Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Biochemistry & Biophysics
Non Technical Summary
Controlled manipulation of cells through the precise intracellular delivery of biologically active materials has been a long-term goal for biotechnological and therapeutic applications. Our ongoing research program aims to enable efficient delivery of macromolecules into animal or plant cells, through control of endosomal membrane permeability. Cellular delivery is a problem that has not yet been solved. Most techniques remain inefficient, are disruptive to cells and can be toxic. Furthermore, no single approach works for all macromolecular cargo, across cell types, or in every context (e.g. cell cultures vs in vivo). This problem is exacerbated by emerging biological applications continually pushing the boundaries of required delivery efficiencies and versatility. For example, effective macromolecular delivery would greatly amplify the therapeutic potentials of CRISPR-Cas9 technologies, wherein a large ribonucleoprotein complex provides challenges to current delivery systems, and of the manipulation of stem or immune system cells. We aim to reveal fundamental mechanisms of how to permeate cellular membranes, enabling precise control of the molecules that achieve this cell permeation, and to develop new platforms for cellular delivery. Thus, our studies will significantly advance both understanding and solutions to the cell delivery problem. In turn, the new delivery tools developed as part of this research program will be useful to perform gene editing in plants or in animals, thereby enhancing agricultural competitiveness.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We have made the discovery of dfTAT, a peptide that enters cells with remarkably high efficiencies and without any toxicity. This reagent has two major benefits. First, it can be used to introduce a variety of molecules into the cytosol and nucleus of cells, including small molecules, peptides, enzymes, transcription factors, and nanoparticles. Second, it is uniquely suited as a molecular probe for revelation of membrane permeation mechanisms in unprecedented detail. In particular, we have established that dfTAT traffics to late endosomes after endocytic uptake, subsequently causing leakage specifically of late endosomes. This leakage occurs via dfTAT interactions with the late-endosome enriched lipid bis(monoacylglycero)phosphate (BMP, aka LBPA) and is critical for the delivery process as it is this step that permits cytosolic entry. Notably, this new mechanism likely extends to other delivery systems (e.g. cationic lipids, viral and polymer nanoparticles).Critically, to rationally design improved delivery agents and to maximize the utility of a variety of delivery systems, it becomes essential for us to understand the mechanisms of endosomal escape. The following specific aims will be pursued:objective 1: Determine how endosomal leakage is achieved by dfTAT and how this impacts the endocytic pathway. The interplay discovered between dfTAT and BMP has revealed that late endosomes can serve as useful gateways for efficient cell entry. First, we will dissect the molecular requirements for dfTAT-mediated endosomal leakage. Second, we will determine how BMP, a poorly characterized lipid selectively enriched in late endosomes, confers leakiness to the late endosomal membrane in response to dfTAT. Third, while endosomal leakage appears relatively innocuous to cells, our preliminary data suggest that endosomal leakage triggers a membrane damage response and transiently impacts endocytic trafficking. We will characterize this response as it pertains to the development of minimally disruptive delivery agents and reveals new membrane biology.objective 2: Determine how small molecule membrane modulators enhance endosomal escape. We have established that a newly discovered small molecule UNC7938 can augment the efficiency of dfTAT-mediated delivery. In particular, UNC7938 appears to prime late endosomal membranes for dfTAT-mediated leakage. Herein, we will uncover the molecular basis for this previously undiscovered synergy between membrane-disrupting compounds. These studies will enable exploration of the chemical space of molecules that can promote cell entry.objective 3: Development of a surface-modified viral delivery platform for macromolecular complexes. Our data indicate that endosomolytic dfTAT-like moieties can be incorporated into viral capsids to improve the cell penetration of virus-like particles (VLP), a potential delivery platform with numerous advantages for tailoring in vivo delivery of cargo. We will decorate the surface of VLP with membrane-active peptides and establish how surface density impacts membrane permeation. We seek to optimize the membrane-permeation of these agents. CRISPR-Cas9 delivery and gene editing activity will serve as a highly attractive model cargo system for manipulating cells, particularly immune cells resistant to current delivery approaches. VLP will be a learning platform for other encapsulation strategies (e.g. liposomes, nanoparticles).A second area of research consists in learning how to exploit exosomes as molecular delivery tools. Exosomes - membrane-bound vesicles secreted by animal cells - signal between cells, alter tissue physiology, and contribute to disease states. Exosomes signal by transferring bioactive lipids, proteins and nucleic acids from one cell to another.[8, 9] Here, we will address this problem by uncovering how exosomes deliver their content into recipient cells and by establishing what controls their transport properties.objective 5: Determine the mechanisms of exosomal trafficking and their modulation by exosome composition We will determine the cellular sites at which exosomes deliver their luminal cargos by using exosomes labeled with probes that report on cytosolic penetration. We will couple monitored exosome delivery with pharmacological or genetic manipulation of the endocytic pathway to identify the step(s) at which contents are delivered for distinct exosomal populations. Furthermore, our studies will determine how subpopulations of exosomes alter their transport properties depending on their membrane composition, their cells of origin, or the recipient cells they enter. These experiments will establish composition-trafficking relationships that control exosomal cell entry and lead to the potential identification of factors that control delivery.objective 6: Determine the mechanisms of exosomal fusogenicity and their modulation by cellular signaling or exosome composition To deliver signaling molecules into cells, exosomal membranes fuse with those of recipient cells. Our working hypothesis is that it takes place within the endocytic pathway and that exosomal membranes respond to the endosomal milieu to initiate fusion. We will determine how exosomal membranes are impacted in vitro by potential triggers of fusion using BAS. The same triggers will also be modulated in the endosomal milieu to determine how exosomal delivery is impacted in live cells. Moreover, because exosome secretion is a dynamic process, we will determine how the membrane and delivery properties of exosomes change under stimulatory conditions known to activate exosomal secretion and signaling of cancer cells. We will then ask how changes in these properties correlate with fusion, delivery efficiency, and trafficking route. These experiments will establish the biological ground rules of exosome function, which we will then manipulate to dissect mechanisms further.
Project Methods
Our methods involve the use of in vitro models (e.g. liposomes), cells (human, animal, plants), and organisms (e.g. mouse).We use a plethora of assays to monitor the process of cell penetration. They include fluorescence microscopy, flow cytometry, and bioactivity assays.