Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy

Abstract

We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.

Development of non-viral vectors for neuronal-targeted delivery of CRISPR-Cas9 RNA-proteins as a therapeutic strategy for neurological disorders

“…These results demonstrate C2-PF14 selective uptake and higher affinity for neuronal cells in agreement with our previous report confirming that the C2 sequence binds specifically to neuronal cells via nAChRs. 46 In a gel-shift assay, C2-PF14 was able to bind and fully complex CRISPR-Cas9 RNP at molar ratio 1 : 20 (Fig. 7a).…”

“…We have shown previously that siRNA can be delivered to neuronal cells successfully using peptides. 46 The peptides designed and engineered by our group to deliver siRNA molecules to neuronal cells comprise the shortest fragment of RVG29 (C2) which binds specifically to the nAChRs expressed on neuronal cells (CDIFTNSRGKRASNK), and a 9r poly-arginine cell-penetrating peptide which binds to siRNA (rrrrrrrrr). 46 We set out to utilize a similar strategy to deliver functional Cas9/sgRNA RNP complexes into neuronal cells.…”

“…46 Furthermore, C2-9r was able to efficiently deliver siRNA to the brain of mice following trans-vascular injection. 46 To confirm the neuronal cell-targeting capability of C2-PF14 peptide, we formed complexes of fluorescently labelled gRNA (gRNA-ATTO 550 ) and C2-PF14 peptide and found that it showed similar binding to C2-9r by a gel-shift assay (ESI Fig. S4dii †).…”

“…To assess MPP+ toxicity, we measured the cellular redox activity with MTT as previously described. 46 SH-SY5Y cells were plated at a density of 10 000 cells per well on a 96-well plate in 100 µl Opti-MEM. The next day, the cells were treated with complexes at the desired peptide molar ratio at a 3 nM RNP final concentration.…”

“…[26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] However, there is a lack of studies showing vectors capable of the neuronal cell-targeting of CRISPR. 46 In particular, peptidebased vectors represent a safe and simple approach for the targeted delivery of CRISPR. [34][35][36][37]39 Studies conducted in our laboratory have recently demonstrated the neural-specific delivery of functional siRNA in vivo using a peptide combining a celltargeting fragment (C2) of the rabies virus glycoprotein (RVG29) and a cell-penetrating D-form nona-arginine (9r) peptide.…”