Background

CRISPR-Cas9 gene-editing technology is considered a key means to treat incurable genetic diseases by correcting specific DNA sequences. However, in vivo applications—where gene-editing tools are injected directly into the body—have repeatedly faced physical and biological barriers. It is extremely difficult for editing complexes to safely reach the interior of target tissue cell nuclei after entering the body.

Immediately after injection, they are exposed to indiscriminate attacks by extracellular degradation enzymes while circulating in the bloodstream and are prematurely cleared by the reticuloendothelial system, including macrophages. Even if they reach the target organ, cell membrane uptake rates are low, and most nanoparticles that enter the cell are destined to be degraded in lysosomes after being trapped in endosomal vesicles.

Previously, viral vectors such as Adeno-associated virus (AAV) were primarily adopted as carriers. However, viral vectors have fatal weaknesses: high immunogenicity, limited gene loading capacity, and the risk of increased off-target cleavage of unintended sequences due to their long-term presence in the body. This is why academia and industry have shifted their focus toward non-viral nanoparticle engineering capable of stepwisely breaking through complex biological barriers.

Key Findings

The researchers systematically classified the biological barriers that CRISPR materials encounter in the body and established a 'barrier-oriented engineering' framework that precisely engineers nanoparticles according to the physicochemical properties of each stage.

In the first hurdle, the blood circulation process, surface modification technology to extend residence time is key. PEGylation processes, which coat the nanoparticle surface with polyethylene glycol (PEG) polymers, or biomimetic coatings using red blood cell or platelet membranes were applied. This method inhibits the phenomenon where serum proteins adsorb onto the particle surface and lead to phagocytosis by macrophages, thereby securing a half-life within the bloodstream.

In the second gate, the target tissue uptake stage, a method of binding cell-specific ligands was presented. The idea is to place peptides or antibody fragments that bind to receptors on the particle surface to induce entry into specific lesion cells while bypassing normal cells.

In the third gate, the endosomal escape stage within the cytoplasm, smart release systems demonstrate their efficacy. Ionizable lipids and pH-responsive polymers were integrated to change shape and rupture endosomal membranes in the low-pH environment inside endosomes. Stimulus-responsive designs that release gene-editing materials in a timely manner in response to external signals or inherent biochemical characteristics of the lesion site, such as reactive oxygen species (ROS) concentrations or specific degradation enzymes, were also highlighted.

In the final hurdle, the nuclear entry stage, strategies were identified to combine an optimized combination of nuclear localization signal (NLS) sequences with the Cas9 protein structure or to manipulate nanoparticles to utilize endogenous intracellular transport pathways. This principle maximizes nuclear membrane passage efficiency, thereby significantly boosting genome-level editing efficiency.

Significance and Outlook

It is assessed that an integrated engineering roadmap has been established, moving beyond previous research trends focused on addressing single barriers to encompass the entire process from systemic entry to nuclear localization. This is because for CRISPR delivery technology to transition from laboratory-scale cell culture to a viable in vivo therapeutic, multiple layered hurdles must be overcome simultaneously.

Practical challenges to overcome for commercialization remain significant. As more diverse surface modifications and complex materials are introduced, the nanoparticle manufacturing process becomes more complex, making it difficult to maintain quality uniformity during mass production. Thorough verification is also required regarding potential toxicity caused by unintended accumulation in the liver or spleen during high-dose administration and the possibility of inducing immune responses in the body. In essence, an optimal balance must be found that satisfies both target accuracy and biocompatibility while increasing intracellular delivery efficiency.

Researchers predict that artificial intelligence (AI)-based molecular modeling and machine learning algorithms will serve as breakthroughs for next-generation nanoparticle design. This is because they can rapidly screen structures exhibiting optimal delivery efficiency from tens of thousands of lipid compositions and surface peptide combinations in virtual space. Furthermore, if customized delivery technology reflecting the individual genetic and pathological characteristics of patients is combined with next-generation CRISPR variants that minimize off-target cleavage risks, the speed of clinical entry for treating incurable genetic diseases is expected to accelerate further.