522. Targeting FVIII-Expression To Liver Sinusoidal Cells By Lentiviral Vectors Corrects the Bleeding Phenotype in Hemophilia A Overcoming Immunological Responses

Hemophilia A (HA) is an X-linked bleeding disorder due to mutations in clotting factor (F) VIII gene. To date the treatment for preventing major bleeding episodes is represented by replacement therapy with recombinant or plasma-derived FVIII. The two major concerns are high cost and development of FVIII neutralizing antibodies in 20-30% of patients.Several studies on gene transfer by direct injection of LV for HA have been recently published. Many efforts were focused on the improvement of LV, to obtain a selective targeting of transgene expression, or on the production of several bioengineered FVIII, in order to overcome some of the issues related to FVIII expression in HA animal models. However, in most cases, the immune responses associated with FVIII remain the major obstacle.We prepared LVs containing the B-domain deleted (BDD) hFVIII under the control of PGK, VEC or CD11b promoters with or without the addition of the miRTs used for initial GFP expression studies, and we then injected HA mice with 109 TU/mouse of these LVs (3 mice for LV PGK-hFVIII ±42; 4-9 mice for the other vectors) and assessed FVIII activity by aPTT assay.All mice injected with LV-VEC-hFVIII ± miRTs and LV-CD11b-hFVIII ± miRTs showed a FVIII activity between 3.5 and 5% one week after injection, while HA mice injected with LV-PGK-hFVIII± 42 showed a FVIII activity £1%. Moreover, starting from 2 weeks after LVs injection we evaluated the presence of anti-FVIII antibodies by a direct ELISA. We detected the presence of anti-FVIII antibodies in the plasma of mice injected with LV-PGK-hFVIII±miRT-142 1 month after LV injection. Interestingly, the antibody titer was significantly lower in mice injected with LV-PGK-hFVIII-miRT-142-3p. In all mice injected with LV-VEC-hFVIII±miRT-122-142-3pwe detected hFVIII activity by aPTT assay up to 52 weeks after injection without production of anti-FVIII antibodies. HA mice injected LV-CD11b-hFVIII±miRT-126 showed hFVIII activity up to 52 w as well; interestingly, 60% of mice injected with LV-CD11b-hFVIII produced anti-FVIII antibodies 10-16 weeks after LV injection, while no anti-FVIII antibodies were detected in plasma of injected mice with LV-CD11b-hFVIII-miRT-126.Genomic analysis on liver samples from mice 24 w after injection of LV-VEC-hFVIII±miRT-122-142-3p and LV-CD11b-hFVIII±miRT-126 demonstrated the presence of LV sequence integrated in the genome of injected mice. Immunofluorescence on liver sections showed that LSECs and KCs were positive for hFVIII. Next, to assess whether EC, in particular LSECs, are able to induce immunotolerance, we immunized mice with Refacto. Mice producing anti-FVIII Ab were then injected with 109 TU of LV-VEC-hFVIII-miRT-122-142-3p. We detected hFVIII activity in all injected mice and, noteworthy, antibody titer decreased over time in the plasma of these mice.In conclusion, LV expressing FVIII under the control of VEC or CD11b promoters combined with miRTs combinations were able to overcome FVIII off-target expression limiting immune responses and providing phenotypic correction in treated HA mice with FVIII expression by sinusoidal cells

An Expanded Self-Antigen Peptidome Is Carried by the Human Lymph As Compared to the Plasma

The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing.A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration.The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance

Age-Related Oxidative Stress Compromises Endosomal Proteostasis

A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response