Development of protein-based nanostructures activated by ultrasound

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Principal Investigator: Sangpil  Yoon
Organization: UNIVERSITY OF OKLAHOMA
Fiscal Year: 2024
Award: $363,919
Funding agency: National Institute of General Medical Sciences

Project Summary / Abstract
The development of site specific nucleases for precise gene engineering has advanced basic understanding of
genes and their connection to phenotype-causing mutations and physiologically relevant endpoints and
treatment strategies to cure human diseases and medical disorders. It is critically important to mention that the
safe and efficient delivery approaches ensure the utilization of these programmable nucleases, thereby
improving the therapeutic potential of gene therapy. There are some terrific delivery systems and vesicles that
include viruses and viral captives that take advantages of their ability to target particular cell types. Nanoparticles
such as lipid nanoparticles, peptide nanoparticles, and gold nanoparticles can allow encapsulation of the
molecules for the delivery. However, still clever ways to try to address challenges in delivery of molecules are
required. We propose a revolutionary approach to realize controlled delivery of Cas9-ribonucleoprotein
(RNP). Our long-term goal is to develop a platform for delivering various types of macromolecules efficiently,
effectively, and safely for cell engineering in vitro and in vivo. The goal of this project is to develop a gas
vesicle (GV) and ultrasound based delivery approach (GVUS) to improve cell viability and cargo delivery
efficiency by conjugating GVs and purified RNP and monomeric streptavidin fusion protein (RNP-mSA-bioGV) to
form protein clouds for precise delivery using optimized ultrasound excitations after the investigation of
ultrasound parameters to induce stable oscillations and cavitation of GVs. GVs are biocompatible and intact for a
long time due to their stability while immediate clearance is available upon brief sonication. Stable oscillations
and cavitation of GVs under ultrasound excitation will be used for controlled disruption of cell plasma membrane
for intracellular delivery. In specific aim 1, we will investigate GV dynamics under different ultrasound excitations
and find optimized ultrasound parameters to generate stable oscillations and cavitation of GVs. We will test
various delivery modes using optimized parameters. We will develop RNP-mSA-bioGV protein clouds for
controlled delivery of RNP for gene editing, followed by the characterization of the protein clouds in specific aim
2. Primary mouse T cells will be engineered using developed protein clouds delivered by GVUS to study gene
editing precision and in vitro anti-tumor activities of engineered T cells to assess the feasibility of the proposed
approach in specific aim 3. An innovative approach to use protein clouds controlled by ultrasound will
revolutionize the current delivery techniques to engineer cells for research in laboratories and clinical
applications. Although many cell therapies have significant challenges in manufacturing and cost, we expect that
GVUS approach combined with protein clouds will contribute to realize simpler and cheaper patient-specific and
cell-based therapy.

Terms: <Acoustics><Address><Antioncogene Protein p53><Area><Assay><Bacteria><Behavior Control><Behavioral Manipulation><Binding><Bioassay><Biological Assay><Biotinylation><Blood - brain barrier anatomy><Blood Plasma Cell><Blood-Brain Barrier><CRISPR><CRISPR/Cas system><Cell Body><Cell Line><Cell Survival><Cell Therapy><Cell Viability><CellLine><Cells><Cellular Tumor Antigen P53><Chimera Protein><Chimeric Proteins><Clinic><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats><Complex><Contrast Agent><Contrast Drugs><Contrast Media><DNA Therapy><Degenerative Neurologic Disorders><Development><Diagnosis><Disease><Disorder><Echography><Echotomography><Encapsulated><Engineered Gene><Engineering><Ensure><Frequencies><Fusion Protein><Future><Gases><Gene Expression><Gene Transfer Clinical><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Goals><Hemato-Encephalic Barrier><Human Figure><Human body><Image><In Situ><In Vitro><Intracellular Membranes><Investigation><Laboratories><Mammalian Cell><Measures><Mediating><Medical><Medical Ultrasound><Methods><Mice><Mice Mammals><Microbubbles><Modification><Molecular Interaction><Murine><Mus><Mutation><Nanostructures><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Oncoprotein p53><P53><Patients><Penetration><Peptides><Permeability><Phenotype><Phosphoprotein P53><Phosphoprotein pp53><Physiologic><Physiological><Plasma Cells><Plasmacytes><Production><Protein TP53><Proteins><Radiopaque Media><Recovery><Research><Resolution><Ribonucleoproteins><Safety><Site><Solid Neoplasm><Solid Tumor><Sonication><Specificity><Strains Cell Lines><Strepavidin><Streptavidin><System><T-Cells><T-Lymphocyte><TP53><TP53 gene><TRP53><Techniques><Testing><Therapeutic><Time><Transfection><Tumor Protein p53><Tumor Protein p53 Gene><Ultrasonic Imaging><Ultrasonic Therapy><Ultrasonic Transducer><Ultrasonogram><Ultrasonography><Ultrasound Diagnosis><Ultrasound Medical Imaging><Ultrasound Test><Ultrasound transducer><Vesicle><Viral><Virus><advanced disease><advanced illness><anti-tumor immune therapy><anti-tumor immunotherapy><behavioral control><biocompatibility><biomaterial compatibility><bloodbrain barrier><cell engineering><cell killing><cell mediated therapies><cell type><cell-based therapeutic><cell-based therapy><cellular engineering><cellular therapeutic><cellular therapy><clinical applicability><clinical application><cost><cultured cell line><customized therapy><customized treatment><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><design><designing><developmental><diagnostic ultrasound><disease diagnosis><effective therapy><effective treatment><engineered T cells><gas vesicle protein><gene editing platform><gene editing system><gene editing technology><gene editing tools><gene repair therapy><gene therapy><gene-based therapy><gene-editing toolkit><genetic therapy><genetically engineered T-cells><genome editing><genome mutation><genomic editing><genomic therapy><gold nano particle><gold nanoparticle><human disease><imaging><improved><in vivo><individualized medicine><individualized patient treatment><individualized therapeutic strategy><individualized therapy><individualized treatment><innovate><innovation><innovative><lipid based nanoparticle><lipid nanoparticle><macromolecule><manufacturability><manufacture><monomer><nano gold><nano particle><nano-sized particle><nano-sized structures><nano-structures><nanoGold><nanoparticle><nanosized particle><neoplasm immunotherapy><neurodegenerative illness><next generation><novel><nuclease><off-target mutation><p53 Antigen><p53 Genes><p53 Tumor Suppressor><patient specific therapies><patient specific treatment><plasmid DNA><plasmocyte><process improvement><protein expression><protein p53><resolutions><scale up><side effect><sonogram><sonography><sound measurement><spatiotemporal><super high resolution><superresolution><tailored medical treatment><tailored therapy><tailored treatment><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><theranostics><thymus derived lymphocyte><tool><transgenic T- cells><translational applications><treatment strategy><tumor><tumor immune therapy><tumor immunotherapy><ultra high resolution><ultrasound><ultrasound energy><ultrasound imaging><ultrasound scanning><ultrasound therapy><unique treatment>