Intracellular small interfering RNA delivery using genetically engineered double-stranded RNA binding protein domain.

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Delivery Systems

Background:

• Synthetic carriers like cationic polymers and lipids for small interfering RNA (siRNA) delivery often cause cytotoxicity, limiting clinical use.
• Existing nonviral vectors for siRNA face challenges with safety and efficacy.
• A need exists for non-cytotoxic, efficient siRNA delivery systems.

Purpose of the Study:

• To develop and evaluate a novel protein-based carrier for siRNA delivery.
• To assess the safety and efficacy of double-stranded RNA binding domain (dsRBD) complexed with siRNA.
• To investigate the potential of dsRBD as a non-cytotoxic alternative to synthetic siRNA carriers.

Main Methods:

• Genetically produced double-stranded RNA binding domain (dsRBD) from human protein kinase R.
• Complexation of siRNA with dsRBD for intracellular delivery.
• Characterization using decomplexation and RNase protection assays.
• Evaluation of cytotoxicity and gene inhibition in human carcinoma cell lines.

Main Results:

• Recombinant dsRBD demonstrated sequence-independent binding to siRNA, protecting it from degradation.
• siRNA/dsRBD complexes showed effective gene silencing of Green fluorescent protein (GFP).
• Enhanced gene silencing was achieved with the addition of a fusogenic peptide (KALA) for endosomal escape.

Conclusions:

• dsRBD-based protein carriers offer a non-cytotoxic platform for therapeutic siRNA delivery.
• These carriers facilitate intracellular gene inhibition with high efficiency.
• dsRBD represents a promising nonviral vector for diverse siRNA-based therapies.