R-3 Repository :: Browsing by Author "Suh, Junghae"

Browsing by Author "Suh, Junghae"

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Adeno-associated virus capsid as a scaffold for metal binding and nanoparticle synthesis
(2014-04-24) Dempsey, Chris; Suh, Junghae; Drezek, Rebekah A.; Biswal, Sibani Lisa
Viruses, natural biological entities that have developed complex and compact mechanisms to deliver genetic material to target cells through natural evolution, can be repurposed for new nanoscale applications in a broad range of fields, including being used as biologically relevant therapeutics. Rationally designed genetic enhancements, chemical modifications, and hybrid linkages to other nanoscale materials can make viral vectors even more attractive as cargo-carrying compounds in cells. The motley array of amino acids on the surface of a virus capsid, which contains different side chains that have certain charge, hydrophobicity, and polarity properties, can be modified in order to bind inorganic metals and other metal ions for the purpose of synthesizing new compounds. Nanoscale metal and composite nanoparticles may have unique nanoscale properties that have relevance in a biological research setting, such as providing high signal over background contrast in crowded tissue compartments, Individual viruses can function as scaffolds, providing a surface for synthesis of these inorganic nanoparticles in order to combine the advantages of each individual element into a single hybrid compound. In this thesis, I first present my efforts to study a type of inorganic nanoparticle, which has been shown to generate high contrast nonlinear optical signal for biological imaging applications. Specifically, I created a hybrid labeling and delivery system by modifying the inorganic nanoparticles with a specific polymer compound, endowing them with the ability to condense DNA as well as to enter cells to deliver a genetic payload. Next, I detail a method of producing gold nanoparticles with variable morphology and dispersity using an adeno-associated virus as a scaffold for precursor nucleation. Finally, I describe how I generated mutant virus capsids that can bind metal ions after responding to an external stimulus that causes a conformational change in capsid subunits, externalizing metal binding domains. These detailed studies of hybrid molecules show that attractive properties of individual components of these nanomaterials can be combined or leveraged in a controlled manner in order to generate new materials for biologically relevant applications in the future.
An open-hardware platform for optogenetics and photobiology
(Springer Nature, 2016) Gerhardt, Karl P.; Olson, Evan J.; Castillo-Hair, Sebastian M.; Hartsough, Lucas A.; Landry, Brian P.; Ekness, Felix; Yokoo, Rayka; Gomez, Eric J.; Ramakrishnan, Prabha; Suh, Junghae; Savage, David F.; Tabor, Jeffrey J.
In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.
Characterization & Application of Immobilized Biomacromolecules using Microcantilever and QCM Sensors
(2014-04-15) Wang, Jinghui; Biswal, Sibani Lisa; Segatori, Laura; Wong, Michael S.; McDevitt, John T.; Suh, Junghae
The structure and function of immobilized biomacromolecules are likely to be altered because of the solid surface. The long-term objective of this thesis is to develop surface-based biosensors for the characterization and application of biomacromolecules at the liquid-solid interface. In this study, two analytical surface-sensitive sensors are utilized: microcantilevers and quartz crystal microbalance with dissipation (QCM-D). Each offers unique information regarding the molecules of interest. In particular, the systems that are covered in this thesis include the detection of target analytes using specific recognition elements and the characterization of supported lipid membranes. This research has led to a better understanding of the effect of solid surfaces on protein structure and function, as well as the ability to engineer biomolecular surfaces with great control. There are two detection systems that were studied: a phage-derived peptide system for the detection of pathogenic bacteria Salmonella and an antibody displacement assay for the detection of an explosive, 2,4,6-trinitrotoluene (TNT). The microcantilever responds to changes in the surface free energy on the sensor surface by monitoring changes in its deflection. The physisorption or chemisorption of molecules to the cantilever surface induces a mismatch in the surface stress, causing the cantilever to bend. The multiplexed measurement is able to quickly determine the binding affinities of various phage-derived peptides, improving the screening efficiency of the peptides derived from phage display libraries for Salmonella detection. The microcantilever-based technique provides a novel biosensor to rapidly and accurately detect pathogens and holds potential to be further developed as a screening method to identify pathogen-specific recognition elements. QCM measures mass changes on the sensor surface by monitoring the frequency change of the crystal. The combination of a competition assay with QCM using an anti-TNT antibody is able to distinguish a TNT molecule among molecules of similar structure at low concentrations, leading a sensitive and selective assay. The reliability of this method was further investigated in more real environments simulated by fertilizer solution and seawater. Furthermore, this method could be also applied in gas phase