CONSTRUCTION OF HUMAN FACTOR-IX EXPRESSION VECTORS IN RETROVIRAL VECTOR FRAMES OPTIMIZED FOR MUSCLE-CELLS

Development of a highly refined human factor IX (hFIX) expression vect or system is critical for establishing a durable hemophilia B gene the rapy. Here we report construction of a series of retroviral vectors an d identification of an optimal basic structure and components for expr essing 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 m inigene (hIXm1 or hIXm2) derived from the hFIX cDNA by insertion of a shortened first intron sequence of the hFIX gene. Regardless of the pr omoter and vector frame used, both hIXm1 and hIXm2 gave 10- to 14-fold higher hFIX expression compared to those with hFIX cDNA. Internal hFI X transcriptional control units of these vectors were composed of vari ous promoters linked with or without the muscle creatine kinase enhanc er (Me) sequence. Promoters tested included those of alpha-actin (alph a A775), beta-actin (beta A280), cytochrome oxidase (CO1250 and CO650) , myogenin (Mg1031 and Mg353), and Rous sarcoma virus (RSV). beta A200 , which was derived from beta A280 by eliminating potential polyadenyl ation sites, was also tested. As extensively examined with the myogeni n promoter, presence of one or multiple copies of Me in the vectors el evated the expression activity in myotubes by 4.5- to 19-fold over tho se without Me, but not significantly in myoblasts. Similar enhancement s in expression activity with Me were also observed with other promote rs, except those of RSV and CO. The latter two showed only modest enha ncements in the presence of Me. As assayed with myotubes in culture, t he general order of hFIX expression activity of various promoters with four copies of Me in the three different vector frames was beta A280 approximate to beta A200 > Mg353 > Mg1031 approximate to RSV approxima te to CO650 approximate to alpha A775 > 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 struct ural components, a group of pdLi vectors consisting of beta A200, two to four copies of Me, and hIXm2 was identified to have the optimal bas ic vector structure to be used in retrovirus for hFIX expression in di fferentiated skeletal muscle cells. The present studies provide the cr itical first step for establishing a highly refined hemophilia B gene therapy based on skeletal muscle-targeted hFIX gene transfer.