Gene Therapy (1998) 5 , 692–699  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http:/ / www.stockton-press.co.uk/ gt Gene transfer into human umbilical cord blood-derived CD34  cells by particle-mediated gene transfer S Verma 1 , C Woffendin 1 , I Bahner 2 , U Ranga 1 , L Xu 1 , Z-Y Yang 1 , SR King 1 , DB Kohn 2 and GJ Nabel 1 1 Howard Hughes Medical Institute, University of Michigan Medical Center, Departments of Internal Medicine and Biological Chemistry, Ann Arbor, MI; and 2 Children’s Hospital Los Angeles, Division of Research Immunology, Bone Marrow Transplantation, Los Angeles, CA, USA Delivery of genes into hematopoietic progenitor cells offers lowing transfection with a neomycin resistance gene, differ- an attractive means for the introduction of corrective or pro- entiation into cells of the myeloid lineage was observed, tective genes into cells of both the myeloid and lymphoid assayed by CFU-GM in the presence of G-418. Both lineage. Previously, investigators have often used murine unstimulated and stimulated cells gave rise to CFU-GM in retroviral vectors for gene delivery which require cells to the presence of G-418, indicating that stable expression be cycling for efficient delivery. We describe a nonviral of the neomycin resistance gene was maintained in early method of gene delivery using particle-mediated gene progenitors. These results demonstrate that particle- transfer to obviate many disadvantages of viral vectors mediated gene transfer into human hematopoietic cells related to safety, production costs and the need for cell from umbilical cord blood can be achieved without affecting cycle proliferation. Using a CMV-CAT reporter plasmid, we their CFU-GM differentiation potential. This gene transfer show transfection of highly purified CD34 + cells isolated method offers an alternative approach to gene therapy from umbilical cord blood. Effective gene transfer was studies involving human hematopoietic progenitor cells. shown in unstimulated and in growth-stimulated cells. Fol- Keywords: gene therapy; hematopoiesis; biolistics; stem cells Introduction Gene therapy offers the potential to contribute to the treatment of many human diseases, including inherited or acquired diseases. The hematopoietic progenitor cell (HPC) represents an attractive target cell for the introduc- tion of corrective or protective genes into multiple lin- eages and can be readily isolated from the bone marrow, peripheral and umbilical cord blood, from which they can be transduced and eventually used to reconstitute the hematopoietic lineage of transplant recipients. Previous studies involving the transfection of CD34 + HPCs have mainly utilized murine leukemia virus (MLV)-derived retroviral vectors to transduce cells. Successful transfer and expression of various genes by these retroviral vec- tors into human bone marrow progenitors and long-term culture-initiating cells aimed at the eventual treatment of various diseases such as ADA deficiency, 1–4 X-linked granulomatous disease, 5 alpha-L -iduronidase deficiency (Hurler syndrome), 6 Gaucher’s disease 7–9 and AIDS 10,11 Correspondence: GJ Nable, Howard Hughes Medical Institute, University of Michigan Medical Center, Departments of Internal Medicine and Bio- logical Chemistry, 1150 W Medical Center Drive, 4520 MSRB I, Ann Arbor, MI 48109–0650, USA The first two authors contributed equally Received 3 March 1997; accepted 5 January 1998 have been reported. Other viral vectors based on the defective parvovirus, adeno-associated virus (AAV), 12–14 and an HIV-1 based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G 15 have also been used to produce stable gene transfer in hemato- poietic cells. A possible method for immune mainten- ance/reconstitution in the pediatric population is via the use of autologous cord blood (CB) stem/progenitor cells collected at birth from the umbilical cord of the mother. For example, in pediatric AIDS, therapeutic anti-HIV genes could be transduced into cord blood-derived CD34 + hematopoietic progenitor/stem cells and engrafted into the HIV-seropositive child. Establishment of these cells may confer protection to the various cells susceptible to HIV infection, especially cells of the myeloid and lymphoid lineages. Previous reports have begun to evaluate the expression, stability and efficacy of introducing HIV protective genes into hematopoietic stem/progenitor cells. MLV-derived retroviral vectors expressing Rev-responsive element (RRE) decoys, 11 hair- pin ribozymes, 16,17 and Rev M10 18 have been shown to confer a degree of resistance to HIV infection in macro- phage-like cells derived from transduced progenitor/stem cells when challenged with diverse strains of HIV in vitro. Given the extensive use of viral vectors for gene deliv-