Construction and characterization of retroviral vectors for interleukin-2 gene therapy.
Several investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was subcloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/10(6) cells/24 h), adult fibroblasts (625 units/10(6) cells/24 h), and embryonic fibroblasts (3,975 units/10(6) cells/24 h) were 150- to 1,000-fold higher than than secreted by the activated PBMC (4 units/10(6) cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/10(6) cells/24 h pLXSN-iIL2 = 375-625 vs. pLXSN-tIL2 = 90-440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7-11% by day 7, 0-29% by day 14, and 25-50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis of SacI-digested genomic DNA from the LXSN-tIL2 producer cell line and SacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.
Fakhrai
H
H
Sidney Kimmel Cancer Center, Clemma A. Hewitt Gene Therapy Laboratory, San Diego, California, USA.
Shawler
D L
DL
Van Beveren
C
C
Lin
H
H
Dorigo
O
O
Solomon
M J
MJ
Gjerset
R A
RA
Smith
L
L
Bartholomew
R M
RM
Boggiano
C A
CA
Gold
D P
DP
Sobol
R E
RE
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin; D007376:Interleukin-2 / Q000235:genetics*; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D013925:Thromboplastin / Q000235:genetics
D007328:Insulin; D007376:Interleukin-2; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D013925:Thromboplastin; D011384:Proinsulin