detection of TNT, as well as the detection of other chemicals, such as environmental pollutants and illegal drugs. In both of these detection assays, a mathematical model was developed to quantify the binding of the target molecules with the molecules of interest. In the second half of the thesis, the microcantilever sensor is applied to characterize supported lipid bilayers (SLBs), an interesting biomacromolecular assembly that holds great importance as a model system for membranes. Through monitoring the cantilever deflection, the formation of the SLB, its temperature induced phase transitions, and its interactions with membrane-active molecules are investigated. With increasing temperature, the lipid acyl chains transition from an ordered state to a disordered state, accompanied by a changes in the surface stress that can be readily detected using microcantilever. The phase transition temperature of SLBs is different from that of a lipid monolayer, indicating that the existence of the solid support aﬀects the monolayer structure. Two amphipathic membrane-active molecules, peptide (PEP1) and a triblock copolymer (Pluoronic), are studied for their associations with SLBs. PEP1’s association with SLBs highly depends on the ratio of peptide over lipid, while the Pluoronic interacts with SLBs as a function of temperature and the length of lipophilic block in the copolymer. Therefore, the microcantilever sensor is capable of measuring the conformational change of surface-bound molecules, as well as characterizing the kinetics of membrane-peptide interactions with great sensitivity.
Data-driven design and prediction of adeno-associated virus tropisms
(2020-03-20) Chen, Jeron; Suh, Junghae
Gene therapy is capable of treating diseases that are “undruggable” by small molecule drugs. At the center of gene therapy is the development of efficient and specific gene delivery vectors. For example, adeno-associated virus (AAV) based vectors are able to deliver gene therapeutics to many different types of cells due to their generally broad tropism. Unfortunately, there are some cell and tissue types that are resistant to AAV transduction, and delivery to off-target organs could lead to undesired side effects. Therefore, it would be valuable if we could engineer AAV vectors to transduce specific desired cells and to reduce delivery to off-target tissues. Furthermore, if we could predict how different AAV vectors will perform in different animal models, it would enable us to select the best virus to be used for certain applications. In this work, I have taken a data-driven approach to modify and engineer AAVs to be capable of transducing specific cells. Additionally, I describe a machine learning approach to predicting AAV in vivo biodistribution. Both approaches will help accelerate the design and screening of AAV for future gene therapy applications.
Design of Caspase and MMP-Activatable Adeno-Associated Virus Vectors and Their In Vivo Application
(2020-08-14) Brun, Mitchell John; Suh, Junghae
Gene therapy is the next evolution in the treatment of diseases, allowing for the correction of genetic mutations, induction of growth to facilitate recovery at injury sites, or the delivery of suicide genes to tumor cells. However, the translation of more gene therapies to the clinic has been hindered by the lack of delivery specificity and efficiency. Many viral vectors have been engineered in an attempt to increase transduction efficiency and targeting capabilities. Adeno-associated virus (AAV) has recently become popular as a gene delivery vector because it has the ability to transduce many cell types efficiently, it is non-pathogenic as well as replication deficient, and it is able to be genetically modified. AAV is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be effective while maintaining safety by minimizing delivery to off target cells is a prevalent challenge in the field of gene therapy. To address this problem, our lab has developed a protease-activatable vector (provector) platform based on AAV9 that responds to extracellular proteases present at disease sites. This thesis details my work expanding the provector platform to target cysteine-aspartic proteases as well as matrix-metalloproteinases as stimuli for provector activation. These caspase-activatable provectors demonstrate up to 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus’ ability to transduce healthy tissue. Following proteolysis by caspase-3, the provector shows a 90-fold increase in transduction compared to the OFF state. This provector has also been characterized in vivo, where compared to AAV9 the provector has significantly decreased delivery to off target organs with similar levels of delivery to the injured heart following a myocardial infarction. This work also details characterization of the of the MMP-activatable provector in disease models of acute heart failure, stroke, and chronic heart disease while also explores the therapeutic efficacy of delivering various therapeutic transgenes in murine MI models. To further increase the control of gene delivery with the provector platform, I also detail designs and in vitro testing of provectors requiring two inputs for activation. Preliminary results indicate the vectors perform as designed in vitro which could provide higher specificity of delivery in vivo. Overall, this thesis diversifies the provector platform to target new diseases and increases our understanding of the provector behavior in vitro and in vivo.
