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Principal Investigator: XUEDONG LIU
Organization: UNIVERSITY OF COLORADO
Fiscal Year: 2024
Award: $327,603
Funding agency: National Institute of General Medical Sciences
Project Summary
Technologies to deliver macromolecules across the plasma membrane and bypass endosome degradation are
not only instrumental for elucidating gene function but also hold enormous potential for therapeutics. Proteins,
nucleic acids, and ribonucleoproteins (RNP) have become indispensable tools for biomedical research, however,
their applications in human therapeutics are largely limited to modulating targets reside in the extracellular space.
Only a few percent of exogenous macromolecules can get through the cellular barriers and make it into the
intracellular space. Extracellular vesicles (EVs) are increasingly being explored as potential vehicles for
intracellular therapeutics delivery since they transport bioactive molecules natively between cells. Cell derived
EVs are heterogeneous in size and composition and, consequently, exhibit low specific activity for delivering
cargo of interest. To address these problems, we developed an innovative macromolecule delivery system
based on engineered extracellular vesicles called gectosomes (G protein ectosomes), designed to co-
encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP
complementation. The reversible tethering of cargo to VSV-G provides efficient cargo loading and endosomal
escape simultaneously. Gectosomes demonstrated efficient delivery of catalytic enzymes, interference RNA,
and Cas9 RNPs to the cytosol and nucleus and successful modifications of cellular phenotypes. We aim to
develop a versatile and broadly applicable platform technology that allows rapid production of highly specific
gectosomes capable of modulating intracellular targets in vitro and in vivo. The objective of this application is to
demonstrate the feasibility of our approach by improving the homogeneity of gectosomes through CRISPR
engineering of the producer cells and by creating gectosomes that deliver engineered nanobodies or ubiquitin
E3 ligase CRBN intracellularly to alter protein aggregation or degradation. We will also examine host immune
responses to gectosomes and elucidate the efficacy window of gectosome delivery in vivo, which will help refine
application areas. The feasibility of proposed studies is supported by our published results showing that active
loading of gectosomes reduces passive incorporation of cellular proteins while CRISPR engineering of producer
cells improves EV homogeneity. Three specific aims are: SA1: Develop new producer cell lines via CRISPR-
mediated cell engineering to improve the homogeneity and specificity of gectosomes; SA2: Develop gectosomes
to deliver antibodies or agents designed for promoting targeted protein degradation in cells, and SA3: Determine
adaptive immune responses to gectosomes and general toxicity profiles of gectosomes. The proposed studies
will overcome current limitations in delivering biologics to the intracellular space. The improved delivery platform
will also provide more accessible research tools for the wider scientific community in their endeavors to elucidate
gene function or develop new therapeutic strategies for treatment of human diseases.
Terms: <Address><Antibodies><Area><Basic Research><Basic Science><Biological Agent><Biological Products><Biomedical Research><Bypass><CD47><CD47 Antigen><CD47 Glycoprotein><CD47 gene><CRISPR><CRISPR/Cas system><Cell Body><Cell Line><Cell Nucleus><Cell membrane><CellLine><Cells><Clustered Regularly Interspaced Short Palindromic Repeats><Communities><Complement><Complement 1><Complement Proteins><Complement component C1><Cytoplasmic Membrane><Cytosol><Diffusion><Dose><E3 Ligase><E3 Ubiquitin Ligase><Effectiveness><Electroporation><Encapsulated><Endosomes><Engineering><Enzyme Gene><Enzymes><Exhibits><Extracellular Space><G-Proteins><GTP-Binding Proteins><GTP-Regulatory Proteins><Gene Delivery><Goals><Guanine Nucleotide Coupling Protein><Guanine Nucleotide Regulatory Proteins><Heterogeneity><Human><Immune response><Immunological response><In Vitro><Integrin-Associated Protein><Intercellular Space><Intracellular Space><Knock-out><Knockout><MER6><Measures><Mediating><Metabolic Protein Degradation><Methods><Microinjections><Modern Man><Modification><Molecular Weight><Nucleic Acids><Nucleus><Pathway interactions><Phenotype><Plasma Membrane><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Production><Protein Turnover><Proteins><Publishing><RNA Interference><RNA Sequences><RNA Silencing><RNAi><Receptosomes><Regulatory Protein Degradation><Research><Resistance><Ribonucleoproteins><Safety><Sequence-Specific Posttranscriptional Gene Silencing><Specificity><Strains Cell Lines><Surface><Surface Antigen Identified by Monoclonal Antibody 1D8><System><Technology><Testing><Therapeutic><Time><Toxic effect><Toxicities><Transfection><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><VSV-TG protein><VSVG protein><Viral><adaptive immune response><biologics><biopharmaceutical><biotherapeutic agent><cell engineering><cellular engineering><complementation><cultured cell line><design><designing><diffused><diffuses><diffusing><diffusions><electroporative delivery><exosome><extracellular vesicles><gene electrotransfer><gene function><host response><human disease><immune system response><immunogenicity><immunoresponse><improved><in vivo><innovate><innovation><innovative><insoluble aggregate><interest><macromolecule><microvesicles><nano engineering><nanobodies><nanobody><nanoengineering><new drug treatments><new drugs><new pharmacological therapeutic><new technology><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutics><new therapy><new therapy approaches><new treatment approach><new treatment strategy><next generation therapeutics><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel technologies><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutics><novel therapy><novel therapy approach><pathway><plasmalemma><protein aggregate><protein aggregation><protein degradation><protein function><resistant><sdAb><single domain antibodies><success><systemic toxicity><technology platform><technology system><tool><translational medicine><treatment strategy><ubiquitin-protein ligase><vesicular stomatitis G protein><vesicular stomatitis virus G protein>