Carrier strategies boost the application of CRISPR/Cas system in gene therapy

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TL;DR

This review summarizes recent advances in viral and nanomaterial-based carriers for delivering CRISPR/Cas9 components (RNP, mRNA/sgRNA, and plasmid) for gene therapy. It highlights that while viral vectors like AAVs are effective in vivo, they face limitations such as immunogenicity and packaging capacity; nanomaterial-based carriers offer safer alternatives but currently have lower transfection efficiency. The clinical significance is underscored by ongoing trials, including LNP-mediated delivery of Cas9 mRNA/sgRNA for transthyretin amyloidosis, which achieved up to 96% reduction in serum TTR levels.

Full summary

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**Background:** Gene therapy has shown potential for treating various diseases, but traditional methods cannot eradicate pathogenic mutations. Genome editing technologies, particularly CRISPR/Cas9, offer precise DNA modification. However, safe and efficient delivery of CRISPR components remains a major challenge. This review focuses on carrier strategies—viral vectors and nanomaterial-based carriers—for delivering CRISPR/Cas9 in three formats: ribonucleoprotein (RNP) complex, Cas9 mRNA/sgRNA, and plasmid DNA (pCas9). The review also discusses tactics to overcome limitations such as immunogenicity and low homology-directed repair (HDR) efficiency.

**Methods:** The authors conducted a narrative review of the literature, summarizing recent developments in viral vectors (lentivirus, adenovirus, adeno-associated virus) and nanomaterial-based carriers (lipid nanoparticles, cationic polymers, inorganic nanoparticles, 2D materials, zeolitic imidazolate frameworks, nanocapsules) for CRISPR/Cas9 delivery. They also reviewed clinical trials and discussed pros and cons of each delivery system.

**Key Results:** - **Viral vectors:** Lentiviral vectors (LVs) can integrate into the host genome, enabling stable expression, but pose risk of insertional mutagenesis. Adenoviral vectors (AVs) have large packaging capacity but high immunogenicity; a trial for ornithine transcarbamylase deficiency (OTCD) using adenoviral gene therapy caused a patient death. Adeno-associated virus (AAV) vectors are low immunogenicity but have limited packaging capacity (<4.5 kb). AAV-mediated CRISPR editing achieved 10% correction in hepatocytes in an OTCD mouse model and restored dystrophin levels from 3% to 90% in a DMD dog model. - **Nanomaterial-based carriers for RNP:** Lipid nanoparticles (LNPs) with ionizable lipids showed gene editing in various cell types. Gold nanoparticles (AuNPs) achieved ~30% knockout efficiency for PTEN and AAVS1 in HeLa cells and ~4% HDR efficiency in DMD mouse model. Black phosphorus nanosheets achieved 78% nuclear transport rate and 80% GFP knockout in 2 hours. Zeolitic imidazolate framework-8 (ZIF-8) achieved 37% GFP knockout in CHO cells. Nanocapsules showed higher editing efficiency than lipofectamine in mCherry-HEK293 cells. - **Nanomaterial-based carriers for Cas9 mRNA/sgRNA:** LNPs with ionizable lipids delivered Cas9 mRNA/sgRNA to liver, achieving 80% PCSK9 knockout in mice. SORT LNPs enabled targeted delivery to lungs, spleen, and liver. iPhos lipids improved endosomal escape and gene editing specificity. Red blood cell extracellular vesicles (RBCEVs) achieved ~90% reduction of targeted gene expression in MOLM13 cells. - **Nanomaterial-based carriers for pCas9:** Cationic lipid-assisted polymeric nanoparticles (CLANs) induced 46.8% indel of BCR-ABL in K562 cells. PEI-β-cyclodextrin nanocomplex achieved 19.1% and 7.0% knockout of HBB and RHBDF1 in HeLa cells. Multifunctional nanoparticles (MUSE) depleted PD-L1 and CD47 in tumors, co-activating innate and adaptive immunity. - **Clinical trials:** As of the review, about 50 clinical trials of CRISPR/Cas systems exist. Notable trials include: AAV-delivered CRISPR for Leber congenital amaurosis type 10 (NCT03872479); LNP-delivered Cas9 mRNA/sgRNA for hereditary transthyretin amyloidosis (NCT04601051), which reduced serum TTR by 87% (max 96% at day 28) with no side effects; and poloxamer 407 gel-encapsulated CRISPR for HPV-related cervical intraepithelial neoplasia (NCT03057912).

**Clinical Implications:** The review emphasizes that while viral vectors remain the most effective in vivo, their immunogenicity and packaging limitations drive the need for nanomaterial-based carriers. LNPs have shown clinical promise for liver-targeted editing, as demonstrated by Intellia Therapeutics' NTLA-2001. However, delivery efficiency for non-liver tissues and HDR remains low. Future directions include improving endosomal escape, reducing off-target effects, and developing targeted delivery systems. The authors conclude that with advances in nanotechnology and biotechnology, CRISPR-based therapies may address currently incurable diseases.