Developing a toolkit for modular adeno-associated virus surface display of peptides and proteins
(2019-04-16) Thadani, Nikki Nisha; Suh, Junghae
Engineering biocomputation in nanotherapeutics is a growing field for the creation of devices that respond to their environment to diagnose diseases or deliver targeted treatments. Viruses are genetically encoded nanoplatforms that come prepackaged with sense-response behaviors allowing them to navigate cellular entry, genome delivery and replication. The field of synthetic virology seeks to enhance viral performance for delivery applications by refactoring viruses into well-characterized domains that can be exchanged or augmented with exogenous functional motifs. Adeno-associated virus (AAV) is a strong candidate for modification through synthetic virology — this vector is relatively well-characterized and has a wide range of potential applications in safe and efficient gene therapy. We sought to develop modular, standardized platforms for the integration of exogenous proteins into the AAV capsid so that biological ‘parts’ identified in other systems can be translated to enhance AAV-based therapies. By applying protein engineering techniques to study and modify AAV’s innate biocomputation, we have developed a series of components that can alter the viral response to external stimulus and expand this platform’s capacity for protein and peptide outputs in addition to gene therapy. To address the challenge of genetically modifying the multifunctional virus capsid while preserving viral assembly and transduction, we have identified computational models that may be able to predict vector formation and function from the modified capsid sequence. These models can potentially accelerate the design process for engineered viral nanoparticles through in silico screening to remove non-functional variants. This toolkit will facilitate the incorporation of a wide range of proteins in AAV, expanding the vector’s capacity for detecting stimuli and responding with a range of diagnostic and therapeutic outputs.
Developing stimulus-responsive adeno-associated virus vectors for cancer-targeted gene therapy
(2018-12-10) Evans, Annicka Carter; Suh, Junghae
The most significant challenge to current gene therapy trials is ensuring delivery to exclusively diseased sites. Both non-viral and viral vectors have broad natural tropisms that elicit off-target side effects when used as a treatment. Adeno-associated virus (AAV) has recently become the most commonly used vector for gene therapy trials because it offers many advantages: it has low pathogenicity in humans, infects most cell types with great efficiency, and can be genetically altered to improve its therapeutic effect. The rapid advancement of viral engineering techniques combined with these innate abilities of AAV serotypes to transduce cells, opens up the possibility for creating recombinant AAV platforms that can act as particles with targeting capabilities. It has therefore been the focus of my research to both understand the innate stimulus-responsive nature of AAV as well as work to develop a cancer-targeted AAV vector through capsid engineering. The designed cancer-targeting platforms utilize known characteristics of the tumor microenvironment and cancer biology - specifically the upregulation of matrix-metalloproteinases and production of reactive oxygen species. The characterization of these stimulus-responsive designs, in combination with the investigation of wild-type n-terminal extrusion in response to temperature and pH, will greatly enhance our understanding of AAV engineering tolerance, and further expand the targeting strategies for the use of this vector for human gene therapy.
Discovering and Calibrating Design Rules for Programming Adeno-Associated Virus Nanoparticles
(2015-12-03) Ho, Michelle Liane; Suh, Junghae; Silberg, Jonathan J; Tabor, Jeffrey J
Effective gene therapy must deliver therapeutic genes to disease sites while avoiding healthy tissue. However, engineering targeted gene delivery vectors to ensure exclusive delivery to diseased sites remains a challenge. Adeno-associated virus (AAV) is receiving increasing attention for its potential as a gene delivery vehicle because it offers several advantages: it is considered the safest viral vector, it infects human cells efficiently, and it can be genetically altered to improve therapeutic efficacy. However, even slight modifications to the virus capsid (the outer protein shell covering its genome) lead to unpredictable outcomes. Thus, a governing set of design rules for virus capsid assembly and function is needed to improve future engineering efforts. To this end, this thesis uncovers some of these rules by applying a computational model, often used in protein engineering, to the AAV capsid. A new strategy to improve AAV targeting was also explored by engineering AAVs to sense and become activated by extracellular proteases found in diseased tissues. The specificity of these protease-activatable viruses can be tuned to recognize a variety of protease profiles to treat a multitude of diseases. Design rules for these platform technologies are unveiled through their development and in-depth characterization. We also explore new motifs in the AAV capsid to further our understanding of AAV basic biology. Ultimately, these studies advance our ability to program virus nanoparticles for many biomedical applications.
