Induction of ER Stress by an AAV5 BDD FVIII Construct Is Dependent on the Strength of the Hepatic-Specific Promoter
Sylvia Fong, Britta Handyside, Choong‐Ryoul Sihn, Su Liu, Lening Zhang, Lin Xie, Ryan Murphy, Nicole Galicia, Bridget Yates, Wesley Minto, Catherine Vitelli, Danielle Harmon, Yuanbin Ru, Guoying Karen Yu, Claudia Escher, Jakob Vowinckel, Jill Woloszynek, Hassib Akeefe, Rajeev Mahimkar, Sherry Bullens, Stuart Bunting
Abstract
Adeno-associated virus 5 (AAV5)-human factor VIII-SQ (hFVIII-SQ; valoctocogene roxaparvovec) is an AAV-mediated product under evaluation for treatment of severe hemophilia A, which contains a B-domain-deleted hFVIII (hFVIII-SQ) transgene and a hybrid liver-specific promotor (HLP). To increase FVIII-SQ expression and reduce the vector dose required, a stronger promoter may be considered. However, because FVIII-SQ is a protein known to be difficult to fold and secrete, this could potentially induce endoplasmic reticulum (ER) stress. We evaluated the effect of two AAV5-hFVIII-SQ vectors with different liver-specific promoter strength (HLP << 100ATGB) on hepatic ER stress in mice. Five weeks after receiving vehicle or vector, the percentage of transduced hepatocytes and levels of liver hFVIII-SQ DNA and RNA increased dose dependently for both vectors. At lower doses, plasma hFVIII-SQ protein levels were higher for 100ATGB. This difference was attenuated at the highest dose. For 100ATGB, liver hFVIII-SQ protein accumulated dose dependently, with increased expression of ER stress markers at the highest dose, suggesting hepatocytes reached or exceeded their capacity to fold/secrete hFVIII-SQ. These data suggest that weaker promoters may require relatively higher doses to distribute expression load across a greater number of hepatocytes, whereas relatively stronger promoters may produce comparable levels of FVIII in fewer hepatocytes, with potential for ER stress.