N. G. Abraham

Adenovirus Mediated Alpha Interferon (IFN‐α) Gene Transfer into CD34+ Cells and CML Mononuclear Cells

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon‐α (IFN‐α) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN‐α to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)‐IFN‐α gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN‐α in these cells. Ad‐cytomegalovirus promoter driven IFN‐α (AdCMV‐IFN‐α) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN‐α produced by CD34+ cells shows that IFN‐α levels increased several fold in transfected cells. Transient expression of the IFN‐α gene did not suppress proliferation of CD34+ progenitors as indicated by BFU‐E or colony forming units‐granulocyte‐macrophage (CFU‐GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU‐GM progenitor cells demonstrated transient IFN‐α mRNA expression. Similarly, CML BMMNC were transfected with AdCMV‐IFN‐α under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN‐α at a substantial level. This in vitro data suggest that Ad‐mediated gene transfer of IFN‐α into hematopoietic stem cells can be achieved and that the IFN‐α gene can be translated into its specific mRNA in CD34 progenitor cells.

Cytochrome P450 Dependent Arachidonic Acid Metabolism in Hemopoietic Cells

This report demonstrates for the first time that human peripheral blood neutrophils and human bone marrow cells metabolize arachidonic acid (AA) by a cytochrome P450 dependent mechanism. The formation of the cytochrome P450 (P450) arachidonate metabolites is dependent on the addition of NADPH, prevented by SKF-525A (100 microM), which is an inhibitor of cytochrome P450 enzymes, but not affected by the addition of BW-755C, a dual inhibitor of cyclooxygenase and lipoxygenase activities. Addition of the Ca+(+)-ionophore A23187 to cell preparations stimulated the release of both cyclooxygenase and lipoxygenase products but did not affect the formation of the P450 metabolites. Incubation of cell preparations with 14C-AA yielded a P450 dependent peak which eluted at 19 min. on reverse phase HPLC and was distinct from 5 and 15-hydroxyeicosatetraenoic acids (HETEs), prostaglandins and other leukotrienes. Recovery of the P450 dependent metabolite(s) was accomplished and preparations were tested in bone marrow clonal culture in order to determine if the substance(s) has/have any effect on the growth and differentiation of bone marrow cells. Results demonstrated that some of the material possessed powerful erythroid colony (CFU-E) enhancing activity at concentrations between 10(-8)-10(-14)M. These results demonstrate that human bone marrow and peripheral blood neutrophils possess a third pathway for AA metabolism which is P450 dependent, and that metabolite(s) of this pathway may have potent stimulatory effects on erythropoiesis.

Effect of Retrovirus and Adenovirus Mediated Interferon Gene Transfer on Hematopoietic Progenitor Cell Growth

Gene therapy following gene transfer into hematopoietic cells (CD34+) is now being investigated for several genetic disorders. We have applied a similar approach using interferon gene transfer for treatment of chronic myelogenous leukemia (CML). IFN-α produces a hematologic and cytogenetic response in CML with a survival advantage for cytogenetic responders. Therefore, we examined the effect of transient over- expression of IFN-α using the adenovirus gene transfer approach. The ability of the adenovirus (Adv)-IFN-α gene construct to transfect normal and CML stem cells, CD34+, was examined. The peripheral blood mononuclear fraction from patients with CML treated with G-CSF or GM-CSF/G-CSF was enriched in CD34+ cells. Adv- cytomegalovirus (pCMV) promoter driven IFN-α at multiple doses was assessed to transfect highly purified CD34+ cells in the presence of matrix protein and in co- cultures with the stromal adherent cell layer. The use of cytokines enhances Adv- mediated IFN-α gene transfer into stem cells. Southern blot analysis demonstrated that the Adv-pCMV-IFN-α construct and IFN-α were expressed in cultured CD34+. Transient expression of the IFN-α gene did not suppress proliferation of CD34+ progenitors, CFU-Meg, BFU-E or CFU-CM growth. Reverse transcriptase/polymerase chain reaction (RT/PCR) analysis of RNA from CFU-GM progenitor cells demonstrated transient IFN-α mRNA expression in CD34+ cells. Immunoassay of IFN-α shows that IFN-α suggests that selective expression of IFN-α may be beneficial for CML therapy. We also report on the establishment of novel conditions which permit high efficiency of the retrovirus IFN-α gene into CD34+ cells. Stem cells transfected with retrovirus were infused intravenously to irradiated mice and spleen colony forming units were evaluated for IFN-α marked clones by Southern blot analysis. These studies demonstrate that the overexpression of IFN-α gene can be used suppression of K562 cell proliferation and suggest its possible use as gene therapy of CML. For permanent transfection, we constructed two IFNα cDNA-containing retroviral vectors LSN-IFNα was constructed by cloning human IFNα cDNA at the downstream of the 5′ long terminal repeat (LTR) promoter of the retroviral vector LXSN. Retrovirus-containing supernatants from PA317 cell line was used to infect NIH3T3 fibroblasts and K562 cells. The IFNα gene expression in transfected cells was confirmed by Northern Blot, RT-PCR, radioimmunoassay (RIA), and biological assay. Retroviral mediated IFN-α gene transfer infection results showed that cell proliferation and cell viability were significantly suppressed in LSN-IFNαa-infected K562 cells as compared to LXSN-infected K562. Furthermore, we found that retroviral mediated IFN-α causes upregulation of adhesion molecules such as VLA-4.