Effective Gene Delivery to Valvular Interstitial Cells Using Adeno-Associated Virus Serotypes 2 and 3
(Mary Ann Liebert, Inc., 2015) Wong, Fergus F.; Ho, Michelle L.; Yamagami, Momona; Lam, Michael T.; Grande-Allen, K. Jane; Suh, Junghae; Systems, Synthetic, and Physical Biology Program
Currently, curative therapies for heart valve diseases do not exist, thus motivating the need for new therapeutics, regenerative and tissue-engineered valves, and further basic research into pathological mechanisms. For studying valve diseases and developing valve therapies, effective methods to manipulate gene expression in primary valvular interstitial cells (VICs), which promote calcification in disease, would be valuable. Unfortunately, there is little information reported about effective gene delivery methods for VICs. Adeno-associated virus (AAV) is a clinically proven gene delivery vector capable of transducing many cell types and tissues, but has not yet been reported to infect valvular cells. In this study, AAV serotypes 1–9 were tested for their ability to deliver a green fluorescent protein (GFP) reporter into VICs in vitro. Flow cytometry results indicate AAV2 and AAV3 are capable of transducing VICs more efficiently than other serotypes. Furthermore, transduction efficiencies can be optimized by increasing the multiplicity of infection (MOI) and using self-complementary, double-stranded genomes, yielding up to 98% successfully transduced cells. Transduction of VICs by AAV2 or AAV3 in the presence of competing soluble heparin significantly reduces delivery efficiencies, suggesting heparan sulfate proteoglycans act as the primary VIC receptors of these two serotypes. Overall, this study establishes AAV2 and AAV3 as efficient gene delivery vehicles for primary VICs. Such effective delivery vectors for valve cells may be broadly useful for numerous applications, including the study of valvular cell biology, development of valve disease therapies, and regulation of genes for tissue engineering heart valves.
Encryption of Adeno-Associated Virus for Protease-Controlled Gene Therapy
(2013-09-16) Judd, Justin; Suh, Junghae; Silberg, Jonathan J.; Segatori, Laura
Gene therapy holds the unprecedented potential to treat disease by manipulating the underlying genetic blueprints of phenotypic behavior. Targeting of gene delivery is essential to achieve specificity for the intended tissue, which is especially critical in cancer gene therapy to avoid destruction of healthy tissue. Adeno-associated virus (AAV) is considered the safest viral vector and, compared to non-viral vectors, offers several advantages: higher efficiency, genetic modification, combinatorial panning, and high monodispersity. Classic viral targeting has focused on engineering ligand-receptor interactions, but many cell surface targets do not support post-binding transduction events. Furthermore, many potential target tissues – such as triple negative breast cancer – may not display a single, unique identifying surface receptor, so new methods of targeting are needed. Alternatively, many pathological states, including most cancers, exhibit upregulation of proteolytic enzymes in the extracellular milieu. The present work describes the development of an AAV platform that has been engineered to activate in response to disease-related proteases. The specificity and sensitivity of these protease-activatable viruses (PAVs) can be tuned to meet the demands of various clinical scenarios, giving the platform some therapeutic versatility. This work represents the first demonstration of a protease-controlled, non-enveloped virus for genetic therapy. These results extend the therapeutic value of AAV, the safest gene vector currently being explored in 73 clinical trials worldwide.
Encryption of adeno-associated viruses with enzymatically decoded peptide locks
(2018-08-14) Judd, Justin; Suh, Junghae; Silberg, Jonathan; Rice University; United States Patent and Trademark Office
The present invention is a peptide lock that comprises at least one peptide that is genetically encoded into the Adeno-associated virus (AAV) capsid that block biologically active domains on the virus capsid surface. The peptide lock, can be processed by biological enzymes to restore biological behavior of the capsid-displayed domains, thus ‘decoding the lock’ or opening the lock. A method of forming the peptide lock comprises providing at least one peptide, providing an Adeno-associated virus capsid and genetically inserting the at least one peptide into the Adeno-associated virus capsid to block the biologically active domains on the virus capsid surface.
