Delivery of RNP complexes for CRISPRi (dCas9) silencing in vitro and in vivo using crystalline ultrasmall particles (CUSP)

Delivery of ribonucleoprotein complexes (RNP) for CRISPR gene editing has emerged as a powerful strategy that minimizes off-targeting risks and simplifies dosing. However, RNP delivery approaches often rely upon bursting of endosomes for RNP release, a strategy that also triggers inflammatory responses, which can be exacerbated by the bacterial Cas9 endonuclease. Here, we describe initial results after encapsulating spCas9 or dead spCas9 (dCas9) RNPs in hepatocyte-targeting CUSP particles, comprising a shell with the glycoprotein ligand Asialoorosomucoid. CUSP particles are ~ 20 nm diameter and enter cells through size-limited lipid raft uptake to reach the nucleus without endosome capture. This design has been shown effective in plasmid delivery and treatment in vivo (1). Presently, we found CRISPR/Cas9 RNPs encapsulated in neutral-charge CUSP particles knocked down target F7 mRNA and protein in murine liver tissue and in 3D hepatocyte cell culture. Surprisingly, knockdown was via CRISPR interference (CRISPRi) and not CRISPR cleavage. A maximum effective dose of ~10e13 particles was observed in vivo (40-85% knockdown of the three F7 protein fragments, 30% F7 mRNA knockdown by qPCR ) beyond which efficacy decreased. This finding corresponds with isotopic biodistribution studies and apparent dysregulation of intracellular trafficking, suggesting optimal dosing of CUSP-RNP particles could consist of repeat dosing at a non-saturating dose. We found no evidence of inflammation at the effective treatment dose. We further investigated CUSP delivery of dCas9 RNP and confirmed dCas9 mechanism, in knocking down F7 mRNA and protein in vitro. We observe that achieving DNA-cleaving activity with nanocapsule spCas9 RNPs may be particularly challenging, possibly due to large conformational changes required for activity (2). Conversely, dCas9 does not require large conformational changes, and CUSP’s repeatable-dosing and nuclear-homing platform can potentially exploit the efficient, inherently short-term activity of dCas9 RNP. We conclude CUSP RNP delivery is a promising strategy for overcoming CRISPR barriers to the clinic.

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