Development of a PML-RARα-IL-2 Recombinant Plasmid DNA for APL.

Abstract

Specific active immunotherapy with DNA vaccines is one of the best approaches to eradicate minimal residual disease in cancer patients, when appropriate tumor-specific antigens are identified for this disease. However, little data are regarding the DNA vaccines in acute leukemia, recent reports has shown that a human PML-RARα breakpoint-drived DNA vaccine can prevent APL in mice model, but the host immune response were weakly, due to the weak immunogenicity. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we develop a vector coexpressing PML-RARα gene fused to human Interleukin- 2 (hIL-2) gene. PML-RARα fusion gene segment and the full-lenght hIL-2 gene were amplified from NB4 cells or Jurkat cells. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hIL-2. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/hIL-2 mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same sequence of PML-RARα gene and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/hIL-2 mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Sal I/Not I) and sequence analysis, the PML-RARα/hIL-2 mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. Our results indicated that the recombinant plasmid expressing PML-RARα and hIL-2 was successfully constructed, which can induce more effective immune response and anti-APL cells effect in animal models. The data suggest that effective vaccination approaches should be possible against APL. It could be farther used in the research as PML-RARα DNA vaccine for APL.