Document text
Principal Investigator: Daniel John Siegwart
Organization: UT SOUTHWESTERN MEDICAL CENTER
Fiscal Year: 2020
Award: $364,500
Funding agency: National Institute of Biomedical Imaging and Bioengineering
Project Summary
Co-delivery of Cas9 mRNA and targeted sgRNA is a promising strategy to achieve CRISPR/Cas gene editing in
vivo with improved safety. Although great advances have been made in the delivery of short RNAs (siRNA,
miRNA), the ideal chemical and formulation composition is largely unknown for longer RNA cargo (mRNA,
sgRNA). We recently overcame this delivery challenge in reporting the first successful NP co-delivery of mRNA
and sgRNA in vivo. In this grant proposal, we aim to fundamentally understand why zwitterionic amino lipid (ZAL)
nanoparticles (ZNPs) are uniquely suitable and particularly efficacious for delivery of long RNAs. We hypothesize
that the molecular balance of zwitterionic and cationic groups within NPs is essential for delivery of long nucleic
acids, especially the noncovalent bonding forces at the interface between carrier molecules and RNAs. This
hypothesis is supported by computational modeling that indicates the role of phospholipids is to solubilize RNAs
inside of aqueous pockets within multi-component NPs. Indeed, we found experimentally that combining the
chemical and structural roles of zwitterionic lipids and cationic lipids into a single lipid compound (ZAL) greatly
improved delivery of long RNAs. In this proposal, we will develop (and fundamentally understand) improved
delivery carriers for co-delivery of Cas9 mRNA and targeted sgRNA to enable safe and efficacious CRISPR/Cas
gene editing in vivo. Completion of the proposed studies will: (1) Determine the functional roles of novel ZAL
headgroups, linkers, and hydrophobic domain tails for co-delivery of mRNA and sgRNA; (2) Identify the
physiochemical properties of efficacious long RNA (mRNA, sgRNA) lipid carriers that are correlated to the
mechanism of intracellular delivery; and (3) Determine how the chemical structure of ZALs mediate cell and
tissue specific CRISPR/Cas gene editing in vivo by utilizing a genetically engineered Lox-Stop-Lox tdTomato
mouse that can reveal editing in any organ or cell. Cumulatively, this will open new avenues for CRISPR/Cas-
based correction of genetic diseases by developing efficacious, safe, and clinically translatable nanoparticle
carriers. Defining the specific interactions is a critical goal that would greatly improve delivery of long RNAs by
numerous existing and future carriers. This broader impact may greatly accelerate the clinical development of
mRNA and CRISPR/Cas therapeutics.
Terms: <Acceleration><Affect><Applications Grants><Binding><Body Tissues><CRISPR><CRISPR based therapeutics><CRISPR therapeutics><CRISPR/Cas system><CRISPR/Cas therapeutics><CRISPR/Cas9 therapeutics><CRISPR/Cas9 therapy><Cas9 based therapeutics><Cations><Cell Body><Cells><Charge><Chemical Structure><Chemicals><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats based therapeutics><Clustered Regularly Interspaced Short Palindromic Repeats therapeutics><Collection><Computer Models><Computerized Models><DNA cassette><Dangerousness><Data><Development><Drug or chemical Tissue Distribution><Equilibrium><Event><Exocytosis><Formulation><Future><GEM model><Genes><Genetic Diseases><Genetic Engineering><Genetic Engineering Biotechnology><Genetic Engineering Molecular Biology><Genetically Engineered Mouse><Goals><Grant><Grant Proposals><Guide RNA><Heart><Hydrogen Oxide><Hydrophobicity><Individual><Knowledge><Libraries><Lipids><Mediating><Messenger RNA><Mice><Mice Mammals><Micro RNA><MicroRNAs><Molecular><Molecular Interaction><Murine><Mus><Non-Polyadenylated RNA><Nucleic Acids><Organ><Outcome><Pathway interactions><Phosphatides><Phospholipids><Polymers><Property><Publishing><RNA><RNA Gene Products><RNA Stability><Recombinant DNA Technology><Reporting><Ribonucleic Acid><Role><Safety><Short interfering RNA><Small Interfering RNA><Structure><Tail><Technology><Therapeutic><Therapeutic Uses><Tissue Distribution><Tissues><Viral Vector><Water><Work><amino group><aqueous><balance><balance function><base><chemical property><clinical development><clinical translation><clinically translatable><computational modeling><computational models><computer based models><computerized modeling><design><designing><developmental><enhancer cassette><expression cassette><functional group><gRNA><gene cassette><genetic cassette><genetic condition><genetic disorder><genetically engineered><genetically engineered mouse model><genetically engineered murine model><immunogenicity><improved><in vivo><integration cassette><lipid nanoparticle><mRNA><mRNA delivery><miRNA><miRNAs><nano particle><nano particle delivery><nano-sized particle><nanoparticle><nanoparticle delivered><nanoparticle delivery><nanosized particle><novel><pathway><physical property><promoter><promoter cassette><promotor><reporter cassette><resistance cassette><selectable cassette><selection cassette><siRNA><social role><stop cassette><tool><transcription cassette><transcriptional cassette><transgene cassette><uptake>