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Principal Investigator: Steven L Armentrout
Organization: PARABON NANOLABS, INC.
Fiscal Year: 2023
Award: $325,000
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
More than 300 million people worldwide are affected by a genetic health condition. Over 4,400 genetic
diseases have been identified; nearly all of which are considered rare, which limits the amount of research
each receives. Gene therapy is an attractive approach for treatment of genetic disease because of its
versatility and broad applicability. Genome editing systems such as CRISPR-Cas9, base editing and prime
editing have revolutionized gene therapy research and other fields of life science, however, few gene editing
treatments have reached the market and clinical translation still faces important challenges. Among them is the
need for safe and effective gene therapy delivery vehicles and platforms for their creation.
In this project, we will design and test a new class of programmable, non-viral gene therapy carriers and cargo
– virus-inspired DNA origami (VIDO) vectors and repair templates – and Essemblix GT, a nanoengineering
platform tailored for their production. In contrast to other gene therapy delivery vehicles, VIDO products are
modular and easily modified for different diseases. Moreover, they are structurally well-defined with little
intermolecular variability, facilitating regulatory approval and clinical translation. To our knowledge, this will be
the first project to investigate the use of DNA origami for encapsulation and delivery of gene editing agents.
In Aim 1, we will demonstrate that CRISPR-Cas9 knock-in efficiency is improved by folding and compacting
homology-directed repair (HDR) templates with DNA origami methods. VIDO-folded reporter templates will be
compared against unstructured controls when delivered via electroporation to HEK293T and Jurkat human cell
lines at two different genome insertion sites. Nuclear entry will be determined by confocal microscopy of
fluorophore-labeled template and knock-in efficiency will be assessed by flow cytometry.
In Aim 2, using the same cell lines and genomic targets, we will demonstrate VIDO vectors can encapsulate
and co-deliver CRISPR-Cas9 editing agents and VIDO templates, are readily taken up by cells and induce
knock-in efficiency that is competitive with delivery of the same agents via virus-like particles (VLP).
Endosomal escape and gene expression will be tracked via confocal microscopy and flow cytometry. In both
aims, correct genomic integration will be confirmed via Illumina sequencing.
Successful completion of these aims will establish VIDO vectors and templates as new, programmable gene
therapy products with key advantages over existing alternatives. By making it practical to rapidly design and
create such VIDO products, the Essemblix GT nanoengineering platform could shift gene therapy research
toward a paradigm of gene therapy engineering, thus enabling researchers to deliver more treatments for rare
diseases to more patients more quickly.
Terms: <Affect><Algorithms><Biologic Sciences><Biological Sciences><Bioscience><Bone Marrow><Bone Marrow Reticuloendothelial System><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Capital><Capsules><Carrying Capacities><Cas nuclease technology><Cell Body><Cell Line><CellLine><Cells><Charge><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Complex><Computer-Aided Design><Computer-Assisted Design><Confocal Microscopy><DNA><DNA Therapy><Deoxyribonucleic Acid><Designing computer software><Disease><Disorder><Dose><Effectiveness><Electroporation><Elements><Encapsulated><Endosomes><Endothelial Cells><Engineering><FDA approved><Face><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Gene Delivery><Gene Expression><Gene Transfer><Gene Transfer Clinical><Genes><Genetic><Genetic Diseases><Genetic Intervention><Genome><Genomics><Goals><Health><Heritability><Human Cell Line><Immune response><Immunological response><Inflammatory><Insertional Mutagenesis><Investigators><Jurkat Cells><Knock-in><Label><Life Sciences><Machine Learning><Marketing><Medical><Medical Technology><Methods><Molecular><Nanostructures><Nanotechnology><Nuclear><Nucleic Acids><Orphan Disease><Outcome><Patients><Peptides><Persons><Phase><Polymers><Production><Rare Diseases><Rare Disorder><Receptosomes><Reporter><Reporting><Research><Research Personnel><Researchers><SBIR><Safety><Services><Shapes><Site><Small Business Innovation Research><Small Business Innovation Research Grant><Software Design><Specificity><Strains Cell Lines><Structure><Surface><System><Testing><Therapeutic Studies><Therapy Research><Transgenes><Vendor><Viral Vector><Virus><Virus-like particle><base editing><capsule><clinical translation><clinically translatable><commercial application><commercialization><cultured cell line><delivery vector><delivery vehicle><design><designing><electroporative delivery><fabrication cost><faces><facial><falls><flow cytophotometry><fluorophore><gene electrotransfer><gene repair therapy><gene therapy><gene-based therapy><gene-editing therapy><genetic condition><genetic disorder><genetic therapy><genome editing><genome editing based therapy><genome editing therapy><genome editing treatment><genome editing-based therapeutics><genomic editing><genomic therapy><host response><human stem cells><immune system response><immunoresponse><improved><in vivo><knockin><lipid based nanoparticle><lipid nanoparticle><machine based learning><manufacturing cost><nano engineering><nano tech><nano technology><nano-sized structures><nano-structures><nano-technological><nanocarrier><nanoengineering><nanofabricate><nanofabrication><nanotech><nanotechnological><nanovessel><non-viral gene delivery><non-viral gene therapy><nonviral gene delivery><nonviral gene therapy><novel><operation><operations><orphan disorder><particle><polymer><polymeric><prime editing><rare genetic disease><rare genetic disorder><repair><repaired><scale up><therapeutic editing><therapeutic genome editing><transgene><vector><virus-like nanoparticles><viruslike particle>