Investigation of the Potency of KALA and REV Cell-Penetrating Peptides for In Vitro/In Vivo Delivery of an HPV Multiepitope DNA Construct.

Developing human papillomavirus (HPV) therapeutic DNA vaccines requires an effective delivery system, such as cell-penetrating peptides (CPPs). In the current study, the multiepitope DNA constructs harboring the immunogenic and conserved epitopes of the L1, L2, and E7 proteins of HPV16/18 (pcDNA-L1-L2-E7 and pEGFP-L1-L2-E7) were delivered using KALA and REV CPPs with different properties in vitro and in vivo. Herein, after confirmation of the REV/DNA and KALA/DNA complexes, their stability was investigated against DNase I and serum protease. Then, their entry into HEK-293T eukaryotic cells was analyzed by qualitative and quantitative methods. Finally, anti-tumor effects of the peptide/DNA complexes were investigated in the C57BL/6 mouse model. Based on the obtained data, the REV/DNA and KALA/DNA complexes at the N/P ratio of 5:1 demonstrated successful penetration into HEK-293T cells. Furthermore, in vivo studies represented that the REV/DNA (survival rate: 75%) and KALA/DNA (survival rate: 50%) complexes provided significant protection against C3 tumors in mice. Indeed, REV CPP exhibited a higher survival rate and lower tumor volume than KALA CPP, 50 days after the C3 challenge. These findings represented the potential of KALA and REV CPPs, especially REV, as promising gene delivery systems for developing HPV therapeutic DNA vaccine candidates.