Up‐regulation of amphotrophic retroviral receptor expression in human peripheral blood CD34+ cells

Kaubisch, Andreas; Ward, Maureen; Schoetz, Stuti; Hesdorffer, Charles; Bank, Arthur

doi:10.1002/(sici)1096-8652(199908)61:4<243::aid-ajh4>3.3.co;2-a

Cited by 3 publications

(2 citation statements)

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“…Reports in humans and mice have associated low transduction efficiencies with the low expression of viral binding proteins, notably the xenotropic receptor GaLV-1 and the amphotropic receptor Ram-1 (Orlic et al, 1996;Sabatino et al, 1997). Cryopreservation and growth of haematopoietic progenitors in phosphate-free medium has been shown to upregulate Ram-1 expression (Orlic et al, 1996;Chien et al, 1997;Kaubisch et al, 1999). Both canine Cf2Th and CD34 + cells express Ram-1 mRNA.…”

Section: Discussionmentioning

confidence: 66%

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

Suter¹,

Gouthro²,

McSweeney

et al. 2006

Vet Res Commun

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We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

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Section: Discussionmentioning

confidence: 66%

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

Suter¹,

Gouthro²,

McSweeney

et al. 2006

Vet Res Commun

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“…One of the alternative mechanisms by which cytokines enhance gene transfer into hematopoietic cells is upregulation of viral receptor expression. In both CD34 1 and CD34 1 38 2 cells, cytokines have been shown to increase expression of the receptor specific for the amphotropic virus envelope PiT-2 (Crooks and Kohn, 1993;Orlic et al, 1996;Kaubisch et al, 1999). Similar results have been obtained for the GaLV-receptor PiT-1 in hematopoietic cell lines (Sabatino et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Optimization of Gene Transfer into Primitive Human Hematopoietic Cells of Granulocyte-Colony Stimulating Factor–Mobilized Peripheral Blood Using Low-Dose Cytokines and Comparison of a Gibbon Ape Leukemia Virus Versus an RD114-Pseudotyped Retroviral Vector

et al. 2002

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Primitive human hematopoietic cells in granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) are more difficult to transduce compared to cells from umbilical cord blood. Based on the hypothesis that MPB cells may require different stimulation for efficient retroviral infection, we compared several culture conditions known to induce cycling of primitive hematopoietic cells. MPB-derived CD34(+) cells were stimulated in the presence or absence of the murine fetal liver cell line AFT024 in trans-wells with G-CSF, stem cell factor (SCF), and thrombopoietin (TPO) (G/S/T; 100 ng/ml) or Flt3-L, SCF, interleukin (IL)-7, and TPO (F/S/7/T; 10-20 ng/ml), and transduced using a GaLV-pseudotyped retroviral vector expressing the enhanced green fluorescence protein (eGFP). Compared to cultures without stroma, the presence of AFT024 increased the number of transduced colony-forming cells (CFC) by 3.5-fold (with G/S/T), long-term culture-initiating cells (LTC-IC) by 4.6-fold (with F/S/7/T), and nonobese diabetic/severe immunodeficiency disease (NOD/SCID)-repopulating cells (SRC) by 6.8-fold (with F/S/7/T). Similar numbers of long-term culture-initiating cells (LTC-IC) and SRC could be transduced using AFT024-conditioned medium (AFT-CM) or a defined medium that had been supplemented with factors identified in AFT-CM. Finally, using our best condition based on transduction with the gibbon ape leukemia virus (GaLV)-pseudotyped vector, we demonstrate a 33-fold higher level of gene transfer (p < 0.001) in SRC using an RD114-pseudotyped vector. In summary, using an optimized protocol with low doses of cytokines, and transduction with an RD114 compared to a GaLV-pseudotyped retroviral vector, the overall number of transduced cells in NOD/SCID mice could be improved 144-fold, with a gene-transfer efficiency in SRC of 16.3% (13.3-19.9; n = 6).

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High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

et al. 2001

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Objectives: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. Methods: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38–CD34++lineage– (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (ΔNGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 µg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10–21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. Results: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.

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Up‐regulation of amphotrophic retroviral receptor expression in human peripheral blood CD34+ cells

Cited by 3 publications

References 0 publications

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

Optimization of Gene Transfer into Primitive Human Hematopoietic Cells of Granulocyte-Colony Stimulating Factor–Mobilized Peripheral Blood Using Low-Dose Cytokines and Comparison of a Gibbon Ape Leukemia Virus Versus an RD114-Pseudotyped Retroviral Vector

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

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