Recombinant Adenosine Deaminase Ameliorates Inflammation, Vascular Disease, and Fibrosis in Preclinical Models of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEGylated adenosine deaminase (PEG-ADA) in preclinical models of SSc. METHODS: The effects of PEG-ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra-2 mice and in a B10.D2→BALB/c (H-2d ) model of sclerodermatous chronic graft-versus-host disease (GVHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness skin model. RESULTS: PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) (P = 0.0079; n = 6), dermal fibrosis by 51.8% (P = 0.0006; n = 6), and intestinal fibrosis by 17.7% (P = 0.0228; n = 6) in Fra-2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) (P = 0.0063; n = 8), and in a human full-thickness skin model. PEG-ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) (P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) (P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) (P = 0.0147; n = 6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) (P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) (P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra-2 mice. CONCLUSION: Treatment with PEG-ADA ameliorates the 3 cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications, as PEG-ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA-deficient severe combined immunodeficiency disease.

Mechanisms of gene therapy for tolerance: B7 signaling is required for peptide-IgG gene-transferred tolerance induction.

LPS-activated B cells, transduced with IgG fusion proteins, are highly tolerogenic APCs. To analyze the mechanisms for this B cell-delivered gene therapy, we first followed the fate of CFSE-labeled B cell blasts. These cells primarily localized to the spleen, where a small population persisted for at least 1 mo after injection. By day 7 after injection, approximately 95% of the transduced cells had divided at least once, presumably an effect of the in vitro LPS activation into the cycle, because resting cells did not divide. B cells from gld donors were not tolerogenic, initially suggesting a role for Fas ligand (FasL) in tolerance. Because transduced normal B cells expressed only low levels of FasL and did not kill Fas-expressing Jurkat or A20 B lymphoma cells in vitro, these data suggest that gld B cells are not tolerogenic due to unique characteristics of these B cells rather than the lack of functional FasL expression. The transduced B cell blasts displayed significant up-regulation of both B7 costimulatory molecules, and B7.2 up-regulation was maintained through day 7 in vivo. When B cells from B7 knockout donors were transduced to express Ig fusion proteins, they were not tolerogenic in two different mouse strains and Ag models. Moreover, anti-B7 Ab blocked tolerance induction in this model, a result consistent with a role for B7 in tolerance induction. We propose that tolerance may be induced in this model by B7-driven negative regulatory signaling, but tolerance is maintained by a lack of signal 2, because expression of B7 is eventually lost in vivo.

Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases.

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.