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 C Minto; Catherine Vitelli; Danielle Harmon; Yuanbin Ru; Guoying Karen Yu; Claudia Escher; Jakob Vowinckel; Jill Woloszynek; Hassib Akeefe; Rajeev Mahimkar; Sherry Bullens; Stuart Bunting

doi:10.1016/j.omtm.2020.07.005

Induction of ER Stress by an AAV5 BDD FVIII Construct Is Dependent on the Strength of the Hepatic-Specific Promoter

Mol Ther Methods Clin Dev. 2020 Jul 9:18:620-630. doi: 10.1016/j.omtm.2020.07.005. eCollection 2020 Sep 11.

Authors

Sylvia Fong¹, Britta Handyside¹, Choong-Ryoul Sihn¹, Su Liu¹, Lening Zhang¹, Lin Xie¹, Ryan Murphy¹, Nicole Galicia¹, Bridget Yates¹, Wesley C Minto¹, Catherine Vitelli¹, Danielle Harmon¹, Yuanbin Ru¹, Guoying Karen Yu¹, Claudia Escher², Jakob Vowinckel², Jill Woloszynek¹, Hassib Akeefe¹, Rajeev Mahimkar¹, Sherry Bullens¹, Stuart Bunting¹

Affiliations

¹ BioMarin Pharmaceutical, Inc., Novato, CA, USA.
² Biognosys AG, Schlieren, Switzerland.

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.

Keywords: 100ATGB; AAV5; AAV5-hFVIII-SQ; ER stress; endoplasmic reticulum; gene therapy; hemophilia A; hybrid liver-specific promoter; molecular chaperone; valoctocogene roxaparvovec.