Bone Marrow Transplantation (2003) 32, 1–7 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00 www.nature.com/bmt Mini review Hematopoietic stem cell gene therapy: progress toward therapeutic targets JL Vollweiler, SP Zielske, JS Reese and SL Gerson Division of Hematology–Oncology, Comprehensive Cancer Center at University Hospitals of Cleveland, Case Western Reserve University School of Medicine, USA Summary: the preclinical and clinical studies, gene therapy studies has also taught us a great deal about stem cell transplant The concept of hematopoietic stem cell gene therapy is as biology. exciting as that of stem cell transplantation itself. The Gene therapy attracted somewhat premature enthusiasm past 20 years of research have led to improved techniques in the mid-1980s, as a targeted and permanent treatment for transferring and expressing genes in hematopoietic for many previously incurable disorders. However, the stem cells and preclinical models now routinely indicate field has met with more clinical disappointment than the ease with which new genes can be expressed in success until very recently. Early results raised more repopulating stem cells of multiple species. Both modified questions than they answered and helped to focus attention murine oncoretroviruses and lentiviruses transmit genes on the elusive prerequisites of identifying an appro into the genome of hematopoietic stem cells and allow priate target cell, establishing efficient transfer of the expression in the host following transplantation. Using therapeutic gene maintaining stable integration and ex- oncoretroviruses, therapeutic genes for severe combined pression of the gene and understanding and explaining immunodeficiency, common variable gamma chain im- the risks involved. munodeficiency, chronic granulomatous disease, Hurler’s and Gaucher’s Disease have all been used clinically with only modest success except for the patients with Bone marrow derived stem cells immunodeficiency in whom a partial T-cell chimerism has been dramatic. Since stem cell selection in vivo Hematopoietic stem cells are arguably the most attractive appears important to the therapeutic success of gene and best-studied target cell population for several reasons. transfer, drug resistance selection, most recently using the They are easily accessible for harvest, and readily delivered MGMT gene, has been developed and appears to be safe. back to the patient by autologous transplant methods that Future trials combining a drug resistance and therapeutic are already familiar and commonplace to transplant gene are planned, as are trials using safety-modified physicians. They are long-lived, and have an enormous lentiviruses. The therapeutic potential of hematopoietic repopulation potential. Furthermore, they are pluripotent stem cell gene therapy, particularly given recent advances and hold the potential to correct defects in all hemato- in stem cell plasticity, remains an exceptionally exciting poietic lineages, and even nonhematopoietic cell lines via area of clinical research. transdifferentiation. Bone Marrow Transplantation (2003) 32, 1–7. doi:10.1038/ Drawbacks of using hematopoietic stem cells as targets sj.bmt.1704081 include historically low rates of successful transduction, Keywords: hematopoietic stem cell; gene therapy; human; given their lack of cell surface receptors and quiescent state. retrovirus; lentivirus; severe combined immune defi- Also, identification of the ideal stem cell has proven elusive. ciency In most clinical gene therapy trials, CD34+ cells are isolated from blood or marrow using immunomagnetic selection and transduced. However, evidence suggests there are CD34À cells that possess the properties of stem cells1,2 and that even a single CD34À cell is capable of long-term 3 Since 1985, when the first studies with stem cell gene repopulation of hematopoiesis. Finally, CD34 has been 4 transfer were reported, we have been tantalized by the shown to be expressed reversibly on murine stem cells. possibilities of gene therapy. It at once captivates us Better identification of the true hematopoietic stem cell, with the promise of curing a vast array of inherited, which appears to be modulated by age, cytokine activation, malignant and even infectious diseases, and challenges us cell cycle state and purification technique, as well as with feasibility, safety and ethical considerations. In both attention to the accessory cells used in the transplantation protocol, may improve transduction efficiency and clinical results in the future. Correspondence: Dr SL Gerson, Case Western Reserve University Other bone marrow-derived cell types of interest include School of Medicine, 10900 Euclid Avenue BRB 3-West, Cleveland, mesenchymal stem cells and multipotent