Optimisation of ultrasound-mediated delivery of mRNA to mammalian cells

Thesis

Gene delivery will cure all. That was the hope of many since the discovery and therapeutic implementation of genetic research. Although some of that hope has been realised, the delivery of nucleotides to cells in the body remains a major barrier in wider application. Ultrasound-mediated delivery of drugs has been investigated in various forms for decades and continues to be an area of engineering and science that is much explored. The body of work presented in this thesis strides to bring the two areas of research endeavour together.

Ultrasound-mediated delivery of drugs and antibody particles, normally in conjunction with cavitation nucleation agents, has been shown to be effective both in vitro and in vivo. In this work, we focus on nucleotide delivery, in particular mRNA, which has yet to be fully explored, especially in the context of achieving delivery of non-encapsulated, ‘free’ mRNA. Data presented here explores the relationship between cavitation and the transfer of, and protein expression from, free mRNA. The protein expression exhibited in various cell lines in vitro was further tested in vivo, using either sub-micron or micron-sized cavitation nuclei and the effects on transfection efficiency and expression time were examined. A luciferase reporter gene assay was used to characterise events in vitro across different cell lines including cancer and non-cancerous types. The impact and varying acoustic exposure parameters and cavitation on expression were assessed, and differences in expression time and transfection rate were observed. These data were used to determine appropriate acoustic parameters for in vivo delivery. Cavitation-mediated delivery was examined in vivo in two types of cancer tumours, and mRNA expression was quantified using in vivo fluorescence imaging and using excised homogenised tumour tissue. The results presented here lay the foundations for cavitation-mediated delivery and transfection of free mRNA both in vitro and in vivo by identifying appropriate acoustic parameters and cavitation nucleation agents, which result in enhanced delivery and by identifying the appropriate time windows for measurements to quantify expression.

Authors: Martin, A

• DOI: 10.5287/ora-0nbmqop25
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: tumour; cancer; gene delivery; high intensity focused ultrasound; cavitation; in vitro ultrasound delivery; in vivo ultrasound delivery; tumour model; ultrasound; mRNA delivery; ultrasound-mediated delivery
• Subjects: Cancer cells; Tumors; Gene therapy; Messenger RNA; High-intensity focused ultrasound