Gang Bao - US grants

The research to be performed on this RIA award will investigate, theoretically, the influence of reinforcement cracking and interfacial debonding on composite flow strength, creep resistance and fracture toughness. Rigorous finite element analyses will be carried out to determine how the overall limit flow stress of a damaged composite depends on the volume fraction and shape of the reinforcing particles, the percentage of these particles which are damaged, and the characteristics of the matrix material.

The exploratory research to be performed on this SGER award will investigate the fundamental relation between crack growth and background plasticity of ductile solids. Rigorous and exceptionally accurate finite element calculations based on an innovative mathematical model will be performed to predict the fracture resistance curves, the crack growth per cycle under sequential stressing, and the interface debond length. These results will be compared to available physical observations for this class of materials. Understanding gained from this research could lead to the development of advanced high-performance composites consisting of a multitude of alternating thin layers of ductile and brittle materials.

*** 9713731 Hemker Fully lamellar two phase TiAl based intermetallic alloys offer a very attractive mix of mechanical properties and are considered to be strong candidates for replacing nickel base superalloys in several structural applications involving temperatures of up to 900' C. At these temperatures, the creep performance of these alloys is of primary concern. Unfortunately, our understanding of the processes that control high temperature deformation in many advanced materials, including TiAl, is currently rather limited. The underlying creep mechanisms in these advanced alloys are often quite different from that in pure metals; the influence of steady-state creep is much smaller than it is in pure metals, and transient deformation processes (i.e.. primary and tertiary creep) have been found to dominate the creep behavior. In these cases, the Dorn description of power-law creep is no longer valid and attempts to characterize the creep behavior with activation energies and stress exponents, derived from minimum creep rates, have met with very limited success. This has profound consequences for the prediction of creep performance, because the FEM codes used for creep analysis require the input of creep laws that characterize the creep behavior of the material. Wherever possible it is desirable to have these laws based on the physical deformation mechanisms. The widely referenced Dorn description of power-law creep is based on diffusion assisted climb in recovery processes that lead to steady state creep. However, as is shown in the PI's RIA related research, in most intermetallic alloys, including TiAl , the diffusion-assisted recovery processes which lead to steady state creep in pure metals are replaced by a gradual evolution of the deformation microstructure. For this reason, the Dorn equation cannot be used to model creep in this set of alloys and it is necessary to develop an alternative set of mechanism-based creep relations for TiAl based lamellar alloys. The primary goal of this work will be to derive a fundamental set of creep laws that are based on observations of microstructural evolution as a function of creep strain. This will require a close integration of mechanics and materials and will involve work at three specific length scales: i) the microscopic deformation mechanisms will be identified and characterized by TEM observations of fully lamellar polycrystalline specimens that have been crept to various amounts of creep strain, ii) the mesoscopic effects of grain size, lamellar spacing, and lamellar orientation will be separated and characterized with single crystal and microsample creep tests, and iii) the macroscopic creep behavior of these alloys will be modeled with constitutive relations that are based on the micro-and mesoscopic measurements. The PI's experience with creep testing, TEM, and TiAl has been teamed with the co-PI's expertise in developing continuum models of multiphase materials in order to assure a bridge between the mechanics and materials issues in this study.***

