Comparison of Retroviral and Adeno-Associated Viral Vectors Designed to Express Human Clotting Factor IX

Abstract

Several different designs for retroviral and adeno-associated virus (AAV) vectors were developed to express human clotting factor IX. Seven separate retroviral vectors were constructed, including chimeric long terminal repeat (LTR)-based designs, vectors containing splice donor/acceptor sites with internal ribosome entry sites (IRES), and vectors with an internal cytomegalovirus (CMV)- or hepatitis B virus (HBV)-derived promoter. Five AAV vectors were produced using the same cassette design where a viral promoter was used to transcribe a bicistronic mRNA containing factor IX and an IRES/neo gene. In the human hepatocyte cell line HepG2, the constructs were tested for factor IX production by ELISA, Northern blot, and Western blot, and for biological activity by normalization of the prolonged activated partial thromboplastin time (APTT) of factor IX-deficient plasma. All of the constructs produced biologically active factor IX in the range of 0.23–152 ng/24 hr per 10⁶ cells (the HBV-promoted factor IX AAV vector was the least effective, and the CMV-promoted retroviral vector was the most active). Primary fibroblasts of both human and rabbit origin were also evaluated for factor IX production following transduction with viral vectors. Fibroblasts produced substantially more factor IX than the HepG2 cell line, with the best AAV vector synthesizing >250 ng/24 hr per 10⁶ cells and the best retroviral vector making >900 ng/24 hr per 10⁶ cells. Generally, we observed lower transduction efficiency and poorer expression with the AAV vectors versus retroviral vectors in these cell types.

Overview

Chen et al. report on the construction and analysis of both retroviral and adeno-associated virus-based vectors for the expression of human factor IX. Expression properties were analyzed in cell culture using the human HepG2 cell line as well as both human and rabbit primary fibroblasts. In general, all vectors were shown to produce biologically active factor IX in cell culture but the amount varied from less than 1 ng to nearly 1 μg/10⁶ cells per day. The study points out the importance of testing multiple vector designs in potential target cells. The study is also the first report concerning the potential use of adeno-associated virus (AAV)-based vectors for hemophilia gene therapy.