DNA nanotherapy for pre-neoplastic cervical lesions.

ABSTRACT

OBJECTIVE: This study aims to test the hypothesis that targeted nanoparticle delivery of DNA encoding HPV16-regulated diphtheria toxin (DT-A) will result in the death of HPV16-infected cells. MATERIALS AND METHODS: Plasmid constructs containing a HPV16 Long Control Region (LCR) DNA sequence upstream of DT-A or luciferase reporter (Luc) DNA sequences were used to formulate poly(ß-amino ester) nanoparticles. The effect on tumor growth of HPV/DT-A-nanoparticle injection directly into HPV16(+) CaSki human cervical cancer cell-derived xenografts in mice was determined. To evaluate the ability of the HPV16 LCR regulatory sequence to activate gene expression specifically in HPV16-infected cells, mice underwent bioluminescent optical imaging following intraperitoneal injection of HPV/Luc-nanoparticles. The use of Lutrol F127, a thermal-sensitive gel, to target delivery of nanoparticles and subsequent gene expression to cervical epithelial cells was evaluated in ex vivo cultures of mouse cervix and following intravaginal delivery of nanoparticle/gel in mice, as well as in ex vivo cultures of surgical LEEP samples. RESULTS: The selected HPV16 LCR regulatory sequence activates gene expression in both HPV16-infected cells and non-infected cells. However, in the cervix, it is specifically active in epithelial cells. Following exposure of cervical cells to HPV/DT-A-nanoparticles mixed with Lutrol F127 gel, DT-A is expressed and cells die. CONCLUSIONS: An HPV16 DNA sequence that targets gene expression specifically to HPV16-infected cells remains to be discovered. Topical application of a Lutrol F127 thermal gel/nanoparticle mix is illustrative of how to restrict exposure of cells to therapeutic nanoparticles, thereby allowing for targeted DNA delivery to cervical pre-cancerous lesions.