The processes of RNA synthesis and DNA repair are fundamental for life. RNA synthesis and DNA repair reactions are carried out by specialized nanomachines, which recognize DNA sequences and structures, catalyze formation and cleavage of chemical bonds, generate mechanical forces, and initiate and respond to biological signals. The principles by which these processes occur are not understood, and coupling of biology and engineering is required to provide quantitative descriptions and models that enable reliable prediction of process outcomes as well as design of interventions and biocompatible nanodevices. One objective of the Georgia Nanomedicine Center (GNC) is to elucidate the engineering principles involved in these nanomachines. Significant technological challenges exist in achieving this goal. Current technologies are unable to measure the behavior of single molecules in living cells. Such measurements are clearly essential, however, to obtain a full description of biological processes in engineering terms. Thus, another part of the GNC mission is to develop technologies capable of providing information about molecular processes at the single-cell and single-molecule levels. Examples of biological problems to be addressed include: maintenance of the unineme chromosome; correction of DNA replication errors; beta-globin transcription factories; and distribution of mRNA during asymmetric cell division. Examples of new nanotechnology tools to be developed include: bioconjugated quantum dots; dual-FRET and peptide-linked molecular beacons; magnetic nanoparticle probes for deep tissue imaging; and STED-4pi light microscope for optical imaging with a spatial resolution of 30 to 50 nm. This planning grant will help the project team to: (a) identify additional problems relevant to RNA synthesis and DNA repair that would benefit from the availability of nanotechnology tools, (b) identify additional tools (including computational/modeling tools), beyond those discussed in this memorandum, that will be needed to mount a comprehensive approach to solving these biological problems, and (c) develop the collaborative structure and mechanisms through which the GNC will function, including interactions with other Nanomedicine Centers. The planning process will include a workshop to be held in Atlanta, which will provide a forum for discussions with potential collaborators in the Southeast and nationwide. Approximately 20 investigators, distributed among the Emory/Georgia Tech/MCG consortium and also outside research centers, will be invited. Some investigators will be experts in biological problems, others in relevant areas of technology and modeling. The detailed plan for the GNC will be developed from results of this workshop and also through further preliminary studies that couple nanotechnology tools and models with the selected biological phenomena.

[unreadable] DESCRIPTION (application abstract) [unreadable] Nucleoprotein machines carry out essential biological processes including synthesis, modification, and repair of DNA and RNA. We propose to establish a nanomedicine development center (NDC) focusing on a model nanomachine that carries out nonhomologous end joining (NHEJ) of DNA double strand breaks. This and other DNA repair machines have relatively simple structures (< 20 components) and significant biological and clinical relevance. DNA damage repair is vitally important to human health, as both normal metabolic activities and environmental factors can cause DNA damage, resulting in as many as 100,000 individual molecular lesions per cell per day. If allowed to accumulate without repair, these lesions interfere with gene transcription and replication, leading to premature aging, apoptosis, or unregulated cell division. We have assembled an interdisciplinary team from eight institutions, with significant expertise in cell and molecular biology of DNA damage repair, protein tagging and targeting, nanostructured probes, cryo-electron microscopy, signal-cell imaging, quantitative image analysis and computational biology, and light microscopy instrumentation. We will develop innovative nanotechnologies and biomolecular approaches to elucidate the structure-function relationships within and among DNA repair nanomachines. General principles emerging from these studies will lay a foundation for precise modification of the information stored in DNA and RNA, leading ultimately to novel therapeutic strategies for a wide range of diseases, including cancer. The NDC has five closely related aims including: (1) to develop orthogonal protein tagging strategies and novel fluorescence probes including quantum dot bioconjugates for nanomachine targeting; (2) to decipher structure-function relationship of components required for the core NHEJ reaction; (3) to characterize the dynamics of nanomachine assembly and disassembly in the context of repair foci; (4) to determine the dimensions and structure of repair foci at high resolution in fixed cells; (5) to establish the engineering design principles underlying DNA double-strand break repair. This NDC will complement existing NDCs that focus on filaments, membranes and protein folding enzymes, and the probes, tools and methodologies developed will be applicable to a wide range of biological and disease studies. Our long-term vision is to provide genetic cures for common human diseases based on the ability to manipulate the somatic human genome using Nanomedicine. [unreadable]

