title: Delayed targeting of cytokine-nonresponsive human bone marrow CD34(+) cells with retrovirus-mediated gene transfer enhances transduction efficiency and long-term expression of transduced genes.
abstract: Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol using neoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34(+) cells. BM CD34(+) cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers of neoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transduced neoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: P
authors.initials: P
authors.lastname: Veena
authors.forename: C M
authors.initials: CM
authors.lastname: Traycoff
authors.forename: D A
authors.initials: DA
authors.lastname: Williams
authors.forename: J
authors.initials: J
authors.lastname: McMahel
authors.forename: S
authors.initials: S
authors.lastname: Rice
authors.forename: K
authors.initials: K
authors.lastname: Cornetta
authors.forename: E F
authors.initials: EF
authors.lastname: Srour
mesh_terms: D000328:Adult; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D002455:Cell Division; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D004352:Drug Resistance, Microbial; D005353:Fibronectins; D005434:Flow Cytometry; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology*; D006801:Humans; D018189:Immunomagnetic Separation; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D019089:Stem Cell Factor / Q000494:pharmacology; D014162:Transfection
chemical_list: D018952:Antigens, CD34; D005353:Fibronectins; D007377:Interleukin-3; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D019868:Kanamycin Kinase