents the only curative treatment of patients affected by the severe form of the disease (5-10% of PKD patients aprox.). Preclinical gene therapy studies conducted in pyruvate kinase deficient mice have shown the safety and the efficacy of a new PGK-coRPK-Wpre therapeutic lentiviral vector that provided the designation by the European Medicinal Agency (EMA) of this PGK-coRPK-Wpre vector as a new orphan drug (EU/3/14/1130). To continue with the preclinical studies required to develop a clinical trial for PKD we have characterized the hematopoietic progenitor's content and the erythroid progenitors’ profile in peripheral blood (PB) from PKD patients, showing an increase in both types of cells. For transduction studies, CD34+ sorted cells from PKD PB were transduced with PGK-coRPK-Wpre viral vector supernatants. Hematopoietic progenitors’ content and final yield of cells and CFCs was evaluated to analyze the toxicity of the vector. Percentage of transduction ranged from 31% to 100%, showing the efficacy of the transduction protocol and of the PGK-coRPK therapeutic vector. Vector copy number was quantified in both, individual CFUs and CD34+ cells maintained in liquid culture for 14 days, ranging from 0.1 to 3.0 VCN/cells. Additional studies are being conducted to demonstrate PGK-coRPK functionality in human PKD cells.

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is a disorder caused by mutations in the PKLR gene that produces chronic nonspherocytic hemolytic anemia. Besides periodic blood transfusion and splenectomy, severe cases require bone marrow (BM) transplant, which makes this disease a good candidate for gene therapy. Here, the normal human R-type pyruvate kinase (hRPK) complementary (cDNA) was expressed in hematopoietic stem cells (HSCs) derived from pklr deficient mice, using a retroviral vector system. These mice show a similar red blood cell phenotype to that observed in human PKD. Transduced HSCs were transplanted into myeloablated adult PKD mice or in utero injected into nonconditioned PKD fetuses. In the myeloablated recipients, the hematological manifestations of PKD were completely resolved and normal percentages of late erythroid progenitors, reticulocyte and erythrocyte counts, hemoglobin levels and erythrocyte biochemistry were restored. Corrected cells preserved their rescuing capacity after secondary and tertiary transplant. When corrected cells were in utero transplanted, partial correction of the erythrocyte disease was obtained, although a very low number of corrected cells became engrafted, suggesting a different efficiency of cell therapy applied in utero. Our data suggest that transduction of human RPK cDNA in PKLR mutated HSCs could be an effective strategy in severe cases of PKD.

Septic shock is a systemic response to severe bacterial infections, generally caused by Gram-negative bacterial endotoxins, with multiple manifestations such as hypotension, tissue injury, disseminated intravascular coagulation, and multi-organ failure. All these effects, are induced by the generation of pro-inflammatory and vasodilator mediators, cell adhesion molecules, coagulation factors, and acute-phase proteins. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two immunopeptides with anti-inflammatory properties exerted through type 1 and 2 VIP receptors (VPAC1 and VPAC2, respectively), and PACAP receptor (PAC1). The present results recapitulate the protective role of PAC1 in an experimental model of lethal endotoxemia using a knockout for the PAC1 receptor. Our results demonstrate that VIP and PACAP decrease lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production, neutrophil infiltration and intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and fibrinogen expression through PAC1 receptor, providing an advantage to design more specific drugs complementing standard intensive care therapy in septic shock.

Toll-like receptor 2 (TLR2) and -4 mediate signals from a great variety of bacterial gut products, giving the host a panel of microbe-recognizing receptors. Under homeostatic conditions, TLRs act as protective receptors of the intestinal epithelium. When homeostasis is disrupted in diseases such as inflammatory bowel disease, TLR2 and -4 are deregulated. Our study demonstrates, by using a trinitrobenzene sulfonic acid-induced colitis model of Crohn's disease, the constitutive expression and the up-regulation of TLR2 and -4 at messenger and protein levels in colon extracts, as well as in macrophages, dendritic cells, and lymphocytes from mesenteric lymphoid nodes. Vasoactive intestinal peptide (VIP) treatment induced a decrease of TLR2 and -4 expressions approaching ethanol control levels. Our results suggest that VIP modulation of TLR2 and -4 could be explained by two possible mechanisms. The first one would be the secondary reduction of TLR2 and -4 caused by the VIP-mediated decrease of inflammatory mediators such as interleukin-1β and interferon- γ, which synergize with bacterial products, contributing to the amplification of TLR presence in the intestine. The other possible mechanism would involve a VIP-mediated decrease of nuclear factor-κB, which would cause a direct down-regulation of TLR expression. In summary, the resultant physiological effect is the decrease of TLR2 and -4 expressions to homeostatic levels. Our study describes for the first time the role of a peptide present in the gut microenvironment as an effective modulator of the initial steps of acute inflammation, acting at local and systemic levels and leading to the restoration of the homeostasis lost after an established inflammatory/autoimmune disease.