Construction of Human Factor IX Expression Vectors in Retroviral Vector Frames Optimized for Muscle Cells

Abstract

Development of a highly refined human factor IX (hFIX) expression vector system is critical for establishing a durable hemophilia B gene therapy. Here we report construction of a series of retroviral vectors and identification of an optimal basic structure and components for expressing hFIX in skeletal muscle cells. These vectors, which are derived from Moloney murine leukemia virus (MoMLV) with its enhancer sequence in the 3′ long terminal repeat (LTR) deleted, contained internal hFIX expression units inserted in forward configuration without or with a viral vector intron sequence (pdL or pdLIn vector frame, respectively) or in inverted configuration without a viral vector intron sequence (pdLi frame). Internal expression units contained a hFIX cDNA or hFIX minigene (hlXm1 or hIXm2) derived from the hFIX cDNA by insertion of a shortened first intron sequence of the hFIX gene. Regardless of the promoter and vector frame used, both hIXml and hIXm2 gave 10- to 14-fold higher hFIX expression compared to those with hFIX cDNA. Internal hFIX transcriptional control units of these vectors were composed of various promoters linked with or without the muscle creatine kinase enhancer (Me) sequence. Promoters tested included those of α-actin (αA775), β-actin (βA280), cytochrome oxidase (CO1250 and CO650), myogenin (Mg1031 and Mg353), and Rous sarcoma virus (RSV). βA200, which was derived from βA280 by eliminating potential polyadenylation sites, was also tested. As extensively examined with the myogenin promoter, presence of one or multiple copies of Me in the vectors elevated the expression activity in myotubes by 4.5- to 19-fold over those without Me, but not significantly in myoblasts. Similar enhancements in expression activity with Me were also observed with other promoters, except those of RSV and CO. The latter two showed only modest enhancements in the presence of Me. As assayed with myotubes in culture, the general order of hFIX expression activity of various promoters with four copies of Me in the three different vector frames was βA280 ≈ βA200 > Mg353 > Mg1031 ≈ RSV ≈ CO650 ≈ αA775 > CO1250. One exception was that CO650 showed significantly less activity in pdLi-type vectors than in the pdLIn vectors. Based on the systematic analyses of various structural components, a group of pdLi vectors consisting of βA200, two to four copies of Me, and hIXm2 was identified to have the optimal basic vector structure to be used in retrovirus for hFIX expression in differentiated skeletal muscle cells. The present studies provide the critical first step for establishing a highly refined hemophilia B gene therapy based on skeletal muscle-targeted hFIX gene transfer. Skeletal muscle cells may serve as an efficient medium for systemic production of transgene products in a gene therapy. Retroviral vector constructs were prepared using human factor IX minigenes, various muscle-specific or nonspecific promoters, and muscle creatine kinase enhancer. These vectors were tested systematically to determine an optimal basic structure for producing factor IX in mouse skeletal muscle cells. The basic retroviral vector structure for an optimal expression of factor IX in skeletal muscle cells may contain an internal factor IX expression cassette composed of a β-actin promoter, muscle creatine kinase enhancer, and human factor IX minigene in a reverse orientation relative to the 5′ long terminal repeat.