Adenovirus mediated alpha interferon (IFN-alpha) 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-alpha (IFN-alpha) 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-alpha to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-alpha in these cells. Ad-cytomegalovirus promoter driven IFN-alpha (AdCMV-IFN-alpha) 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-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells. Transient expression of the IFN-alpha 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-alpha mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-alpha under similar conditions as described 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 into hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.
Feldman
E
E
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Ahmed
T
T
Lutton
J D
JD
Farley
T
T
Tani
K
K
Freund
M
M
Asano
S
S
Abraham
N G
NG
D000256:Adenoviridae / Q000235:genetics; D018952:Antigens, CD34 / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology; D003114:Colony-Forming Units Assay; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics* / Q000627:therapeutic use / Q000633:toxicity; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000628:therapy; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D013234:Stem Cells / Q000187:drug effects / Q000276:immunology / Q000502:physiology; D014162:Transfection
D018952:Antigens, CD34; D018076:DNA, Complementary; D016898:Interferon-alpha; D012333:RNA, Messenger