A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery

Abstract

Adeno-associated virus (AAV) has become a leading gene transfer vector for striated muscles. However, the AAV vectors also exhibit broad tropisms after systemic delivery. In an attempt to improve muscle tropism, we inserted a 7-amino-acid (ASSLNIA) muscle-targeting peptide (MTP) in the capsids of AAV2 at residue 587 or 588, generating AAV587MTP and AAV588MTP. In vitro studies showed that both viruses diminished their infectivity on non-muscle cell lines as well as on un-differentiated myoblasts, however, preserved or enhanced their infectivity on differentiated myotubes. AAV587MTP, but not AAV588MTP, also abolished its heparin-binding capacity and infected myotubes in a heparin-independent manner. Furthermore, in vivo studies by intravenous vector administration in mice showed that AAV587MTP enhanced its tropism to various muscles and particularly to the heart (24.3 fold of unmodified AAV2), whereas reduced its tropism to the non-muscle tissues such as the liver, lungs and spleen, etc. This alteration of tissue tropism is not simply due to the loss of heparin-binding, since a mutant AAV2 (AAVHBSMut) containing heparin-binding site mutations lost infectivity on both non-muscle and muscle cells. Furthermore, free MTP peptide, but not the scrambled control peptide, competitively inhibited AAV587MTP infection on myotubes. These results suggest that AAV2 could be re-targeted to the striated muscles by a muscle-targeting peptide inserted after residue 587 of the capsids. This proof of principle study showed first evidence of peptide-directed muscle targeting upon systemic administration of AAV vectors.