Cell-penetrating peptides for efficient siRNA delivery

Abstract

Arginine-rich peptides such as TAT or oligo-Arg can deliver siRNAs non-specifically into several cell types because of their cell-penetration property. However efficiency of siRNA delivery into primary cells, like T cells and neurons is extremely poor as these cell types are recalcitrant to nucleic acid uptake. We chemically conjugated oligo-9R to a recombinant human T cell-specific single-chain antibody (scFv) which enabled siRNA delivery into and efficient gene silencing within human T cells1. Strikingly, delivery was highly specific to the targeted cell type. Surprisingly, scFv similarly conjugated to the TAT peptide was very inefficient in siRNA delivery. Moreover, target gene silencing was also achieved with scFv-9dR in vivo in humanized mice without affecting T cell viability, function or miRNA biogenesis. However, for efficient silencing scFv-9dR:siRNA ratios of atleast 3-5 was required owing to the typical conjugation efficiencies of ~70%. To obviate the need for chemical conjugation and improve siRNA delivery, we have expressed the scFv-9R as a fusion protein, which results in the production of the natural L-isoform of arginine. We find that scFv-9dR mediated siRNA delivery more efficiently than scFv-9LR into T cells. Silencing efficiencies were also at least 30% higher indicating that the 9dR isoform is a better carrier of siRNA. The greater efficiency of the D isoform appears attributable to a more stable complex formation with siRNA.

1 Kumar, et. al., . T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell. 2008; 134(4):577-86.