This application aims to establish the Center for Translational Cardiovascular Nanomedicine, a highly collaborative and multidisciplinary Program of Excellence in Nanotechnology (PEN). The broad and long-term vision of this PEN is to develop and apply nanotechnology and biomolecular engineering tools and approaches to address compelling medical needs in the detection and treatment of atherosclerosis and the repair of damaged vasculature and heart tissue. Cardiovascular disease remains the leading cause of death in the US, and there is an un-met need to inform the state and activity of atherosclerotic plaques, to deliver therapeutics directly to lesions, and to guide the evaluation of therapy. Nanotechnology has the potential to address these challenges. We will develop and apply: (1) nanoparticle based PET and dual-modality PET/MR contrast agents for in vivo imaging of atherosclerotic plaques, (2) fluorescence imaging probes and gold-nanoparticle sensors for in vitro detection of atherosclerosis, (3) nanocarriers for targeted delivery of siRNA and drug molecules to lesions in vivo, and (4) nanotools in stem cell research for personalized repair of damaged tissue. Our specific milestones include the ability to deliver 20% injected dose/gram in diseased tissue, achieve RNA knockdown exceeding 70% in primary cell lines and generate patient-specific endothelial cells and cardiomyocytes with a 10-fold increase in long-term viability. The goals of this PEN are to establish the nano-toolbox and nano-cardiology knowledge-base, to translate the nano-scale tools and technologies to clinical applications in the detection and treatment of cardiovascular disease and to train the next generation of leaders in cardiovascular nanomedicine.

1606794-Bao

NSF has made an award in support of student, junior faculty and speaker travel to the ASME 2016 NanoEngineering for Medicine and Biology Conference (NENB) being held in Houston, TX, Feb. 21-24, 2016. The goal of the conference is to bring together leading experts in bioengineering, nanomaterials, biology and medicine to review recent advances in nanoengineering and nanomedicine, including new experimental methods, synthetic approaches, modeling and computational efforts, analyses and innovative methodologies for sensing, quantifying, diagnosing and treating medical disorders using nanoengineering approaches. The program is organized under 5 tracks: Nano-imaging, Nanoparticle-based delivery, Nano and Microfluidics, Modeling of nano-phenomenon, and Nano-materials design. The goal of the conference is well matched to programs supported by Chemical, Bioengineering, Environmental and Transport Systems (CBET) and Emerging Frontiers in Research and Innovation (EFRI), which is particularly focused on the development of new technologies.

PROJECT SUMMARY Nuclease-based somatic genome editing, including approaches that use CRISPR/Cas9 and DNA Base Editor (BE), is a transformative technology that has the potential to cure many human diseases. However, to translate genome editing into widespread clinical use, there is an unmet need for safer and more effective technologies to deliver genome editing machinery into disease-relevant somatic cells and tissues in vivo. Although Adeno- Associated Viral (AAV) vectors are capable of delivering CRISPR/Cas9 systems in vivo with high editing efficiency, they have limited packaging capacity, lack the specificity in targeting cells/tissues, and can induce genotoxicity and immune responses due to persistent expression of Cas9. Further, most nonviral methods for in vivo delivery of CRISPR/Cas9 using systemic administration remain ineffective. To address these challenges, we have developed the Velcro AAV platform ? AAV vectors with Leucine Zippers (LZ) inserted strategically onto the capsid surface such that vector production and transduction efficiencies are minimally impacted. The LZ adaptors can then be used for modular and versatile attachment of proteins onto the capsid, such as cell-targeting nanobodies or peptides as well as genome editing reagents. Our central hypothesis is that Velcro AAVs will provide improved cell-targeting specificity and increased packaging capacity without affecting transduction efficiency, enabling safer and more robust somatic genome editing in vivo. During Phase 1 (UG3), Velcro AAV vectors will be constructed, characterized and optimized for nanobody-based endothelium-targeting (Aim 1a) and Cas9/BE protein attachment (Aim 2a). The effects of nanobody/nuclease attachment on viral titers and transduction efficiency will be quantified. Mouse studies will be carried out in Aims 1b, 2b and 2c to test the ability of Velcro AAV vectors to specifically target the endothelium with increased packaging capacity for gene editing in vivo. The targeting specificity and gene editing efficiency of Velcro AAV vectors will be further determined in Phase 2 (UH3) through pig studies in Aim 3. If successful, the proposed studies will yield strong preclinical demonstration of a new delivery platform technology that can provide specific cell/tissue targeting, larger cargo capacity, and transient nuclease activity, enabling safe and efficient somatic genome editing in humans.