adult progenitor OH 44106-4937, USA cells, or MAPCs. Mesenchymal stem cells isolated from the Hematopoietic stem cell gene therapy JL Vollweiler et al 2 bone marrow have the ability to differentiate into not only Table 1 Therapeutic targets of corrective stem cell gene therapy hematopoietic support stroma, but osteocytes, chondro- (A) Primary immunodeficiencies cytes, tenocytes, adipocytes and skeletal myocytes. In their ADA-deficient SCID role as support stroma, mesenchymal stem cells have shown X-linked SCID promise in improving the transduction efficiency of Common variable immunodeficiency hematopoietic stem cells, immuno-downmodulation of Chronic granulomatous disease [CGD] X-linked agammaglobulinemia graft-vs-host disease after an allograft and potential Wiskott–aldrich syndrome engraftment.5,6 Mesenchymal stem cells also express glucocerebrosidase, alpha-l-iduronidase, arylsulfatases (B) Hemoglobinopathies and adrenoleukodystrophy protein, raising the possibility Sickle cell anemia of a therapeutic application in lysosomal storage disea- Beta-thalassemia sescharacterized by loss of expression of these proteins, and (C) Other single-gene disorders in other diseases requiring replacement therapy. Although Hurler’s disease allogeneic bone marrow transplantation has failed to show Gaucher’s disease engraftment of donor mesenchymal stem cells,7 preclinical Hemophilia A, B models using autologous mesenchymal stem cells and gene Alpha-1 antitrypsin deficiency 8 transfer have had early success. (D) Stem cell defects MAPCs are a unique, newly described class of stem cells, Fanconi anemia characterized as CD34-, c-kitÀ, HLA-DRÀ, but AC133+, VEGFR2/Flk1/KDR+, and VEGFR1/Flt1+. They have demonstrated a superior proliferative potential with cyto- genetic and telomeric stability, while maintaining multi- been the most widely studied. These vectors are based on potentiality. They can differentiate in vitro into simple type C murine oncoretroviruses such as murine mesenchymal lineage cells, neural cells, hepatocyte-like leukemia virus. An appealing property of retroviral vectors cells and angioblasts.9,10 A potential application involves is that after reverse transcription of their genomic RNA early preclinical work using marrow-derived MAPCs to into proviral DNA, it is integrated into the host cell’s neovascularize infracted myocardium.11 genome at random locations. This means that all progeny derived from a transduced cell will contain the provirus, and therefore potentially express the therapeutic gene. Therapeutic targets Hence, an integrating vector is needed when expression of the transgene is required after many cell divisions, such Diseases of hematopoietic stem cells and their progeny are as when a gene needed in terminally differentiated blood obvious choices for gene therapy. Classically, this has cells is transferred to HSC. The oncoretroviral genome involved providing a therapeutic gene to patients with a is simple in that three genes are encoded. The gag, pol single locus genetic deficiency such as any of the genetic and env genes code for structural, replicative and disorders that make up severe combined immunodeficiency envelope proteins, respectively. Each end of the integrated (SCID), adenosine deaminase (ADA) deficiency, hemoglo- provirus contains long terminal repeat (LTR) sequences binopathies, glycogen storage diseases such as Gaucher’s that have promoter activity for driving the expression and Hurler’s, and disorders of stem cell proliferation such of viral genes. as Fanconi’s anemia (Table 1). More recently, there has Most current viral gene therapy vectors targeting been interest in not just correcting a deficit, but also hematopoietic stem cells are modified in a number of key providing stem cells with new or enhanced properties. This ways to make them safe for clinical use. First, the vectors has typically been investigated in the setting of malignant are ‘gutted’ of the endogenous viral genes necessary for disease. Approaches include chemotherapy resistance viral replication and budding from the cell. Only the genetic genes, and expression of tumor-specific antigens or information necessary for reverse transcription of the viral cytokines to stimulate immune responses against tumors RNA into DNA, its subsequent transport to the nucleus among differentiated progeny such as dendritic cells and and its integration into the genome are retained. Second, lymphocytes. In addition, the ability of hematopoietic stem the vectors are modified to accept the therapeutic gene. cells to promote cardiac muscle healing suggests a possible Third, the vectors are modified to enhance expression role in the delivery of vascular endothelial growth factor, of the new gene in the target cell and its progeny for many VEGF.