There is currently an estimated 350 million people worldwide chronically infected with the Hepatitis B virus (HBV). These individuals, over the course of their life, are at an increased risk of developing liver cancer. While there are currently antiviral drugs that can suppress HBV replication, latent HBV so far has proven difficult to eradicate. Homing endonucleases, enzymes that recognize long DNA sequences and induce DNA double strand breaks, could be a means by which latent HBV is targeted and inactivated. In particular, we aim to modify the recognition site of the wild type I-GzeII homing endonuclease (HE) into a HBV specific DNA targeting enzyme that will recognize specific sequences in the HBV genome and introduce double-strand DNA breaks. By exploiting the error prone nature of non-homologous end joining, the process cells undergo to repair double-strand breaks, these modified HEs will have a mutagenic effect on target sequences in the HBV genome. Repeated HE activity will eventually cripple the replicative ability of the latent virus. To selectively modify the structure of homing endonucleases, the amino acid residues that interact with the DNA substrate are identified and randomized. A library of variant forms of I-GzeII is generated. Variants that encoded for active forms with the appropriate HBV specific recognition site are selected for by in vitro compartmentalization. After several rounds of selection, bacterial selection assays and in vitro cleavage assays using a reporter cell line will be utilized to select for structurally stable variants that functions in vitro. The final step is to test the HE product on an in vitro HBV cell line. Through our research, we hope to produce a proof of concept that latent viruses can be targeted and inactivated through mutagenesis by homing endonucleases and that specificity and target of the enzyme can be modified and assessed through this protocol.

We are interested in the effects of naturally secreted defensins on viral replication. Defensins are small, cysteine-rich, antimicrobial peptides produced by the innate immune system. Although most studies of defensins have focused on their antimicrobial activity, we have shown in vitro that growth of enteric mouse adenovirus 2 (MAdV-2) is unexpectedly enhanced by defensins. Since this observation was made using purified defensins, we sought to determine if naturally secreted defensins also enhance enteric viral infection. Mouse defensins are exclusively produced by Paneth cells (PCs) in the small intestine. As isolated PCs are not culturable, we utilize a novel 3D small intestinal organoid model, which allows us to grow primary small intestinal epithelial cells in culture. The organoid lumen is topologically equivalent to the apical side of the small intestine. Thus, to mimic oral infection, I directly microinjected the virus into the organoid lumen. I first demonstrated that MAdV-2 replicates in wild-type organoids. Then, I compared the growth of MAdV-2 in wild-type organoids to growth in knockout organoids that do not produce functional defensins. I found that, consistent with the enhanced infection, replication was accelerated in the wild-type organoids as measured by plaque forming units. To confirm these results, and to examine any effect of defensins on viral tropism, I created and characterized a fluorescently tagged construct of MAdV-2, in which I fused green fluorescent protein (GFP) to a minor capsid protein. After microinjection, more green cells were observed in the wild-type organoids. Enteric viruses are routinely exposed to defensins during natural infection. Therefore, the observed enhancement of MAdV-2 may represent a novel mechanism by which enteric viruses hijack a host defense peptide and use it to augment their own infection. Future studies will look at enhancement of other enteric viruses and use additional models with altered levels of defensin expression.

As our understanding of the genetic basis for the pathophysiology of many diseases has broadened, gene therapy, which is the delivery of genetic material into cells in order to supplement or alter defective genes, is being explored as a potential treatment option. Despite its promise, no gene therapies have been approved for clinical use due to the lack of safe and effective vectors. There are many obstacles that must be overcome in order to successfully transfect a cell with exogenous DNA. First, the vector-DNA complex must navigate through the extracellular environment and be uptaken into the target cell. Then, the genetic material must achieve endosomal escape, be released from the complex, and then translocate into the nucleus where it can be transcribed and translated into functional or therapeutic proteins. In the Pun Lab, we previously synthesized a block-statistical copolymer comprised of different hydrophilic and hydrophobic segments aimed at providing a plethora of functionalities to the formed polyplexes. Due to this polymer’s unique architecture, it exhibits transfection efficiencies higher than branched polyethyleneimine, the gold standard for nonviral gene delivery, but still fails to reach the same level of transfection efficiency as seen with viral vectors. In this project, two additional modifications will be made to the copolymer and a library of well-defined polyplexes with varying formulations and structures will be synthesized and evaluated in vitro and in vivo. These modifications, guanidinylation and conjugation of a targeting peptide, aim to increase uptake into cells through electrostatic interactions and increase localization to the target cell type. From these studies, the ideal method to incorporate both modifications into one system to improve transfection efficiency and the structure-function effects on transfection efficiency will be evaluated.