PROJECT SUMMARY Heart failure (HF) remains a serious public health concern despite recent advances in medicine. Currently available drugs only address symptoms of HF, thus new approaches for curative therapies are sorely needed. Gene therapy has been proposed as one such promising approach; unfortunately, many candidate genes would lead to serious clinical side effects if delivered systemically. Additionally, considerable loss of delivery vectors to off-target organs require high vector doses to be used in order to achieve therapeutic effect at diseased tissue sites. To address these challenges, we propose to engineer adeno-associated virus (AAV) vectors that can specifically target cardiac tissue damaged after a myocardial infarction (MI). The key scientific premise of this project is the observation that extracellular proteases, specifically matrix metalloproteinases (MMPs) are elevated in damaged cardiac tissue post-MI. We have developed a platform of protease- activatable AAV vectors that can deliver genes in response to the MMPs elevated post-MI. Promisingly, upon intravenous injection, our engineered AAV vectors are able to achieve significantly improved targeted gene delivery to the high MMP region of the diseased heart in vivo, and this targeted delivery is accompanied by decreases in delivery to non-target organs. In this R01 project, we aim to design, build, and characterize an improved panel of protease-activatable AAV vectors for HF treatment. In aim 1, we will create AAV vectors that can target different disease stages post-MI. In aim 2, we will use molecular modeling and structural approaches to study the AAV capsid variants and to further improve our vector designs. Then in aim 3, we will use in vivo molecular imaging to characterize the in vivo specificity of the engineered vectors in relation to elevated MMP levels in the heart post-MI. Finally, in aim 4 we will test the therapeutic efficacy of using the protease-activatable AAV vectors in in vivo models of MI-induced HF. Overall, by improving the specificity of AAV vectors for target cardiac tissues, we aim to (i) overcome the need to use invasive administration strategies; (ii) minimize delivery to off-target organs, leading to decreased side effects as well as decreased overall vector dosage needed to achieve therapeutic effect, and (iii) reduce any dose-dependent immune responses against the vector.

PROJECT SUMMARY Delivery of nucleic acids (e.g. genes or RNAi) to combat metastatic ovarian cancer is a highly promising therapeutic approach. Tumor cells may be killed by nucleic acids encoding either pro-apoptotic factors or enzymes that can convert prodrugs into toxic molecules. Invariably, however, the success of any gene therapy approach hinges on the ability to deliver the nucleic acid-based effector molecules to target tumors with high specificity and efficiency, a feat that has been largely difficult to achieve. Most vector targeting approaches to date have relied on cell surface receptors overexpressed on some subpopulation of target cancer cells. Unfortunately, there is no unique cell surface biomarker that specifically identifies all cells in a tumor. To overcome this limitation, we propose to develop a platform of protease-activatable viral vectors that we call Provectors. The Provectors cannot deliver transgenes until they become activated by extracellular proteases present at high levels in ovarian tumor microenvironments. In particular, the Provectors are designed to detect matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, whose overexpression is correlated to ovarian cancer progression and death. Our Provector technology is based on the clinically promising adeno-associated virus (AAV), which has recently been approved as the first human gene therapy product in Europe. We have key pilot data demonstrating our ability to build Provectors whose transduction capabilities are activated by MMPs. In an orthotopic ovarian cancer model, a Provector prototype is able to significantly increase transgene delivery and expression in tumors with decreased off-target delivery to liver and spleen. Furthermore, after just a single intravenous injection of Provector encoding HSV-tk, metastatic ovarian tumor bearing mice treated with ganciclovir had significantly better therapeutic outcomes compared to controls. The proposed project will support the design and characterization of the 2nd generation of Provectors with improved features and enable further in vivo testing. In specific aim 1, we will construct a modular platform of high efficiency Provectors for targeting metastatic ovarian tumors. In specific aim 2, we will characterize the developed Provectors via a panel of in vitro assays. Finally, in specific aim 3, we will test the performance of Provectors in preclinical models of ovarian cancer. The proposed project, if successful, will generate a suite of protease-activatable Provectors with improved properties that can target and eradicate metastatic ovarian tumors in vivo.

