Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Hana Totary-Jain, Ph.D.

Committee Member

Hua Pan, Ph.D.

Committee Member

Jerome Breslin, Ph.D.

Committee Member

Thomas Taylor-Clark, Ph.D.

Keywords

atherosclerosis, endosome, microRNA switch, nucleotide modification

Abstract

The field of synthetic mRNA therapeutics is a rapidly expanding arm of gene therapies. The use of mRNA provides multiple benefits over viral or DNA vectors. synthetic mRNA vectors are immediately translated into protein after entering the cytoplasm of cells in contrast to DNA vectors that must first be transcribed to mRNA in the nucleus. This allows synthetic mRNA to produce a therapeutic protein in any cell type, including non-dividing cells. In addition, the non-replicative nature of mRNA means that insertional mutagenesis or generation of escape mutants is not a concern. However, the stimulation of innate immune responses by unmodified synthetic mRNA prevented widespread clinical applications.

The discovery that incorporation of modified nucleotides, such as pseudouridine or 5-methylcytosine, prevents the recognition by innate immune sensors has renewed interest in the use of synthetic mRNA as a therapeutic. In conjunction, numerous post-transcriptional regulatory elements have recently been described in mRNA. Adding these regulatory elements to synthetic mRNA allows control of the expression of the encoded protein in tissue-, cell-, or environmental-specific conditions. However, the influence that the modified nucleotides commonly incorporated in synthetic mRNA have on the regulatory capacity of these elements has not been examined.

In this study we investigated what effects modified nucleotides have on the regulation of synthetic mRNA by microRNA (miRNA switch). We found that nucleotide modifications that increase the translation of the synthetic mRNA tended to decrease the regulatory capacity of microRNA switch. Inclusion of multiple microRNA target sites at the 3’ UTR of the synthetic mRNA was able to minimize the loss of miRNA-dependent regulation of the miRNA switch, but microRNA target sites complementary to the six-nucleotide microRNA “seed” sequence were more affected to nucleotide modification. We found the effect of nucleotide modifications varied between microRNA species and was not determined by the proportion of modified nucleotides present in the microRNA target sites. Finally, we observed that utilizing a single microRNA target site at the 5’ UTR of the synthetic mRNA completely ameliorated the loss of regulation due to nucleotide modifications.

Because synthetic mRNA are easy to produce and can be made to encode any protein of interest, they are ideal for clinical development. Currently there are over 45 clinical trials underway utilizing synthetic mRNA as a monotherapy or in conjunction with other therapeutics. Most of these clinical trials are focused on cancer immunotherapy, particularly autologous T-cell therapy. This therapeutic modality is well suited for synthetic mRNA as the target cells are transfected ex vivo. This avoids the major obstacles that synthetic mRNA therapeutics must still overcome: delivery to target organs.

The sensitivity of synthetic mRNA to extracellular ribonucleases requires encapsulation of the mRNA in a protective nanoparticle. Numerous such nanoparticles have been reported, but nearly all are variations on either lipid nanoparticles or polymeric nanoparticles. The advances made thus far with these two mRNA delivery platforms have significantly reduced their toxicity, however the endosomal escape rate of these particles remains well below 5%. Furthermore, when administered systemically these nanoparticles are avidly taken up by sentinel macrophages of the liver and spleen or hepatocytes. The accumulation of particles in the liver has thus far limited the applications of mRNA therapeutics to diseases and disorders that are liver-specific or that can be treated by using the liver as a biosynthetic depot. Expanding the clinical application of synthetic mRNA may require the discovery of novel delivery platforms that are capable of targeting other organs.

In this study we also tested the delivery of synthetic mRNA using a small cell penetrating peptide, called p5RHH, that is derived from bee venom protein melittin. We showed that in the presence of mRNA, p5RHH self-assembles into spherical nanoparticles that display a high degree of RNase resistance. These nanoparticles were consistently sized regardless of the length of the mRNA payload. Furthermore, after uptake by cells, p5RHH-mRNA nanoparticles displayed a high degree of endosomal escape that was dependent upon the acidification of endosomes, which disassembles the nanoparticles. The high concentration of p5RHH in the lumen of the endosome led to efficient endosomal disruption and produces minimal cytotoxic effects. When the p5RHH-mRNA nanoparticles were injected intravenously into an atherosclerotic mouse, we observed robust expression of the payload mRNA in only the atherosclerotic plaques. The lack of expression in typical depot organs, such as the liver, spleen, lungs, or kidneys, was also confirmed in a normal mouse. The simplicity and specificity of p5RHH-mRNA nanoparticles makes them an ideal candidate for further pre-clinical development as an mRNA delivery platform.

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