HUMAN GENE THERAPY 6:873-880 (July 1995) Mary Ann Liebert, Inc. Expression of Biologically Active Human Factor IX in Human Hematopoietic Cells after Retroviral Vector-Mediated Gene Transduction QIAN-LIN HAO,i PUNAM MALIK,^ ROBERTO SALAZAR,^ HUI TANG,^ ERLINDA M. GORDON,^ and DONALD B. KOHN' ABSTRACT Gene therapy is a potential treatment for hemophilia, wherein cells transduced with a normal factor IX gene could provide a continuous in vivo source of circulating factor IX. In this study, we examined the potential use of hematopoietic cells as a target for factor IX gene therapy. Human myeloid leukemia cells (HL-60) were transduced by retroviral vectors carrying a normal human factor IX cDNA under control of either the Moloney murine leukemia virus long terminal repeat (MoMuLV LTR) (LIXSN), the SV40 promoter (LNSVIX), or a cytomegalovirus (CMV) promoter (LNCIX). Factor IX production was measured in the transduced cells both in the uninduced state and after induction of granulocytic differentiation [with dimethylsulfoxide (DMSO)] or monocytoid differentiation [with phorbol myristic acetate (PMA)]. Transcription of factor IX from the MoMuLV LTR was seen in all cells, with a two-fold increase upon differentiation. Induction with PMA led to an 8- to 15-fold increase in factor IX transcripts from an internal CMV promoter. No factor IX transcripts from the internal SV40 promoter were detected. Immunoreactive factor IX protein was identified by Western blot from induced HL-60 cells transduced by either LIXSN or LNCIX. Factor IX production by HL-60 cells transduced by LNCIX ranged from 38-93 ng/10* cells/24 hr following induction of monocytic differentiation. The factor IX antigen titer was directly related to factor IX coagulant titer (r = 0.98; p < 0.001). These data indicate that human myelomonocytic cells are capable of performing the necessary post-translational modi- fications to produce functional factor IX. These studies suggest the potential of autologous transplantation of factor IX-transduced bone marrow cells for gene therapy of hemophilia B. OVERVIEW SUMMARY Hemophilia B accounts for 10% of the cases of hemophiUa and has clinical severity similar to classic hemophiUa (hemophilia Hemophilia B results from genetic deficiency of factor IX. A, factor VIII deficiency) (Thompson, 1986). The mainstay of In this study, Hao et al. explore the possibility of using current therapy for hemophilia B is administration of plasma- hematopoietic cells as the source of factor IX protein after derived factor IX concentrates. Although thistieatmentallevi- gene therapy. The results demonstrate that some of the vec- ates bleeding, factor replacement must be given repeatedly tors that were examined produce significant amounts of fac- throughout life, is costiy, and carriesrisksof vims transmission tor IX, which possesses functional clot-promoting activity. (Eyster et al, 1987; Troisi et al, 1993), thromboembolism Thus, gene therapy for hemophilia B may be performed us- (Lusher et al, I99I), and inhibitor formation (Kasper, 1989). ing bone marrow cells. Gene replacement therapy is a potential altemative tteatment for hemophiUa B. Modification of a patient's cells by insertion of a normal factor IX gene could provide a continuous in vivo INTRODUCTION source of circulating factor IX. Because the normal site of fac- tor IX synthesis is in hepatocytes, initial attempts to express EMOPHiLiA B IS AN X-LINKED HEMORRHAGIC DISORDER factor IX focused on hcpatocytcttansduction.These studies caused by deficiency of functional clotting factor IX. have shown that inttoduction of a normal human factor IX Divisions of 'Research Immunology/Bone Marrow Transplantation and ^Hematology/Oncology, Childrens Hospital Los Angeles, Departments of Pediatrics and Microbiology, University of Southem Califomia School of Medicine, Los Angeles, CA 90027. 873