HIV/AIDS is a pandemic that affects over 33 million people globally, and it is estimated that the HIV virus has claimed over 22 million lives. It has been shown that HIV therapeutics are most efficacious when delivered in combinations that target multiple points of the HIV life cycle. Small molecule therapeutics must remain within a narrow range of concentrations in the blood and tissues known as the therapeutic window to be effective. With traditional drugs the patient will spend a large amount of time out of the therapeutic window as concentrations of drug rise, peak, and then fall as the drug is metabolized, leading to inefficient treatment. Therefore to maximize efficacy it is necessary to develop technologies that are able to stabilize the serum concentration of multiple small molecule drugs simultaneously. Controlled combination delivery of antiretroviral agents promises to vastly improve the quality of care for HIV positive patients. However modern biomaterial platforms have fallen short of this goal. I propose the use of an adapted lipid-based, nanoscale biomaterial platform (ICMV) for the controlled and simultaneous delivery of multiple small molecule antiretrovirals as well as an antiviral protein. I further propose future work for the evaluation and optimization of the ICMV platform for sustained drug release, in hopes of developing a long acting HIV treatment. The ICMV system will significantly improve antiviral drug regimes, as well as the scope of deliverable medications. Controlled release platforms also play an essential role in maintaining systemic drug concentrations within the therapeutic window. These advances in therapeutic technology will lead to significant improvements in the treatment and prevention of HIV, which will directly impact millions of human lives.

Sexually transmitted infections are a major health concern, both in the developing and developed world. Despite the fact that vaccines have been developed and are under development for many STIs, there is currently no efficient method of topical delivery for these vaccines. The barrier posed by the vaginal epithelium and the difficultly of achieving intracellular delivery hinder their ability to elicit immunity. The optimal topical drug delivery system would ideally transport vaccines through the epithelium and into immune system cells. There is a naturally-occurring pathway in the vaginal epithelium in which the FcRn receptor, which is present on epithelial cell surfaces, binds and bidirectionally translocates IgG antibodies across the epithelium. We hypothesize that a nanoparticle decorated with IgG could be transported by the same mechanism. Loading the nanoparticle with the desired vaccine would then enable delivery to cells populating the vaginal submucosal tissue, providing the topical delivery that is desired. Several methods of conjugating fluorescent polystyrene nanoparticles to IgG have been investigated. I have selected two methods to pursue. The first of these is a passive adsorption method, in which the antibody was allowed to stick to the nanoparticle surface through hydrophobic interactions. The second of these employed commonly used (thiol-maleimide) conjugation chemistry. Now that these methods have been identified as potentially viable, I will assay them to determine if the IgG protein can still bind FcRn while conjugated to the nanoparticle. If this yields positive results, I will move on to animal models to determine the biodistribution of the particles. This will allow me to see if the IgG modification has increased the particles’ ability to reach immune system tissues. If this project’s aim is successful, it may become as useful system for the delivery of vaccines against a variety of diseases.

