Daniel G. Anderson, Ph.D. - US grants

An effective non-viral gene delivery agent must 1) bind DNA, and 2) facilitate various steps necessary for transfection. While much effort has gone into the design of such agents, one basic question remains unclear, namely: how tightly should these compounds bind DNA in order to achieve the maximum efficiency of gene delivery? One possible answer is that the optimal DNA- binding stability of a compound depends on which specific step in delivery it mediates. Thus, I propose to quantitatively and systematically examine how DNA-binding stability affects three crucial steps in gene delivery, namely: cell-specific uptake, endosomal translocation, and nuclear transport. In many medically relevant human cell lines, all of these steps can be mediated by protein sequences - a fact that has led to the development of modular, protein-based delivery systems. I will fuse protein sequences known to catalyze all three of these steps to DNA binding domains derived from two well-characterized repressor proteins, LacR and TetR(D). Both of these proteins bind to their respective operators with a high affinity, and more specifically, with a high degree of stability. The stability of the protein-DNA complexes will then be varied from weak to covalent with little change in overall chemistry by simply modifying their specific DNA binding sites. Furthermore, since small, non-toxic molecules induce the dissociation of Lac and Tet proteins, these proteins will also contain a novel triggered release mechanism. The long-term goal of this project is to create a safe synthetic delivery system with high transfection efficiency, and a useful framework with which other gene delivery agents can be designed.

DESCRIPTION (provided by applicant): This application addresses broad challenge area (14) Stem Cells and the specific challenge topic 14-EB-101 Synthetic Delivery Systems for Generating Pluripotent Stem Cells The recent advent of cell reprogramming as a means of producing induced pluripotent stem (iPS) cells from somatic cells has produced great excitement in the biological and medical communities due to its potential to circumvent the immunological and ethical issues surrounding traditional embryonic stem cells. For this technology to prove useful as a therapeutic and/or experimental tool one must first develop a technique that can reprogram cells in a rapid, efficient and repeatable manner. Most importantly, one must find alternatives to the potentially cancer-causing retroviruses that are currently used for this process. In this proposal, we request funding to further develop a current, proof of concept, cell injection prototype and to utilize it as an enabling tool to conduct reprogramming studies. The device is capable of delivering pico liters of materials across the cell membrane in a high throughput, efficient manner. The microfluidic system utilizes a micron-scale nozzle to inject a jet of liquid into cells passing through a channel. This jet pierces the cell membrane, without causing cell lysis, and is capable of delivering the factors necessary to reprogram somatic cells. By further developing this device, we hope to use the system to produce iPS cells at a rate and efficiency comparable to viral transfection, while avoiding the issues of mutagenesis and toxicity. The efficacy of micro-injector based cell reprogramming will be thoroughly tested using published methods of inspecting iPS cells. Tissue cultures produced by the system will also be used in animal studies for verification of pluripotency in reprogrammed cells. Eventually we will use the device in tandem with our novel polymeric delivery mechanism as a means of further enhancing performance. In addition, the quantitative, high throughput nature of the device will allow us to conduct studies on the biological aspects of the reprogramming process itself. More specifically, we can determine what optimal combination of genes and factors will result in maximum reprogramming efficiency as well as the specific role of individual genes in the overall process. Due to the physical nature of the delivery system, our device can also explore the use of proteins as a means of enhancing reprogramming (or even replacing DNA) in the production of iPS cells. Over the past years, embryonic stem cells have revolutionized the field of regenerative medicine;their potential for producing entire organs and tackling genetic disorders, such as Multiple Sclerosis (MS), has made them a favorite among researchers. The cell injection device described herein will have the capability of producing stem cells in a rapid, safe and efficient manner by reprogramming a patient's existing adult cells. The system can thus not only circumvent the ethical and technical issues that surround embryonic stem cells, but also avoids many of the toxicity and cancer risks associated with existing virus-based reprogramming techniques.