Document Type


Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Kevin G. Rice


Diseases of the liver have a large impact on human health. Genetic disorders, metabolic disorders, alcoholism, cancer, or infections can all impair liver function. If serious enough, a liver transplant may be necessary, a major surgical procedure which requires life-long immune suppression and relies on the availability of donor livers.

Gene therapy is being intensively studied as a potential method to treat many disorders, including disorders of the liver. While viral gene therapy has seen some success, possible side effects make it risky, so nonviral gene delivery vectors are being developed. Unfortunately, these nonviral vectors do not yet have the efficiency of the viral vectors.

Nonviral gene delivery vectors face many chges in vivo. The vectors must protect DNA from nucleases while it moves through the bloodstream, they must avoid nonspecific uptake, they must be enter the correct cells, and must enter the nucleus before the DNA can be expressed. If any step of this process fails, there will be very little, if any, expression, and it may be impossible to determine what went wrong.

One impediment to nonviral gene delivery research is the transition from in vitro studies to in vivo studies. The cancer derived cell lines most often used for in vitro transfections are rapidly dividing, which makes nuclear entry much easier than in the whole animal. While primary cells would be a more accurate model of the in vivo environment, the number of cells that can be obtained from tissues is small, and primary cells usually cannot be cultured for long. This limits the number of experiments that can be done with each preparation of cells. To overcome this, we have miniaturized transfection assays, including the transfection of mouse primary hepatocytes with luciferase in 384 well plates. Because fewer cells are needed, more experiments can be performed with each liver preparation.

Another issue introduced by the differences between in vitro and in vivo research is circulatory stability. In vitro, large particles with strong positive charges are desired, because they sink down onto the cells and are attracted to the negatively charged cellular membranes. However, in vivo these particles will aggregate serum proteins and become lodged in narrow capillary beds in the lungs or other organs, often causing toxicity. While this behavior can usually be overcome through PEGylation, improving a particle's circulatory half-life will still improve its chances of finding the correct target. Scavenger receptors found on liver nonparenchymal cells are very efficient at removing negatively charged particles from the bloodstream. We have shown that dosing large amounts of PEGylated polyacridine DNA polyplex can saturate the scavenger receptors and improve circulatory half-life. We have also shown that large doses of PEGylated peptide with or without acridine groups can inhibit scavenger receptor uptake through the formation of peptide-protein nanoparticles. By inhibiting scavenger receptor uptake, DNA can be successfully hydrodynamically stimulated at times up to 12 hours post-delivery, demonstrating a longer circulatory half-life and suggesting a mechanism to explain how delayed hydrodynamic stimulation can achieve full level gene expression in the liver after the DNA has had time to circulate throughout the whole animal.

Once a nonviral vector finds its target cell, it must still enter the cell through endocytosis and then escape the endosome before it becomes digested in the lysosome. Before the DNA cargo can be expressed, it must enter the nucleus. Nuclear entry in nondividing cells is a major barrier to efficient gene delivery. One method to over come this barrier is to avoid the need for

nuclear entry altogether by delivering mRNA instead of DNA. mRNA can produce protein in the cytoplasm by finding a ribosome and initiating translation. However, it is even less stable in the bloodstream than DNA. We have produced an mRNA construct capable of high-level expression in the liver through hydrodynamic delivery. The PEGylated polyacridine peptides used to protect DNA were applied to mRNA and shown to enhance expression, allowing a 1μg dose of mRNA peptide polyplex to produce higher expression than an equal dose of DNA. The peptides were also shown to provide some protection against nuclease digestion in serum. This suggests that efficient, if transient, protein expression can be achieved through peptide protected mRNA delivery.

However, DNA delivery is still desired for longer term expression, and the nuclear entry of DNA is still a problem. In an effort to help facilitate nuclear entry, the membrane disrupting enzyme phospholipase A2 was modified in several ways. The enzyme was conjugated with DNA binding peptides, nuclear localization peptides, and hepatocyte targeting oligosaccharides. Additionally, mutant forms of the enzyme were prepared in bacterial expression systems to achieve site-specific conjugation. Unfortunately, none of these efforts produced a useful tool for nuclear entry.

The research presented in this thesis represents some progress toward the goal of nonviral gene delivery to the liver. Hopefully, some of this work will be useful in the development of new treatments and therapies to improve human health.

Public Abstract

Gene therapy has the potential to treat many diseases and disorders, including genetic disorders, diabetes, cancer, viral infections, and more. However, gene therapy requires that DNA is efficiently delivered to the correct cells in the correct tissue in the body. This is not easy, because enzymes in the blood and other fluids can destroy unprotected DNA on its way to these cells. Several chemicals have been created to bind to DNA and protect it during delivery.

A particularly effective group of these chemicals are known as PEGylated polyacridine peptides. These peptides have positive charges that bind to the negative charges on DNA, and acridine structures that fit in between the base pairs of the DNA double helix, allowing much tighter binding. The long polyethylene glycol, or PEG, chains attached to these peptides help to keep dangerous enzymes away from the DNA while it's moving through the blood.

This work not only further studied how PEGylated polyacridine peptides protect DNA, but also studied how they might be used to protect and deliver messenger RNA, or mRNA. mRNA is the chemical that carries information from DNA to make proteins, and may be used to treat some diseases like DNA can. However, mRNA is much harder to protect than DNA. Despite this challenge, low doses of protected mRNA were given to mice, and were able to produce more protein than an equal dose of DNA.


publicabstract, Gene Delivery, Gene Therapy


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Copyright 2015 Samuel Thomas Crowley