Engineering Adeno-Associated Virus for Protease Targeted Gene Therapy and Immune Avoidance
(2019-04-19) Robinson, Tawana M; Hartgerink, Jeffrey D.; Suh, Junghae
Adeno-associated virus (AAV) has earned significant attention as a safe and efficient gene therapy tool. AAV has been used in over 100 clinical trials to treat a variety of human diseases. However, non-specific targeting to diseased cells and activation of the host immune response hinder its therapeutic efficacy. To address these challenges, genetic modification of the AAV capsid can lead to an improved gene delivery platform. Therefore, capsid-engineering strategies may be necessary to develop optimized vectors for clinical progress. This present work reveals design rules governed by amino acid properties for engineered AAV to become activated by upregulated proteolytic biomarkers in diseased sites. AAV constructs with varying chemical properties were synthesized and characterized for functional behavior. In parallel, a Nature-inspired strategy was employed to create an immune-evasive AAV vector. A panel of AAV vectors with inserted stealth peptides in the AAV capsid was generated to study immune cell uptake. Finally, to gain a better understanding of AAV intracellular trafficking, we found several amino acid residues that are necessary for viral infectivity. The ultimate goal for my research contributions is to develop and to advance AAV vectors for future clinical applications.
Engineering protease-activatable adeno-associated virus (AAV) for targeting of pancreatic ductal carcinoma
(2020-12-02) Butler, Susan; Suh, Junghae
Viruses have evolved genetically encoded responsive behaviors that allow for specific cell targeting, cell-entry, genome delivery, and replication. Harnessing and reprograming these behaviors for desired treatments is the goal of the burgeoning field of synthetic virology - allowing for the creation of novel virus-based nanoparticles to diagnose diseases or deliver targeted therapies. Adeno-associated virus (AAV) is a strong candidate for synthetic virology due to its relatively simple structure, broad tropism, and lack of pathogenicity. However, the broad tropism of AAV can be detrimental to the development of gene therapy treatments since off-target delivery can lead to cytotoxic effects. To address this problem, our lab has previously developed a protease-activatable vector (provector) platform based on AAV serotype 9 that responds to extracellular proteases present at disease sites. Building on this platform, I sought to develop a high-throughput pipeline for future provector development and engineer a provector targeting membrane-bound enzymes, such as MMP-14, that are upregulated in sites of pancreatic ductal carcinomas. Overall, this thesis details the exploration of the provector design using rational and library-screening techniques to develop a pathway for further high-throughput provector development.
Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles
(Elsevier, 2014) McConnell, Kellie I.; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E.
There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticleﾖnanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells.
Evidence for nuclear internalisation of biocompatible [60]fullerene1)
(Walter de Gruyter GmbH, 2013) Huang, Feiran; Mackeyev, Yuri; Watson, Erin; Cheney, Matthew A.; Wilson, Lon J.; Suh, Junghae; Richard E. Smalley Institute for Nanoscale Science and Technology
Many types of nanoparticles (NPs) have been shown to internalise within mammalian cells (1), but only a few have been observed to internalise within the cell nucleus-most likely due to the tightly-regulated nuclear membrane (2). Internalisation of NPs into the nucleus is desirable for several reasons, including their use as 1. transfection agents (3), 2. drug delivery platforms for drugs that act on DNA (4), and 3. hyperthermia-inducing agents for cancer therapy using non-invasive stimulation by radiofrequency irradiation (5), magnetic-field cycling (6), or photonic activation (7). For example, derivatised NPs, including protein-functionalised quantum dots (8) and peptide-functionalised gold NPs (9), have been shown to internalise into the nucleus. For underivatised NPs, single-walled carbon nanotubes (SWNTs), have been observed by direct transmission electron microscopy (TEM) imaging to also localise in the nucleus of human macrophage cells with dose-dependent cytotoxicity (10). Fullerene C60ﾠis another classic carbon-based NP, however it was not been shown to enter the cell nucleus until recently. In particular, a water soluble derivative of C60ﾠfluorescently labelled with a small molecule fluorophore was shown to enter cell nuclei through nuclear pore complexes in liver cancer cells (11). Here, we validate the nuclear internalisation ability of the C60derivative in several other cell types, further supporting the unique intracellular biodistribution property of this specific fullerene compound.
