Antitumor effect of antibiotic resistance gene-free plasmids encoding interleukin-12 in canine melanoma model.

The electrotransfer of interleukin-12 (IL-12) has been demonstrated as an efficient and safe treatment for tumors in veterinary oncology. However, the plasmids used encode human or feline IL-12 and harbor the gene for antibiotic resistance. Therefore, our aim was to construct plasmids encoding canine IL-12 without the antibiotic resistance genes driven by two different promoters: constitutive and fibroblast-specific. The results obtained in vitro in different cell lines showed that following gene electrotransfer, the newly constructed plasmids had cytotoxicity and expression profiles comparable to plasmids with antibiotic resistance genes. Additionally, in vivo studies showed a statistically significant prolonged tumor growth delay of CMeC-1 tumors compared to control vehicle-treated mice after intratumoral gene electrotransfer. Besides the higher gene expression obtained by plasmids with constitutive promoters, the main difference between both plasmids was in the distribution of the transgene expression. Namely, after gene electrotransfer, plasmids with constitutive promoters showed an increase of serum IL-12, whereas the gene expression of IL-12, encoded by plasmids with fibroblast-specific promoters, was restricted to the tumor. Furthermore, after the gene electrotransfer of plasmids with constitutive promoters, granzyme B-positive cells were detected in the tumor and spleen, indicating a systemic effect of the therapy. Therefore, plasmids with different promoters present valuable tools for focused therapy with local or systemic effects. The results of the present study demonstrated that plasmids encoding canine IL-12 under constitutive and fibroblast-specific promoters without the gene for antibiotic resistance provide feasible tools for controlled gene delivery that could be used for the treatment of client-owned dogs.

Magnetic field contributes to the cellular uptake for effective therapy with magnetofection using plasmid DNA encoding against Mcam in B16F10 melanoma in vivo.

AIM: We explored the distribution and cellular uptake of intratumorally injected SPIONs-PAA-PEI-pDNA (magnetofection complexes), and antitumor effectiveness of magnetofection with plasmid DNA encoding short hairpin RNA (shRNA) against Mcam (pDNA(anti-MCAM)). MATERIALS & METHODS: Analyses were made based on the histology, ultrastructure and quantitative measurements of magnetofection complexes, and quantification of the antitumor effectiveness in B16F10 melanoma in vivo. RESULTS: Injected magnetofection complexes were distributed around the injection site. Exposure of tumors to external magnetic field contributed to the uptake of magnetofection complexes from extracellular matrix into melanoma cells. Three consecutive magnetofections of tumors with pDNA(anti-MCAM) resulted in significant reduction of tumor volume. CONCLUSION: Magnetofection is effective for gene delivery to melanoma tumors, but requires a magnetic field for cellular uptake and antitumor effect.