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-DE-103: Enhancing Human Embryonic Stem (ES) Cell Culture Systems. The overall objective of this proposal is to develop and optimize a feeder-free in vitro culture system with a controlled culture microenvironment for human pluripotent stem cell growth and directed differentiation. In particular, high-throughput approaches will be used to develop culture materials with optimized surface properties incorporating defined extracellular matrix (ECM) components that allow for accurate control of oxygen partial pressure of the cell surface (pO2cell) to provide more physiological conditions and allow strategies to better mimic normal development processes. The developing embryo is exposed in vivo to very low oxygen levels, and there is growing evidence that oxygen concentration, together with cell-protein interactions, are important factors in differentiation and proliferation. In typical culture systems, pO2cell is unknown because of gradients in the culture medium. Currently, there are no commercially available methods or equipment for culturing cells under known, hypoxic conditions. Membranes of silicone rubber, which have very high oxygen permeability, will be used as a culture substrate so that cells are exposed to the same oxygen level as in the gas phase. Silicone rubber surfaces will be investigated in various forms: (1) unmodified, (2) chemically modified and functionalized with synthetic polymers, and (3) chemically modified with synthetic polymers to which ECM components are attached by physical adsorption or covalent linkage, including defined proteins and mixtures thereof. Cell compatible, ECM protein microarrays on functionalized silicone rubber will be created with a high-throughput microarray platform that we previously developed for culture of cells in order to screen a large number of cell-ECM-biomaterial interactions. The various cell substrates will be examined for cellular attachment, proliferation, and gene expression patterns at various levels of pO2cell using combinatorial techniques. Confirmatory experiments in macro-scale culture using culture vessels will be carried out with the most promising combinations. Similar experiments with the high-throughput microarray platform and culture vessels will be carried out for differentiation of human pluripotent stem cells to cardiomyocytes, which has been shown to benefit from hypoxic culture, as a model system. Accomplishment of the goal of the proposed studies will lead to elimination of conventional feeder layers and undefined xenogenic proteins and to the development and validation of a more well-defined and physiological culture platform for simultaneous variation and study of soluble factors, ECM-cell interactions, and pO2cell, thereby enabling enhancement of our understanding of the role of these factors in maintenance and differentiation of human pluripotent stem cells. This novel, generally applicable platform, will upgrade the capability and quantity of research in this field. Pluripotent stem cells hold enormous promise for drug screening, in vitro modeling of genetic disorders, and cell therapies and the proposed research will have wide impact on human health. At its most basic level, this research will provide information about the interaction of biomaterials, ECM components, and undifferentiated or differentiating human pluripotent stem cells at different known values of pO2cell that mimic physiological conditions, data for which has not previously been available except under normoxic culture. This information will advance our knowledge in the fields of developmental and stem cell biology as well as human pluripotent stem cell technology. In particular, we expect to learn how the choice of ECM proteins and pO2cell levels interact to aid directed differentiation. From a broader standpoint, the proposed high-throughput platform and macro-scale culture vessels that derive therefrom are tools that constitute enabling technology to culture human pluripotent stem cells under defined physiological conditions. They will improve the research infrastructure in terms of the capability for culturing human pluripotent stem cells. The knowledge acquired and tools developed may have profound effects on a wide variety of applications in many fields of human health. This will accelerate developments in applications such as drug screening, in vitro models of genetic disease, therapeutic replacement of diseased cells in major diseases such as heart disease, diabetes, and neural diseases such as Parkinson's, which annually affect millions of people in the United States. Indeed, differentiation of human pluripotent stem cells to cardiomyocytes, the proposed model system, is itself of high interest for generating cells and tissues for repair of cardiac tissues following ischemic heart disease. Furthermore, the culture vessels to be tested will be useful in culturing cardiac tissue. This collaborative study will integrate recent progress in the fields of bioengineering, biomaterials, and developmental biology to design a new culture platform and protocols that will facilitate cell-based therapies for treatment of a multitude of human diseases and other ES cell applications. PUBLIC HEALTH RELEVANCE: This project concerns improved methods to work with human stem cells so that they can develop into medically useful cells and tissues. For example, this can lead to ways to grow functioning heart muscle cells that can be used to treat heart disease.

