Systemic RALA/iNOS Nanoparticles: A Potent Gene Therapy for Metastatic Breast Cancer Coupled as a Biomarker of Treatment

Abstract

This study aimed to determine the therapeutic benefit of a nanoparticular formulation for the delivery of inducible nitric oxide synthase (iNOS) gene therapy in a model of breast cancer metastasis. Nanoparticles comprising a cationic peptide vector, RALA, and plasmid DNA were formulated and characterized using a range of physiochemical analyses. Nanoparticles complexed using iNOS plasmids and RALA approximated 60 nm in diameter with a charge of 25 mV. A vector neutralization assay, performed to determine the immunogenicity of nanoparticles in immunocompetent C57BL/6 mice, revealed that no vector neutralization was evident. Nanoparticles harboring iNOS plasmids (constitutively active cytomegalovirus [CMV]-driven or transcriptionally regulated human osteocalcin [hOC]-driven) evoked iNOS protein expression and nitrite accumulation and impaired clonogenicity in the highly aggressive MDA-MB-231 human breast cancer model. Micrometastases of MDA-MB-231-luc-D3H1 cells were established in female BALB/c SCID mice by intracardiac delivery. Nanoparticulate RALA/CMV-iNOS or RALA/hOC-iNOS increased median survival in mice bearing micrometastases by 27% compared with controls and also provoked elevated blood nitrite levels. Additionally, iNOS gene therapy sensitized MDA-MB-231-luc-D3H1 tumors to docetaxel treatment. Studies demonstrated that systemically delivered RALA-iNOS nanoparticles have therapeutic potential for the treatment of metastatic breast cancer. Furthermore, detection of nitrite levels in the blood serves as a reliable biomarker of treatment.

RCC2, a regulator of the RalA signaling pathway, is identified as a novel therapeutic target in cisplatin‐resistant ovarian cancer

“…In the downstream signaling pathway, Ral small GTPases (RalA), a member of the Ras branch of the Ras superfamily (19), has been shown to be significantly activated in colorectal cancer tissues and cell lines positively regulating cell growth and proliferation in a study by Martin et al (20). RalA is also involved in multifarious cancer progression, such as cervical cancer (21), hepatocellular carcinoma (22), chronic myelogenous leukemia (23), lung cancer (24), colorectal cancer (20), breast cancer (25) etc. In addition, Ral proteins mediate their activities through multiple downstream effectors, of which Ral binding protein-1 (RalBP1) is an important downstream effector, which is expressed at higher levels in cancer cells (26,27).…”

Gene therapy with RALA/iNOS composite nanoparticles significantly enhances survival in a model of metastatic prostate cancer

“…The improvement in survival in this prostate cancer model was much higher (56% increase in median survival in the PC-3 model versus a 27% increase in median survival in the breast cancer model), although an explanation for this has not been ascertained. MDA-MB-231 breast cancer cells are a notoriously aggressive model of breast cancer, and the progression of disease was faster in that model (the median survival of control mice in this study was 90.5 days, but only 31.5 days in the breast model), which may account for the observed differences (McCrudden et al 2017 ).…”

“…It was previously reported that RALA/iNOS nanoparticulate gene therapy produced ·NO production in vitro, and improved survival following systemic administration in a model of breast cancer metastasis (McCrudden et al 2017 ). The results reported here reinforce those findings in prostate cancer.…”

“…Indeed, previous research has reported abrogation of the growth of ZR-75-1 breast cancer xenografts when treated with RALA/pFKBPL (Bennett et al 2015 ), and the potency of RALA-delivered inducible nitric oxide synthase (iNOS) in a model of breast cancer metastasis. Survival of mice bearing aggressive MDA-MB-231 micrometastases was significantly increased by treatment with nanoparticles comprising RALA and a constitutively active or a transcriptionally regulated iNOS plasmid (McCrudden et al 2017 ).…”

Delivery across the blood-brain barrier: nanomedicine for glioblastoma multiforme

“…2) [97]. RALA has been utilised to deliver a range of cargo including plasmids encoding reporter genes, small interfering RNA (siRNA), messenger RNA (mRNA), and therapeutic plasmids, in addition to anionic small molecule (such as bisphosphonates) and incorporation into polymeric microneedles [97,[101][102][103][104][105]. This demonstrates the immense potential of RALA as a CNS delivery agent, highlighting the versatility of fusogenic peptides in general.…”