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

Abstract

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.