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) and their application to tissue engineering and disease modeling have great potential to change current medical practices. Current research is largely focused on devising efficient virus-free protocols to produce large numbers of iPSCs. Direct delivery of proteins obviates the risk of mutagenic insertion and enables more accurate control of the highly sensitive reprogramming process. However, cell-penetrating peptide methods currently provide reprogramming efficiencies that are too low for clinical use. The microfluidic delivery technology proposed has demonstrated its ability to deliver proteins at high efficiencies to human fibroblasts and it eliminates the need fo chemical modification or the use of exogenous compounds. Moreover, preliminary results indicate that the technique can be developed into a universal delivery method capable of delivering a range of macromolecules to different cell types underserved by current technologies. The current prototype is capable of delivering high throughput rates of 10,000-20,000 cells/s and can yield up to 1 million delivered cells per run. This combination of single-cell level control and macro-scale throughput places this device in a unique position relative to existing delivery methods. Aim 1: The mechanism of protein delivery and cell recovery will be investigated to better understand the system and direct its optimization. Preliminary results indicate macromolecular delivery occurs through a pore formation mechanism. To validate this hypothesis, model fluorescent macromolecules and proteins will be used in experiments designed to control against endocytosis and image membrane pores directly. Results will be used to develop a predictive model of the delivery system and conduct optimization studies to improve delivery efficiency, uniformity and cell viability. The design of future device generations will be guided by the gained mechanistic understanding and will aim to incorporate features such as coupling with electroporation. A streamlined version of the system will also be developed for use in collaborating laboratories. Aim 2: The intracellular delivery method will be optimized for protein-based reprogramming of fibroblasts to iPSCs. The robust delivery capabilities of the device will allow studies on the biological aspects of the reprogramming process itself, such as the optimal combination of transcription factors to produce maximum reprogramming efficiency and identification of the role of individual factor in the overall process Moreover, the device will be used to investigate potential improvements by combining other macromolecules, such as microRNA and mRNA, with protein-based reprogramming. In addition to reprogramming applications, such a high throughput microfluidic device platform capable of delivering a range of macromolecules with minimal cell death could enable unprecedented control over cellular function. Hence, in the future, it can be implemented in studies of disease mechanisms, identification of macromolecular therapeutic candidates, stem cell differentiation, and diagnostic applications with reporter cell lines.

The problem. The last decade has seen unprecedented progress in understanding the immune processes that drive atherosclerotic lesion initiation, maturation and complication. In particular, macrophages emerged as key cells that promote disease progression. If the number of inflammatory macrophages in plaque increases, atherosclerosis advances towards life-threatening complications. Plaque macrophages derive from circulating monocytes, which in turn arise from myeloid progenitors and hematopoietic stem cells. When released into circulation, monocytes follow chemokine gradients towards atherosclerotic plaque, where endothelial adhesion molecules aid their extravasation into the vessel wall. Once in plaque, inflammatory macrophages destabilize matrix via proteases and may die locally. Alternatively, if the local environment permits, macrophages may obtain less inflammatory phenotypes promoting cholesterol removal and tissue repair. These insights have been difficult to translate into clinically useful therapeutics, partly because broad anti-inflammatory therapy may compromise beneficial functions of immune cells and host defense. The goal. In this application, we aim to create a new class of macrophage-targeted atherosclerosis therapeutics using in vivo RNA interference (RNAi). We will design small interfering RNA (siRNA) targeting discrete proteins which are key decision nodes for macrophages' fate. We propose to interfere with the life cycle of macrophages with the goal to support inflammation resolution in atherosclerotic plaque. We will test the central hypothesis that RNAi can be harnessed to design precision therapeutics for inflammatory atherosclerosis. We will test this hypothesis by targeting proteins that are essential for macrophage birth (silencing transcription factors MTG16 and PU.1 that influence activity of hematopoietic stem cells and endothelial targets in the hematopoietic niche), migration (silencing chemokine receptors in monocytes and adhesion molecules in endothelial cells), maturation (silencing the M-CSF receptor essential for differentiation of monocytes into macrophages) and polarization (silencing the essential transcription factor IRF5 that gives rise to M1 macrophages with inflammatory functions). Innovation. We will use new nanomaterials for delivery to myeloid, progenitor and endothelial cells, newly and yet-to-be identified siRNA sequences, and target innovative biological targets important in the macrophage life cycle. Impact. We will develop new therapeutics to dampen inflammatory macrophage activity in the arterial wall. We will identify a winning therapeutic strategy in mice with post-MI acceleration of atherosclerosis, a scenario that simulates the vulnerable patient in need of aggressive therapeutic intervention. Our ultimate goal is to bring these materials into the clinic, improving the currently insufficient standard of care by enabling better secondary prevention of myocardial infarction and stroke.

