ADENOVIRUS-MEDIATED ALPHA-INTERFERON (IFN-ALPHA) GENE-TRANSFER INTO CD34(-CELLS() CELLS AND CML MONONUCLEAR)

Gene transfer or gene therapy has advantages in the treatment of a var iety of disorders due to its selective expression within specific mamm alian cells. Interferon-alpha (IFN-alpha) has been used in the managem ent of leukemia but its diverse adverse activities with multiple poten tial side effects, possibly unrelated to therapeutic targets, may nega tively influence the ability of IFN-alpha to treat this disorder. Ther efore, we examined the ability of adenovirus (Ad)-IFN-alpha gene const ruct to transfect normal (CD34(+) cells) and chronic myelogenous leuke mia (CML) bone marrow mononuclear cells (BMMNC) and the transient over expression of IFN-alpha in these cells. Ad-cytomegalovirus promoter dr iven IFN-alpha (AdCMV-IFN-alpha) at multiple doses was assessed to tra nsfect highly purified CD34(+) cells in liquid culture, and optimal tr ansduction of CD34(+) cells was achieved using 120 plaque forming unit s. Flow cytometric determinations revealed that there was no significa nt difference in cell viability for the 4 h or 24 h transfection perio ds. Immunoassay of IFN-alpha produced by CD34(+) cells shows that IFN- alpha levels increased several fold in transfected cells. Transient ex pression of the IFN-alpha gene did not suppress proliferation of CD34( +) progenitors as indicated by BFU-E or colony forming units-granulocy te-macrophage (CPU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34(+) harvested CFU-GM progenitor cell s demonstrated transient IFN-alpha mRNA expression. Similarly, CML BMM NC were transfected with AdCMV-IFN-alpha under similar conditions as d escribed for CD34(+) cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-alpha at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-alpha int o hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.