Dr. Joel Sunshine

Dr. Sunshine

BIODEGRADABLE NANO- AND MICROPARTICLES FOR GENE DELIVERY AND IMMUNE ACTIVATION

This thesis discusses the development of biodegradable polymers, nanoparticles, and microparticles for gene delivery and immune activation. The bulk of this thesis focuses on trying to understand basic principles important to the development of polymer-based gene delivery nanoparticles and acellular artificial antigen presenting cells (aAPC) for CD8+ T cell activation, In addition to the basic work, these technologies can be applied to many areas of human health and I have focused on applications in cancer and ophthalmology.

The root cause of many diseases has a genetic component, from single gene disorders like hemophilia to multifactorial disorders like cancer. As a result, gene therapy has enormous therapeutic potential if it can be done safely and efficiently. The vast majority of the effort into gene therapy has been directed at co-opting viruses as vectors for gene delivery, as viruses have evolved to be supremely efficient at getting their genetic information into foreign cells, but viral gene therapy has been hampered by high profile setbacks in clinical trials, owing to the potential for insertional mutagenesis and overly aggressive host immune responses to the vector. Theoretically, non-viral gene therapy should overcome these limitations by reducing the host response, enabling unlimited cargo capacity, and should be easier to produce and standardize. However, non-viral gene therapy has thus far been unable to achieve the required high transfection efficacies seen with viruses. A class of synthetic cationic polymers, poly(ß-amino)esters (PBAEs), have shown promise, but in order to develop next-generation synthetic polymer vectors for gene delivery, a deeper understanding of the relationship between polymer design and functional outcomes is required. In this thesis, I investigated structureiii function relationships within a library of PBAEs that we developed, with an eye towards investigating the impact of polymer properties on critical barriers to intracellular delivery. To extend this work, we looked to develop PBAEs for non-viral gene delivery to the eye, by investigating how different particle formulations might enable differential delivery to ocular cell types, and by performing a pilot study looking at in vivo gene delivery to the mouse retina via subretinal injection. We found that polymer hydrophobicity was a critical dimension that significantly effected polymer vector performance. We also found that the amine termini of PBAEs were critical to vector function at the level of nanoparticle uptake even though they did not substantially alter any putative key nanoparticle properties. We found that polymer formulations that worked well for one cell class worked well for another cell type within that class but may not work well for a different cell class, and demonstrated that PBAEs could engender high levels of gene expression in the mouse retina.

Tumor immunotherapy requires the activation of cytotoxic T lymphocytes (CTLs) against tumor-specific targets. This activation process occurs in vivo through the interaction of activated antigen presenting cells (APCs) with CD8+ T-cells in the lymph nodes. As an alternative to inducing biological APCs to create the targeted response of interest in the CD8+ T cell population, acellular artificial antigen presenting cells (aAPC) have been designed that mimic biological APC by presenting proteins for signal 1 (antigen specificity) and signal 2 (costimulation) on the surface of spherical particles. When activated, biological APCs undergo significant changes to surface composition and surface morphology; however, in the quest to mimic this process and engender immune responses with aAPCs, the focus has been squarely on the changes to surface protein composition. We hypothesized that artificial antigen presenting cell (aAPC) shape (or morphology) is a critical parameter that modulates T-cell activation and proliferation. We hypothesized that high aspect ratio ellipsoidal aAPCs, rather than spherical aAPCs, might enable increased contact between aAPCs and T-cells, result in enhanced T-cell activation in vitro, and mediate enhanced aAPC-based tumor killing in vivo in melanoma mouse models. To this end, we fabricated ellipsoidal aAPCs using PLGA microparticles, and tested the effects of shape on T-cell activation and tumor prevention by aAPCs in vitro and in vivo. We found that ellipsoidal aAPCs were substantially more efficient than their spherical counterparts at CTL activation, that increased aspect ratio resulted in increased activation, and that ellipsoidal aAPCs reduced / delayed tumor growth and increased mouse survival as compared to spherical and non-cognate controls in a melanoma tumor prevention study in vivo.

May, 2013