Sequentially Self-Assembled Supramolecular Nanocomplexes Enable Systemic Cas9 RNP Delivery and In Vivo Tumor Genome Editing

Technical Summary: Sequentially Self-Assembled Supramolecular Nanocomplexes for Systemic Cas9 RNP Delivery and In Vivo Tumor Genome Editing

Problem Statement
The clinical translation of CRISPR–Cas9 ribonucleoprotein (RNP) therapeutics for in vivo genome editing is currently hindered by two intrinsic barriers: rapid clearance from the bloodstream and the lack of delivery systems capable of systemic tumor targeting. Free RNPs (~12 nm, slightly anionic) suffer from poor cell-membrane translocation, nuclear import limitations, and rapid enzymatic degradation by RNases and proteases in circulation. While existing carriers (e.g., cationic lipid nanoparticles, gold nanoparticles) improve cytosolic delivery, they often lack precise control over intracellular release and sufficient blood retention for systemic targeting. Consequently, most in vivo studies rely on local injection or hepatic accumulation, and a quantitative correlation between pharmacokinetics and on-site editing efficiency remains an unmet need.

Methodology
The authors developed a supramolecular ternary complex platform utilizing sequential self-assembly to encapsulate Cas9/sgRNA RNPs. The system consists of three components:

1. Cargo: Cas9/sgRNA RNP complexes.
2. Core Stabilizer: Tannic acid (TA), a biodegradable polyphenol that engages proteins via hydrogen bonding and hydrophobic interactions.
3. Shell Stabilizer: Phenylboronic acid (PBA)-conjugated block copolymers. Two specific polymers were synthesized and tested: P[Glu(DET)/Glu(FPBA)] (containing ethylenediamine moieties) and P[Lys/Lys(FPBA)] (containing primary amine moieties).

Assembly Process:
RNPs were first mixed with TA to form RNP/TA complexes. These were subsequently mixed with PBA-conjugated polymers. At physiological pH (7.4), the galloyl groups of TA form reversible boronate esters with the PBA side chains, creating a ~30 nm core–shell ternary nanocomplex. The system is designed to be pH-responsive; the boronate esters dissociate under endosomal acidity (pH 5.5), triggering the release of the RNP cargo.

Key Contributions and Results

• Characterization and Stability:

  • The ternary complexes (specifically RNP/TA/PBA-polymer) exhibited a hydrodynamic diameter of approximately 21–28 nm, significantly smaller than the micro-aggregated RNP/TA complexes (~49 nm).
  • Serum Stability: In the presence of fetal bovine serum (FBS), the ternary complexes maintained stable particle sizes, whereas RNP/TA complexes destabilized. The PEG shell of the polymers prevented protein corona formation and protected the core RNP.
  • Enzymatic Protection: The ternary complexes successfully protected sgRNA from degradation by RNase and serum components, whereas free RNPs and RNP/TA complexes showed significant degradation.
  • pH Responsiveness: The complexes remained stable at pH 7.4 but dissociated and released the RNP cargo at pH 5.5, mimicking endosomal conditions.

• Cellular Uptake and Subcellular Distribution:

  • Flow cytometry and confocal laser scanning microscopy (CLSM) demonstrated that the polymer-coated ternary complexes facilitated endocytosis-mediated internalization.
  • Unlike free RNPs, which largely remained on the cell membrane, the ternary complexes escaped endosomes and translocated to the nucleus.
  • FCS analysis indicated that the RNP dissociated from the polymer carrier within the cell, allowing the RNP to function independently.

• In Vitro Gene Knockout:

  • In HeLa-Luc cells, free RNPs and RNP/TA complexes showed negligible gene editing (luminescence reduction similar to controls).
  • The ternary complexes achieved significant gene knockout: RNP/TA/P[Glu(DET)/Glu(FPBA)] reduced luciferase activity by ~13%, while RNP/TA/P[Lys/Lys(FPBA)] reduced it by ~40%.
  • T7 Endonuclease I (T7EI) assays confirmed DNA cleavage, and in vitro cleavage assays demonstrated that the complexes retained editing activity specifically at acidic pH (5.5), confirming stimulus-responsive release.

• In Vivo Biodistribution and Pharmacokinetics:

  • Following intravenous administration in tumor-bearing mice, the ternary complexes exhibited prolonged blood circulation compared to free RNPs or RNP/TA complexes.
  • Tumor Accumulation: Both ternary complexes achieved 2–4% ID/g tumor accumulation, significantly higher than the ~1% ID/g observed for free RNPs. This is attributed to the Enhanced Permeability and Retention (EPR) effect.
  • Organ Distribution: The two polymer variants showed distinct biodistribution profiles. P[Glu(DET)/Glu(FPBA)] accumulated more in the kidneys, while P[Lys/Lys(FPBA)] accumulated more in the liver, likely due to differences in structural stability and surface charge density.

• In Vivo Gene Editing and Anti-Tumor Efficacy:

  • Luciferase Knockout: In HeLa-Luc tumor models, the ternary complexes achieved a gene editing efficiency (indel frequency) of 37.2% at the tumor site, compared to only 1.5% for free RNPs. This resulted in a significant suppression of tumor luminescence.
  • Oncogene Knockout: The platform was used to target "undruggable" oncogenes.
    • PLK1: Knockout of Polo-like kinase 1 (PLK1) in HeLa-Luc tumors significantly suppressed tumor growth.
    • KRAS: In HCT116 tumor models (bearing the KRAS G13D mutation), the ternary complexes targeting the mutant KRAS gene significantly impeded tumor growth compared to controls.
  • Safety: The treatment showed negligible toxicity, with no significant changes in body weight or blood parameters (liver/kidney function markers) compared to controls.

Significance and Claims
The authors claim to have established, for the first time, a quantitative pharmacokinetics–editing relationship for an RNP carrier in a solid-tumor model. The study demonstrates that a supramolecular ternary complex, formed via sequential self-assembly of TA and PBA-conjugated polymers, can overcome the barriers of systemic RNP delivery.

The significance of this work lies in:

1. Systemic Delivery: It enables the systemic administration of Cas9 RNPs, moving beyond local injection or liver-specific targeting.
2. Stimuli-Responsive Release: The platform utilizes endosomal pH to trigger cargo release, ensuring the RNP is active only after cellular internalization.
3. Therapeutic Potential: The system successfully knocks out difficult-to-target oncogenes (mutant KRAS, PLK1) and suppresses tumor growth in vivo without systemic toxicity.
4. Generalizable Framework: The authors posit that this strategy provides a generalizable framework for developing systemically administered in vivo CRISPR therapeutics, addressing the tissue-targeting limitations of conventional delivery systems.

The paper concludes that while complete remission was not observed in the anti-tumor studies, the significant delay in tumor growth and the high editing efficiency (37.2%) validate the platform as a promising strategy for cancer gene therapy. Future optimization is suggested to focus on enhancing tumor accumulation and retention, potentially through the addition of tumor-targeting ligands.