[Frontiers in Bioscience 4, e26-33, May 1, 1999] 26 STEM CELL DIRECTED GENE THERAPY Barbara C. Engel and Donald B. Kohn Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Department of Pediatrics, University of Southern California, School of Medicine TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Phenotype of hematopoietic stem cells 4. Sources of hematopoietic stem cells 5. Anti-HIV genes 6. Gene transfer into hematopoietic stem cells 6.1. Retroviral vectors 6.2. Lentiviral vectors 7. Gene expression 8. Clinical trials 8.1. Transfer of an anti-HIV-1 ribozyme into CD34 + PBSC from HIV-1-positive adults 8.2. Transfer of an RRE decoy gene into CD34 + cells from the bone marrow of HIV-1-positive children 8.3. Transfer of an anti-HIV-1 ribozyme into CD34 + PBSC from adults with HIV-1 and lymphoma 8.4. Umbilical cord blood cell trial 9. Perspective 10. References 1. ABSTRACT A potential therapeutic approach to HIV-1 infection is the genetic modification of cells of a patient to make them resistant to HIV-1. Hematopoietic stem cells are an attractive target for gene therapy of AIDS because of their ability to generate a broad repertoire of mature T lymphocytes, as well as the monocytic cells (macrophages, dendritic cells and microglia) which are also involved in HIV-1 pathogenesis. A number of synthetic "anti-HIV-1 genes" have been developed which inhibit HIV-1 replication. However, current methods for gene transfer into human hematopoietic stem cells, using retroviral vectors derived from the Moloney murine leukemia virus, have been minimally effective. Clinical trials performed to date in which hematopoietic cells from HIV-1-positive patients have been transduced with retroviral vectors and then reinfused have produced low to undetectable levels of gene-containing peripheral blood leukocytes. New vector delivery systems, such as lentiviral vectors, need to be developed to ensure efficient gene transfer and persistent transgene expression to provide life-long resistance to the cells targeted by HIV-1. 2. INTRODUCTION Although the recent breakthroughs in anti-viral chemotherapy have produced great benefits to many people infected with human immunodeficiency virus-1(HIV-1) (1- 3), there is still room for additional, complementary modalities to try to sustain immune function and health. As the field of gene therapy has developed, applications to treatment of infectious diseases, such as HIV-1 infection, have been explored (4-9). The question about the optimal target for gene therapy for HIV has not yet been resolved. Initial clinical trials used CD4 + T lymphocytes (7) which are relatively easy to obtain, isolate and transduce. As mature T cells have a limited life span, in the long run, repeated transfers of gene-modified cells would be required. In addition to CD4 + T lymphocytes, other cells like T cell precursors in the thymus and lymph nodes as well as monocytes/macrophages, dendritic cells and microglia in the brain are also infected during the course of the acquired immunodeficiency syndrome (AIDS). Hematopoietic stem cells (HSC) give rise to the full spectrum of cells involved in AIDS pathogenesis (10, 11). Therefore, the hematopoietic stem cells contained in either bone marrow, peripheral blood or the umbilical cord from newborns represent a logical target cell for gene therapy of AIDS. HSC are long-lived, producing new progeny cells for the life of the recipient after transplant. Theoretically, insertion of a gene capable of conferring resistance to HIV-1 into hematopoietic stem cells would result in that gene being present in the descendant mature T lymphocytes and other HIV-1 susceptible cells. Despite more than 15 years of research in the field of gene therapy using hematopoietic stem cells, the major hurdle remains the inability to efficiently and stably insert genes into these cells. Retroviral vectors based upon the Moloney murine leukemia virus (MLV) have been used most extensively, but yield relatively low gene transfer into pluripotent human HSC and gene expression which is often unsatisfactory. 3. PHENOTYPE OF HEMATOPOIETIC STEM CELLS Stem cells as well as lineage-committed hematopoietic progenitor cells share the sialomucin CD34