The authors chose HIV envelope protein (Env) as a model receptor for targeted delivery of siRNA. This was achieved with a fusion protein (F105-P) consisting of an Env-specific antibody Fab fragment (comprising the non-immunogenic antigen-recognition domains), fused to protamine, a nucleic acid-binding protein that normally nucleates DNA in sperm. Incubation with siRNAs resulted in stable fusion protein/RNA complexes, which were internalized by cells carrying the respective surface antigen and efficiently mediated the downregulation of the siRNA-targeted gene product.
In in vitro experiments, F105-P complexed with siRNA targeting HIV gag (encoding an essential viral capsid protein) significantly reduced viral replication and release of viral particles in HIV-infected primary CD-4 T cells compared with cells treated with F105-P alone. Using the same fusion protein construct, these studies were extended to in vivo mouse tumour models with melanoma cells that were transfected with vectors for HIV Env before their implantation in the host. Here, intratumoural or intravenous injection of F105-P complexed with a cocktail of siRNA directed against several oncogenes significantly slowed down tumour growth in Env-expressing tumours, without exerting any effect on non-transfected tumours. Furthermore, the complex seemed to be completely non-immunogenic, and was not trapped by any cells of the reticuloendothelial system that could interfere with systemic delivery. Demonstrating the flexibility of the targeting strategy, further siRNA/fusion proteins were generated that consisted of a single-chain antibody directed against ErbB2 (an antigen on many breast cancer cell lines), which also mediated targeted delivery of siRNAs.
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