Gene Therapy With RALA/iNOS Composite Nanoparticles Significantly Enhances Survival In A Model Of Metastatic Prostate Cancer

Abstract Background Recent approvals of gene therapies by the FDA and the EMA for treatment of inherited disorders have further opened the door for assessment of nucleic acid pharmaceuticals for clinical usage. Arising from the presence of damaged or inappropriate DNA, cancer is a condition particularly suitable for genetic intervention. The RALA peptide has been shown to be a potent non-viral delivery platform for nucleic acids. This study examines the use of RALA to deliver a plasmid encoding inducible nitric oxide synthase (iNOS) as an anti-cancer treatment. Methods The physiochemical properties of the RALA/DNA nanoparticles were characterized via dynamic light scattering and transmission electron microscopy. The nanoparticles were labelled with fluorophores and tracked over time using confocal microscopy with orthogonal sections to determine cellular location. In vitro studies were employed to determine functionality of the nanoparticles both for pEGFP-N1 and CMV-iNOS. Nanoparticles were injected intravenously into C57/BL6 mice with blood and serum samples analysed for immune response. PC3-luc2M cells were injected into the left ventricle of SCID mice followed by treatment with RALA/CMV-iNOS nanoparticles to evaluate the tumour response in a metastatic model of prostate cancer. Results Functional cationic nanoparticles were produced with gene expression in PC-3 prostate cancer cells. Furthermore, repeated administrations of RALA/DNA nanoparticles into immunocompetent mice did not produce any immunological response: neutralization of the vector or release of inflammatory mediators. RALA/CMV-iNOS reduced the clonogenicity of PC-3 cells in vitro, and in an in vivo model of prostate cancer metastasis, systemically delivered RALA/CMV-iNOS significantly improved the survival of mice. Conclusion These studies further validate RALA as a genetic cargo delivery vehicle and iNOS as a potent therapy for the treatment of cancer

DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer