The endothelium of the cerebral microcirculation is a critical component of the blood-brain barrier (BBB),
which protects neural tissue through the presence of tight junctions between endothelial cells and efflux
transporters that extrude many compounds back into the bloodstream. When developing drug delivery strategies
for brain pathologies, these efflux transporters [e.g. multi drug resistance 1 (MDR1)] present a considerable
challenge to effective drug delivery, as they limit the exposure of central nervous system (CNS) pathologies to
many systemically administered agents. Meanwhile, nutrient transporters [e.g. glucose transporter 1 (GLUT1)]
are critical for maintaining normal brain function. Indeed, genetic mutation(s) of GLUT1 can cause recurrent
epileptic seizures, microcephaly, intellectual disability, spasticity, ataxia, and dysarthria.
Given this central role in health and disease in the brain, the endothelium of the BBB represents a rich
target for therapeutic genomic manipulation. In this proposal, we will engineer a platform technology capable of
genome editing the BBB in a safe, endothelial cell-selective, and non-invasive manner, with precise loco-regional
targeting provided by MR image-guidance. We call this approach, wherein very low pressure focused ultrasound
is used to activate plasmid-coated microbubbles, “sonoselective” gene delivery. This is because, instead of
employing a cell-specific promoter, ultrasound (i.e. “sono”) alone “selects” which cell type is transfected. Since
endothelial cell-specific promoters are unnecessary, a vast array of genetic manipulations may be employed.
In Aim 1, we will engineer acoustically-activated delivery agents that sonoselectively edit the genome of
blood-brain barrier endothelium. This will entail testing CRISPR-Cas9 “nickase” plasmids with varying guide RNA
(gRNA) pair sequences for their ability to sonoselectively delete GLUT1 and MDR1 from BBB endothelium. We
will also test whether efficiency can be improved by incorporating plasmids into non-viral polymer nanoparticles
(NPs) that are coupled covalently to MBs with non-immunogenic linkers. The most promising compositions will
then be examined functionally using positron emission tomography (PET) [i.e. 18F-FDG for brain metabolism
changes after GLUT1 deletion and (R)-[11C]verapamil for drug efflux changes after MDR1a deletion]. Further,
single cell RNA sequencing (scRNAseq) will be used to assess the cell selectivity and efficacy of gene deletion.
In Aim 2, we will augment the efficiency and control of sonoselective genome editing by rationally manipulating
focused ultrasound parameters. We will test whether increasing FUS burst duration improves plasmid delivery
and subsequent gene (GLUT1 and MDR1) deletion efficiency. We will also test whether we can control
sonoselective genome editing using a feedback control system based on acoustic emissions. Once completed,
we will have established a safe, non-invasive, MR image-guided, platform for genome editing of endothelium in
the BBB. We submit that such an approach will have multiple applications in pre-clinical neuroscience research
and considerable potential as a therapeutic approach to treating many diseases of the CNS.