Despite the advent of biological therapies for the treatment of rheumatoid arthritis, there is a compelling need to develop alternative therapeutic targets for nonresponders to existing treatments. Soluble receptors occur naturally in vivo, such as the splice variant of the cell surface receptor for vascular endothelial growth factor (VEGF)--a key regulator of angiogenesis in rheumatoid arthritis. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, generating proteins--many of which may have regulatory functions. The objective of the present study was to identify alternative splice variants (ASV) from cell surface receptor genes, and to determine whether the novel proteins encoded exert therapeutic activity in an in vivo model of arthritis.
To identify novel splice variants, we performed RT-PCR using an mRNA pool representing major human tissue types and tumors. Novel ASV were identified by alignment of each cloned sequence to its respective genomic sequence in comparison with full-length transcripts. To test whether these ASV have biologic activity, we characterized a subset of them for ligand binding, and for efficacy in an animal model of arthritis. The in vivo study was accomplished using adenoviruses expressing secreted ASV.
We cloned 60 novel human ASV from 21 genes, encoding cell surface receptors--many of which are known to be important in the regulation of angiogenesis. The ASV were characterized by exon extension, intron retention and alternative exon utilization. Efficient expression and secretion of selected ASV--corresponding to VEGF receptor type 1, VEGF receptor type 2, VEGF receptor type 3, angiopoietin receptor Tie1, Met (receptor for hepatocyte growth factor), colony-stimulating factor 1 receptor, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, Kit, and RAGE--was demonstrated, together with binding to their cognate ligands. Importantly, ASV derived from VEGF receptor type 1 and Tie1, and to a lesser extent from VEGF receptor type 2 and fibroblast growth factor receptor 1, reduced clinical signs of arthritis in vivo. The reduction was paralleled by decreased joint inflammation and destruction.
The present study shows that unique ASV derived from receptors that play key roles in angiogenesis--namely, VEGF receptor type 1 and, for the first time, Tie1--can markedly reduce arthritis severity. More broadly, our results demonstrate that ASV are a source of novel proteins with therapeutic potential in diseases in which angiogenesis and cellular hyperplasia play a central role, such as rheumatoid arthritis.