Enhancement of adeno‐associated virus serotype 6 transduction into T cells with cell‐penetrating peptides

Abstract Background Adeno‐associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell‐penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. Methods The present study evaluated five CPPs (Antp, TAT‐HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. Results Vector incubation with peptides TAT‐HA2 and LAH4 at a final concentration of 0.2 m m resulted in an approximately two‐fold increase in transduced cells. At the lowest MOI tested (1.25 × 10 4 ), using LAH4 resulted in a 10‐fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6‐LAH4 complexes, their internalization does not appear to depend on macropinocytosis. Conclusions Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT‐HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.