Expanding the toolkit of protease activatable viruses to improve their versatility and modularity
(2018-04-16) Guenther, Caitlin Marie; Suh, Junghae
Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viral vectors. However, its adoption as a safe and effective gene therapy treatment for disease relies on targeting the vector to deliver transgenes to desired cell populations. To this end, our lab has developed a protease-activatable virus (PAV) model based on AAV that responds to elevated protease activity commonly found in diseased tissue microenvironments. This thesis explores the expansion of this platform from the original AAV2-based prototype into AAV serotype 9 to allow for greater clinical translation, specifically for cardiac disease applications. These AAV9-based PAVs have been characterized in vitro, and the activatability of these PAVs ranges from 2.5x to 5.4x differences between the “locked” vs. “unlocked” states. The PAVs have also been characterized in vivo in a murine MI model. Compared to wild-type AAV9, PAV-L001 is able to deliver a reporter transgene site-specifically to the injury region of the heart, with decreased delivery to off-target organs. We are currently investigating the therapeutic efficacy of the PAV packaging the YAP5SA transgene in the murine MI model. This work also explores a new PAV lock format that incorporates different protease cleavage motifs besides MMPs in an attempt to make the PAV response to MMPs more modular and predictable. While this lock format does not display the desired behavior, it reveals the need for further characterization of the PAV lock behavior. To answer some of the remaining questions about the PAV lock behavior, I also demonstrate a method for the quantification of the kinetics of PAV cleavage by MMPs through the development of a simplified virus-like particle model, and preliminary results suggest that the C-terminal motif of the lock may be cleaved slightly faster than the N-terminal motif by MMP-9. Preliminary results also suggest that both sides of the lock must be cleaved off AAV9-based PAVs for the virus to resume binding. Overall, this thesis expands the protease-activatable virus toolkit for new disease applications and advances our understanding of PAV behavior.
Gold Nanoparticle Dendrimer Conjugates for Gene Therapy
(2015-04-24) Figueroa, Elizabeth Raquel; Drezek, Rebekah; Suh, Junghae; Wong, Michael; Foster, Aaron
Gene therapy is a promising treatment that has enormous potential for the management of numerous diseases of acquired and innate origin. Viral delivery vectors are successful in delivering therapeutic DNA, but their efficacy is circumvented by immunogenicity and cost. Non-viral vectors face other issues of inflammatory response, colloidal stability, and low transfection efficiency. Gold nanoparticles (AuNPs) have emerged as attractive nanocarriers for gene delivery. AuNPs are bioinert, easily synthesized, and possess rich surface chemistry that facilitates versatile functionalization. Therefore, AuNPs provide an excellent platform for gene delivery. Polyamidoamine (PAMAM) dendrimers are commercially available cationic branched polymers in which growth branches from a core molecule. Their physiochemical properties make PAMAM dendrimers well suited for gene delivery applications. In this thesis, PAMAM dendrimers are functionalized on the surface of small AuNPs yielding a unique class of gene delivery vectors termed AuPAMAM vectors. We begin by establishing the synthesis and characterization of AuPAMAM vectors, showing that AuPAMAM colloidal stability and DNA condensation ability are dependent on the PAMAM conjugation reaction rate, and that this reaction rate can be altered to enhance transfection efficiency in vitro. Then, we further investigate the influence of each chemical component of the bottom-up AuPAMAM synthesis process by systematically probing each step of the reaction and analyzing its effect on the overall transfection efficiency and cytotoxicity. Finally, in order to clarify the mechanism underlying the differential transfection efficiency seen across many cell lines and tissues, the AuPAMAM vectors are tracked intracellularly over time in vitro using confocal imaging, cellular TEM and flow cytometry. Together, this thesis demonstrates that AuPAMAM conjugates present attractive candidates for non-viral gene delivery due to their commercial availability, ease of fabrication and scale-up, high yield, high transfection efficiency and low cytotoxicity. Additionally, this thesis demonstrates the need to characterize the tissue-specific transfection hurdles vectors face in order to improve application-specific non-viral vector design.
