Adult mice are unresponsive to AAV8-Gremlin1 gene therapy targeting the liver.

OBJECTIVE: Gremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects. METHODS: Adult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time. RESULTS: Liver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation. CONCLUSION: GREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered.

Inhibition of T-cell activation by the CTLA4-Fc Abatacept is sufficient to ameliorate proteinuric kidney disease.

Diabetic nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria, and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.