Nanobody-siRNA Conjugates for Targeted Delivery of siRNA to Cancer Cells

Area of Science:

• Biotechnology
• Molecular Biology
• Therapeutics

Background:

• Targeted extrahepatic delivery of small interfering RNA (siRNA) is crucial for nucleic acid therapeutics but remains challenging.
• An ideal delivery system should specifically internalize siRNA into target cells without impairing its function.

Purpose of the Study:

• To develop and evaluate anti-Epidermal Growth Factor Receptor (EGFR) Nanobodies as tools for targeted siRNA delivery.
• To assess the efficacy and specificity of Nanobody-siRNA conjugates (Nb-siRNAs) for gene silencing.

Main Methods:

• Site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on anti-EGFR Nanobodies.
• Assessment of EGFR binding and receptor-mediated endocytosis of Nb-siRNAs.
• In vitro evaluation of mRNA knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells.

Main Results:

• Nb-siRNAs retained EGFR binding and were internalized into EGFR-positive cells.
• Nb-siRNAs demonstrated in vitro activity, inducing mRNA cleavage in the targeted cell line.
• Comparison of different linker types (cleavable and non-cleavable) for Nb-siRNA construction.

Conclusions:

• Anti-EGFR Nanobodies provide a viable strategy for targeted siRNA delivery.
• Nb-siRNAs show promise for specific gene silencing in EGFR-expressing cells.
• Linker choice impacts the performance of Nanobody-mediated siRNA delivery systems.