Adenovirus and retrovirus mediated interferon alpha gene transfer into CD34+ cells maintains regeneration capacity and enhances adhesion molecules in K562 cells.
BACKGROUND
Systemic administration of interferon-alpha (IFN-alpha) results in cytogenetic remissions and enhanced survival in a significant percentage of patients with chronic mylogenous leukemia (CML) and lymphoma. However, this treatment is associated with deleterious toxic effects. Gene transfer of the IFN-alpha gene into hematopoietic progenitors represents a novel strategy to deliver high concentrations of IFN-alpha to a local area.


METHODS
We compared the effect of the transfer of the IFN-alpha gene on the cell growth and differentiation of several CD34+ cells in culture and in a NOD/SCID animal model, using adenovirus and retrovirus constructs.


RESULTS
Transient local expression of the IFN-alpha gene using an adenovirus vector was associated with normal proliferation of CD34+ progenitors as measured by a colony forming unit of granulocyte-macrophage (CFU-GM) growth. Flow cytometric determination revealed that there was no significant difference in viability of these cells for 24-hour transduction periods. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of RNA from CD34+ harvested CFU-GM progenitors demonstrated expression of IFN-alpha mRNA; radioimmunoassay (RIA) revealed that transduced cells secreted substantial levels of IFN-alpha protein. Furthermore, we constructed a retroviral vector in which IFN-alpha cDNA was driven by a viral LTR promoter to evaluate the effect of permanent IFN-alpha gene expression on cell growth. Retroviral packaging cells PA317 with high titers of retrovirus were produced and used to infect CD34+ and K562 cells. RIA showed that IFN-alpha-transduced CD34+ cells (with the aid of fibronectin fragment CH-296) produced approximately 400 units of IFN-alpha protein compared to CD34+ cells, or cells transduced with empty vector. IFN-alpha transduced CD34+ generated similar numbers of CFU-GM colonies as compared to control CD34+ cells. Engraftment of CD34+ cells transduced with IFN-alpha gene in NOD/SCID mice was successful for the first 30 days. Additionally, we studied the effect of local IFN-alpha expression on the cellular adhesion molecules, VLA-4, Mac-1, ICAM-1, and L-selectin in K562 cells, and human umbilical endothelial vein cells. K562 cells transduced with the IFN-alpha gene expressed a significantly elevated level of VLA-4, Mac-1, and ICAM-1.


CONCLUSIONS
We conclude that expression of the IFN-alpha gene using retrovirus vectors results in an adequate localized expression of IFN-alpha mRNA and protein.
Seiter
K
K
Department of Medicine, New York Medical College, Valhalla 10595, USA.
Kancherla
R
R
Yang
L
L
Quan
S
S
Farley
T J
TJ
Abraham
N G
NG
Ahmed
T
T
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism; D020014:K562 Cells / Q000378:metabolism*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018952:Antigens, CD34; D015815:Cell Adhesion Molecules; D016898:Interferon-alpha; D012333:RNA, Messenger