Project Summary The ability to correct disease gene mutations in vivo has broad potential utility for both therapy and basic research. CRISPR/Cas9 is a powerful RNA-guided tool for genome editing. Our recent discovery that CRISPR/Cas9 delivery can cure genetic disease in adult mouse liver provided proof-of-concept of gene correction therapy in vivo. The main goal of this proposal is to establish innovative delivery technologies to maximize the efficiency of CRISPR delivery and gene correction in disease-relevant lung cell types. The impact of this project is a novel paradigm of lung-targeted delivery tools for CRISPR-mediated gene correction. The development of safe and effective delivery vehicles and genome editing tools will guide future studies for CRISPR-mediated gene therapy. This project has three aims that focus on different aspects of lung-directed somatic genome editing: Aim 1: Optimize NP+AAV combination for delivery and gene correction in mouse lung; Aim 2: Identify the best AAV capsid for lung delivery and optimize AAV genome as donor template for HDR; Aim 3: Characterize NP+AAV developed in the UG3 phase in macaque models. This project will develop innovative designs of adeno-associated virus and nanoparticles to significantly improve the efficiency, cell specificity, and safety of CRISPR delivery in vivo.

Many HIV vaccine candidates have failed clinical trials, as they were unable to elicit a potent and durable response to HIV viral challenge. Broadly neutralizing antibodies (bnAbs) have been identified in a number of HIV+ individuals with well-controlled viral levels, and these bnAbs target epitopes that contain residues that are relatively conserved across viral strains. It is thought bnAbs may have efficacy against various strains of HIV pathogen. It is therefore widely believed that systems which induce a potent immune response that includes the generation of broadly neutralising antibodies (bnAbs) in humans could be effective HIV vaccines, and help to mitigate the wide genetic diversity in envelope proteins and relatively high mutation rate of HIV. However, developing a vaccine which can elicit the production of these bnAbs in vivo has proven to be extremely challenging. This is likely due to the complex affinity maturation process that is required to produce bnAbs. Immunization protocols typically administer a single dose of antigen (prime dose), which is sometimes followed by a ?boost? dose delivered several weeks later. In a traditional bolus immunization, the half-life of the antigen present in lymph nodes is generally shorter than the time scale over which germinal centres start producing higher affinity IgG antibodies relative to the initial IgM response (~18 hrs). In contrast, natural infections expose the immune system to escalating antigen and inflammation over days to weeks, resulting in the formation of a germinal centre with dynamic antigen presentation. This germinal centre niche also supports activation of antigen presenting cells, T follicular helper cells, and appropriate cytokine signalling to generate bnAbs. It is likely that to develop effective bnAbs, sophisticated vaccination techniques which can more closely mimic natural infections and natural bnAb formation may be required. We believe that to develop a successful HIV vaccine, researchers must aim to engineer more sophisticated and biomimetic vaccines. Bioengineered vaccines should therefore consider three key parameters in parallel; 1) delivery of an appropriately selected antigen, with 2) favourable kinetics of antigen expression, and 3) control of the immune response in the germinal centre. We believe lymph node targeted delivery of computationally designed mRNA antigens inside immunostimulatory lipid nanoparticles (mRNA LNPs) administered with computationally optimized immunization protocols will address these three aspects in a unique way. Additionally,Translate Bio will provide expertise in manufacturing considerations for mRNA therapeutics. As modifications to mRNA structure may impact the mRNA antigen translation, stability, and immunogenicity, the input of our translational partner (Translate Bio) will allow us to develop vaccines with a potential avenue for commercial development. This R61/R33 proposal combines our expertise in computational antigen design, HIV immunology, combinatorial chemistry, and the commercialisation of mRNA therapeutics to develop a new class of HIV mRNA vaccine candidates.