Gold Nanoparticle Platforms for Antigen and Adjuvant Delivery in Cancer Immunotherapy
(2014-09-03) Mattos Almeida, Joao Paulo; Drezek, Rebekah; Suh, Junghae; Harrington, Daniel
Cancer immunotherapy is a growing treatment modality with the promise to yield systemic and targeted treatments for cancer. Major modalities such as radiation, chemotherapy, and surgery are limited in that they are either too localized--as is the case with radiation and surgery—and cannot be effective in metastatic disease, or they are not targeted-- as with chemotherapy--thus leading to severe toxicities. Immunotherapy aims to stimulate the body’s immune system against disease, allowing one to circumvent such challenges because the immune system can act systemically and can have specific activity against cancer cells expressing antigens of interest. The development of an effective cancer vaccine and subsequent immune response requires the delivery of an antigen for immune recognition and of an adjuvant for an inflammatory response. Gold nanoparticles (AuNPs) are promising vaccine carriers because they are generally non-toxic, can be synthesized in the optimal sizes for lymphatic drainage and cell uptake, and can be readily conjugated with antigens and adjuvants for delivery. This thesis project characterizes AuNP distribution in the immune system and details the development of AuNP mediated delivery of antigens and adjuvants for cancer immunotherapy. In our work, we have detailed AuNP distribution within immune cells of the spleen and the tumor microenvironment, thereby identifying that AuNPs associate with a range of immune populations, including B cells, dendritic cells, and macrophages, all of which can be potentially targeted for immune modulation. Next we developed AuNP complexes capable of delivering the CpG oligonucleotide adjuvant, demonstrating that AuNP delivery promotes the therapeutic effect of CpG in vitro and in vivo. In addition, we studied AuNP mediated delivery of the ovalbumin (OVA) peptide antigen and showed that AuNP delivery enhances vaccination with the antigen in vivo, subsequently causing tumor inhibition and prolonged survival in both prophylactic and established tumor models. The thesis thus elucidates AuNP interactions with the immune system and demonstrates that the technology is an effective platform for delivery of immune modulatory agents.
Identification of Novel Short BaTiO3-Binding/Nucleating Peptides for Phage-Templated in Situ Synthesis of BaTiO3 Polycrystalline Nanowires at Room Temperature
(American Chemical Society, 2016) Li, Yan; Cao, Binrui; Yang, Mingying; Zhu, Ye; Suh, Junghae; Mao, Chuanbin
Ferroelectric materials, such as tetragonal barium titanate (BaTiO3), have been widely used in a variety of areas including bioimaging, biosensing, and high power switching devices. However, conventional methods for the synthesis of tetragonal phase BaTiO3 usually require toxic organic reagents and high temperature treatments, and are thus not environment-friendly and energy-efficient. Here, we took advantage of the phage display technique to develop a novel strategy for the synthesis of BaTiO3 nanowires. We identified a short BaTiO3-binding/nucleating peptide, CRGATPMSC (named RS), from a phage-displayed random peptide library by biopanning technique and then genetically fused the peptide to the major coat protein (pVIII) of filamentous M13 phages to form the pVIII-RS phages. We found that the resultant phages could not only bind with the presynthesized BaTiO3 crystals but also induce the nucleation of uniform tetragonal BaTiO3 nanocrystals at room temperature and without the use of toxic reagents to form one-dimensional polycrystalline BaTiO3 nanowires. This approach enables the green synthesis of BaTiO3 polycrystalline nanowires with potential applications in bioimaging and biosensing fields.
Investigating the role of adeno-associated virus capsid in virus function, targeted gene delivery, and the host immune response
(2020-12-03) Chen, Maria; Suh, Junghae
Adeno-associated virus (AAV) is a promising gene therapy vector that is currently approved for use in humans. However, areas of improvement have been identified that can expand the success and applications of AAV. This thesis addresses several of these areas, with a particular focus on the role of the virus capsid. To provide further insight on the role of the virus capsid in the infection pathway, we characterized a serine/threonine rich region in the N-terminal region of VP1 and VP2 capsid proteins and found this region to play an essential role in virus transduction in multiple AAV serotypes. To improve capsid engineering for targeted vector delivery, we discovered a novel design criterion for the protease-activatable provector platform which significantly reduced off-target transduction without affecting on-target activity. To contribute to a solution for evading or escaping from the immune system, we studied the immune response to the AAV capsid in a panel of immune-deficient mice, leading to the identification of B cell deficiency as being sufficient for AAV re-administration. Furthermore, we showed that several components of the innate immune system were necessary for generating a robust antibody response against the AAV capsid. Altogether, this work contributes to our understanding of AAV behavior and the interactions of the viral capsid with its target cells and the host immune system. This information will advance the development of novel AAVs and AAV-based therapies that have improved efficacy, targeting, and longevity for the treatment of a broad range of diseases.

Browsing by Author "Suh, Junghae"

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