Microbial growth can be monitored by changes in the optical density of a culture. In the past, these measurements were generally made by hand, one at a time at regular intervals, in order to produce a growth curve. Currently no such device is suitable for monitoring the growth of fastidious anaerobes. This presented problems for both efficiency and resolution of experiments requiring analysis of multiple cell lines and different growth conditions. For growth periods greater than a work day, researchers needed to take alternating shifts in order to capture data. This is a very tedious process which is draining on the tester. The result of such a worker demand was infrequent measurements to make the minimal curve, resulting in inherent tester bias. Naturally, the desire to automate the process arose. I began development on the ROGR system began in order to implement a microcontroller operated test setup using LEDs and phototransistors to replace the manual measurements made with a spectrophotometer. Previous work has shown that LEDs of these wavelengths do not interfere with cell growth. As this is an ongoing experiment, it is imperative that the method used to measure growth does not change the testing environment. For example, the anaerobic cultures have long been incubated with constant shaking with the culture tubes oriented in a horizontal plane, but measurements were made in the upright position in order to create an accurate reading. The ROGR is a multi-disciplinary project that addresses these project needs. The electronic design ensures system isolation and achieves high precision and dynamic range. Design also ensures mechanical stability, reducing error in LED phototransistor pairs readout originating from changes in their physical orientation. Presently the system is undergoing the physical implementation phase with its circuitry at a working state. This reproducable system will allow for data colection across multiple teams to be connected with confidence of small error.

Current medical diagnostic gold standard, the Enzyme-Linked Immune-Sorbent Assay (ELISA), requires additional labels and amplification to determine the existence of interested molecules. Such process is extremely time consuming and expensive, and thus a fast and low-cost alternative is in demand. Our project aims to develop a rapid and affordable diagnostic biosensor platform that can be administered in the Point-Of-Care (POC) setting. In our silicon photonic sensor chip, we exploit a unique aspect of light when it is confined into a waveguide much smaller than its own wavelength. Since different molecules have different refraction index, the portion of light travels outside the waveguide, called evanescent field, is capable of sensing molecules binding on the sensor surface. While the design and fabrication of these biosensing devices can be done quickly and affordably, characterizing and experimenting the devices remains challenging because of the complexity of testing submicron-scale components. Chips require careful alignment of the optical I/O fibers with micron-tolerances. Microfluidics must be integrated to deliver reagents to specific regions of the chip. Real-time processing of optical spectra is needed to facilitate user operation. Hence we have customized a low-cost test bench to perform experiments and characterizations on the silicon photonic biosensors. I have designed the software, which employs industrial standard Model-View-Controller (MVC) architecture, for instruments integration, experiments orchestration as well as data acquisition and analysis. This software has greatly enhanced the efficiency of the biosensor platform development and future improvement is foreseeable because of the ongoing progress of full automation of experiment preparation. I have characterized multiple sensor devices through optical data acquisition, analysis, model fitting and key parameters’ estimation this software provides. Characterization results show great promise on the sensing capability of the platform and we will soon proceed to biomolecules (e.g. protein), and even cell (e.g. cancer cell) sensing.

Low intensity non-thermal non-ionizing electromagnetic fields (EMF) in the ranges of extremely-low to low frequency (< 200 kHz) have been shown to kill cancer cells in various studies. The external electromagnetic field cannot be concentrated to focus enough energy in the tumor site to be effective. We are designing an EMF-emitting microchip that can easily be surgically implanted into a tumor. The microchip emits electromagnetic fields when it is activated by an ‘activator’ outside of the body. Since the chip is in close proximity to cancer cells within the tumor, the cells would be exposed to a very high local field to achieve cytotoxic effect. A prototype of microchip (cylindrical, ~12 mm in length, ~2 mm in diameter) will be tested with Molt-4 cells (human leukemia). Molt-4 cells will be incubated for 24 hours at 37^oC in a humid atmosphere of 5% CO₂/and 95% air. After initial cell count, one ml of cells each will be put in four microfuge tubes. A microchip will be placed in two of the tubes. Two tubes (one containing a microchip and other not) will be exposed using an activator. The other two (with and without microchip) will be sham exposed, i.e., they will be subjected to the same experiment procedures as the ‘exposed’ samples except that the activator will not be turned on. During exposure, cells will be maintained in a water bath at 37^oC. Viable cell count will be performed at 1, 24 and 48 hours after exposure. Our preliminary results using the above-mentioned protocol are encouraging. We anticipate to show that a microchip can be used to kill cancer cells and produce data to proceed to in vivo (experiment within a living organism) study.