Efficient adenovirus-mediated gene transduction of normal and leukemic hematopoietic cells

Abstract

We evaluated the efficiency of adenovirus-mediated gene targeted cells. A novel protocol for the efficient transduction transfer into normal and malignant human hematopoietic of adenovirus into B-CLL cells was presented. We showed cells. An E-1 and E-3 deleted, replication-defective recomthat anti-CD40 mAb or CD40 ligand acts in synergy with binant Ad.RSV␤gal vector was used and the transduction rhIL-4 to enable the transduction of approximately 50-75% efficiency was studied at a multiplicity of infection of 13 of B-CLL cells (B-CLL, n = 6). Expression of ␤-galactop.f.u. per cell. Approximately 40-50% of normal monocytes sidase in transduced CML cells and B-CLL cells was were transduced, whereas purified normal resting T cells detected for at least 15 days after transduction. The and B cells were resistant to infection. We showed that 50-present studies underline the utility of adenovirus vectors 80% of primary chronic myeloid leukemia cells (CML, n = for the construction of cytokine gene-modified tumor vac-12) were efficiently transduced. In contrast to CML, succines for the treatment of hematopoietic malignancies such cessful transduction of resting primary chronic B lymphoas CML and B-CLL. cytic leukemia cells required appropriate preactivation of for 3 days with rhGM-CSF (100 U/ml) before infection. Activation of cells with GM-CSF did not influence the (CD14 + , 90-98%) were readily transduced and stained transduction rate (data not shown). strongly positive for ␤-galactosidase (five separate experiments). Unlike the experience with retrovirus, we rhIL-4 together with hCD40 ligand are efficient and showed that efficient transduction of normal peripheral necessary for the transduction of primary B-CLL cells blood monocytes can be achieved with adenovirus withMononuclear cells from chronic B lymphocytic leukemia out preactivation of target cells. In contrast, purified (B-CLL, n = 6) patients were studied for adenoviral vector resting T lymphocytes (CD3 + , Ͼ96%) and B lymphocytes transduction. B-CLL cells were Ig-light chain restricted (CD19 + , Ͼ95%) were, however, not permissive to transand displayed CD5 (100%) and CD19 (80-95%) on the duction (three separate experiments).cell surface. Similarly to normal resting B lymphocytes (Figure 1), Efficient transduction of primary CML cells without resting primary B-CLL cells were resistant to adenovirus preactivation of target cells infection. A number of activation protocols [15][16][17][18] were Unselected mononuclear cells from chronic myeloid leutested to improve the transduction efficacy of B-CLL kemia patients (CML, n = 12) were included in the cells. We found that neither phorbol ester induction nor present studies. Although all the CML cells were Ph + , but the combination of SAC + thioredoxin + IL-2, forskolin represented heterogeneous cell populations. CML cells nor TNF␣ were efficient in improving the transduction expressed CD13 (40-80%), CD33 (40-81%) and other cell rate. In marked contrast, approximately 70-75% B-CLL surface markers (Table 1). No sign...

Efficient infection of primitive hematopoietic stem cells by modified adenovirus

“…28 Only 40% of the total bone marrow, 15% of CD34 + stem cells, 9 and a small proportion of T 29 and B lymphocytes express CAR, while integrins are generally absent on hematopoietic cells and cell lines. Mitogens and IL2 induce expression of integrins 30 and CAR, respectively, on T cells, 29 while CD40 cross-linking 31 and IL4 stimulation 32 up-regulate integrins on B cells and allow them to be infected by adenovirus. Similarly, CD34 + cells appear to be more permissive to Ad5 infection after culture with various cocktails of cytokines including SCF, IL3, G-CSF, Flt3 ligand, Tpo, and c-Kit ligand.…”

Efficient gene transfer in CLL by mRNA electroporation

“…Successful adenoviral transduction of CLL cells was achieved, but only when the virus was administered at a very high multiplicity of infection (MOI). [10][11][12][13] Adeno-associated viral vectors were used as an alternative, but appropriate prestimulation of the CLL cells by CD40 ligand (CD40L) expressing feeder cells or IgM crosslinking appeared to be necessary for transgene expression. [14][15][16] Viral gene transfer methods suffer from additional drawbacks: the production of vectors is laborious and time-consuming and needs specialized equipment, laboratory costs are elevated due to safety requirements, clinical applications are currently restricted and target cells need to express specific receptors to allow binding and entry of the virus.…”

Recombinant adenovirus-mediated cytotoxic gene therapy of lymphoproliferative disorders: is CAR important for the vector to ride?

“…7,28,[43][44][45] These initial studies were discouraging for the low infectivity shown by the cells and fresh tissue tested. [43][44][45] In one study, high moi of adenoviral particles were necessary to infect cells derived from CLL.…”

“…7,28,[43][44][45] These initial studies were discouraging for the low infectivity shown by the cells and fresh tissue tested. [43][44][45] In one study, high moi of adenoviral particles were necessary to infect cells derived from CLL. 28 In recent years, other investigators and we have shown that the expression of CAR that was detected by flow cytometry analysis of cells stained with monoclonal antibody anti-CAR, RmcB, varies among cells derived from several lymphoproliferative malignancies as compared with normal cell counterparts.…”