Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases

Abstract

The rapid advancements in gene therapy have opened up new possibilities for treating genetic disorders, including Duchenne muscular dystrophy, thalassemia, cystic fibrosis, hemophilia, and familial hypercholesterolemia. The utilization of the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has revolutionized the field of gene therapy by enabling precise targeting of genes. In recent years, CRISPR/Cas9 has demonstrated remarkable efficacy in treating cancer and genetic diseases. However, the susceptibility of nucleic acid drugs to degradation by nucleic acid endonucleases necessitates the development of functional vectors capable of protecting the nucleic acids from enzymatic degradation while ensuring safety and effectiveness. This review explores the biomedical potential of non-viral vector-based CRISPR/Cas9 systems for treating genetic diseases. Furthermore, it provides a comprehensive overview of recent advances in viral and non-viral vector-based gene therapy for genetic disorders, including preclinical and clinical study insights. Additionally, the review analyzes the current limitations of these delivery systems and proposes avenues for developing novel nano-delivery platforms.

Lipid nanoparticles: The game-changer in CRISPR-Cas9 genome editing

“… LNP System CRISPR/Cas9 cargo Advantages Limitations ref Lipid Nonoparticles (LNP) Chemically modified sgRNA/Cas9 mRNA and sgRNA/Cas9 RNP Low immunogenicity, high biocompatibility Low toxicity, very simple for large-scale manufacture CRISPR-off-target Cas9's effects might be decreased by a temporary release. Endosomal degradation of cargo, Specific cell tropism [ 13 , 92 , 93 ] LNP System/liposomes/lipoplexes Cas9 mRNA sgRNA/RNP Virus-free, Simple manipulation, Low cost Endosomal degradation of cargo, Specific cell tropism [ 94 ] Ionizable LNPs Cas9 mRNA and co-delivered sgRNA targeting PCSK9 (for selective organ targeting (SORT) High endosomal escape, Biodegradable, Cumulative gene editing upon repeated dosing in vivo N/A [ 94 ] Ionizable LNPs Cas9 mRNA and co-delivered sgRNA targeting DMD1 Restoration of gene expression Hepatotoxicity increase in plasma [ 95 ] NTLA-2002 biodegradable LNP Cas9 mRNA and co-delivered an sgRNA targeting targeting KLKB1 Tissue-specific delivery N/A [ 96 ] Cationic arginine functionalized Gold Nonoparticles sgRNA/Cas9 glut (+NLS) RNP targeting AAVS1 gene (or PTEN gene) FDA-approved, Low stress to the cells Variable efficiency depends on cell types, Requires extensive optimization [ 97 ] LNP−pDNA delivery containing DLin-KC2-DMA and unsaturated PCs pDNA targeting EGFP N/A Moderate cellular toxicity [ …”

“…LNPs offer advantages like biodegradability, biocompatibility, and protection of genome-editing systems. They are also easily modifiable to enhance delivery efficiency and achieve cell- or tissue-specific targeting [ 93 ]. LNP formulations for CRISPR/Cas9 gene editing must be tailored to the specific experimental application.…”

An Outline on the Advancements in Surgical Management of Osteoporosis-Associated Fractures

“…Gene editing technologies like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer a different approach by potentially allowing for the correction of genetic defects that contribute to osteoporosis. Although still in the early stages, experiments have successfully targeted and modified genes in animal models that are crucial for bone density regulation, suggesting a future where genetic predispositions to osteoporosis could be mitigated or even eliminated [92].…”