LAUSR

Diabetes mellitus, caused by loss or dysfunction of the insulin producing beta cells of the pancreas, is a promising target for rAAV-mediated gene therapy. To target potential therapeutic payloads specifically to beta cells, a cell-type specific expression control element is needed. Here, we tested a series of rAAV vectors designed to express transgenes specifically in human beta cells using the islet-tropic rAAV-KP1 capsid. A small promoter, consisting of only 84 bp of the insulin core promoter was not beta cell-specific in AAV, but highly active in multiple cell types, including tissues outside the pancreas. A larger 363 bp fragment of the insulin promoter (INS) also lacked beta cell specificity. However, beta cell-specific expression was achieved by combining two regulatory elements, a promoter consisting of two copies of INS (INSx2) and microRNA (miRNA) regulatory elements (MREs). The INSx2 promoter alone showed some beta cell preference, but not tight specificity. To reduce unspecific transgene expression in alpha cells, negative regulation by miRNAs was applied. MREs that are recognized by miRNAs abundant in alpha cells effectively downregulated the transgene expression in these cells. The INS2x-MRE expression vector was highly specific to human beta cells and stem cell derived beta cells.