Sickle cell disease (SCD) is a genetic disease that affects millions of people worldwide, with significant morbidity and a median life expectancy in the mid-forties. Although SCD can be cured by allogeneic hematopoietic stem cell transplantation (HSCT), this treatment strategy has substantial limitations and is only available to ~15% of patients. We have developed a genome-editing based strategy for treating SCD by correcting the sickle mutation in ?-globin (HBB) gene in patient?s hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 and corrective single-stranded oligonucleotide (ssODN) donor template, demonstrated that up to ~37% of mutant HBB alleles can be gene corrected. Injection of gene-edited SCD HSPCs into immunodeficient NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment. We further demonstrated that cells differentiated from gene-edited SCD HSPCs produced high levels of normal hemoglobin A (HbA), resulting in a significant reduction of the amount of sickle hemoglobin (HbS) present in the red blood cells. In particular, delivery of Cas9/gRNA RNP into SCD CD34+ cells without ssODN template (i.e. only with Cas9 cutting of HBB) resulted in a large increase in fetal hemoglobin (HbF) induction and significant decrease in the amount of HbS, leading to prevention of sickling even under hypoxic conditions. However, the mechanism underlying HbF induction by Cas9 cutting is poorly understood, the clinical implications of large deletions/insertions at the HBB on-target cut-site and chromosomal rearrangements need to be determined, and the risk of inducing ?-thalassemia by HBB indels needs to be evaluated. The central hypothesis of the proposed research is that a quantitative understanding of HBB gene editing consequences will increase the efficacy and safety of gene-editing based treatment of SCD. In Aim 1 studies we will determine the mechanism(s) of Cas9-cutting induced HbF induction in SCD HSPCs by assessing the effect of Cas9 cutting of HBB on HSPCs in erythroid culture, and measuring the impact on relative expression of HBB and HBG. In Aim 2 we will quantify large deletions at HBB on-target site and chromosomal rearrangements in SCD HSPCs using new PCR and next-generation sequencing tools. In Aim 3 we will determine the potential of inducing ?-thalassemia due to HBB gene editing in SCD HSPCs by quantifying the total hemoglobin protein levels and the complete hemoglobin profile using our sickle HUDEP-2 cell-line and cells from gene-edited SCD HSPCs, and engrafted edited cells in a sickle mouse model. These studies will facilitate the translation of genome editing based SCD treatment into clinical practice.

ABSTRACT Genetic differences among individuals ultimately trace to single nucleotide polymorphisms, the majority of which affect traits, including disease susceptibility, by influencing the expression of nearby genes. Over the past decade, tens of thousands of loci of this type have been mapped to what are called credible intervals, namely sets of anywhere from a handful to over one hundred polymorphisms that have similar statistical signals. The next step is to resolve the identities of the causal variant(s) within these credible intervals. Similarly, there is a parallel pressing need to validate that de novo and ultra-rare variants in the promoter of a gene thought to contribute to a congenital abnormality actually disrupt expression of the gene. This project proposes a systematic effort to fine map causal variants for hundreds of genes that influence autoimmune and other immune conditions. It utilizes a combination of genome engineering and single cell genomics, in a recently developed assay called expression CROP-seq, or eCROPseq. The idea is to knock out each of the variants in a credible interval with a pool of CRISPR-Cas9 guide RNAs, each targeting one SNP for microdeletion of mutation, and taken up by about 100 cells. The expression of the gene in those cells is then compared with the expression in all other cells that receive different guide RNAs in the same experiment. Aim 1 is to use eCROPseq to identify causal variants in up to 600 credible intervals associated with 350 immune loci, measured in both a myeloid (HL60) and lymphoid (Jurkat) cell line. Aim 2 is to perform a similar analysis of up to 500 rare variants in the promoter regions of these genes to evaluate whether new mutations can cause extreme levels of aberrant gene expression. With these results in hand, Aim 3 utilizes a more precise genome editing tool, search-and-replace CRISPR (also known as prime editing) to substitute one allele for another instead of just evaluating mutations. Then Aim 4 asks whether the effects observed in the cell lines are also seen in primary T cells from eight different people, and whether the magnitude of effect differs among people. Collectively the proposed experiments will systematically map the causal regulatory variants for hundreds of autoimmune genes, and establish a tool that should be readily adapted by even small labs to test the function of new genome-wide associations.