Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb

Abstract

Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5′ or 3′-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5. Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5′ or 3′-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5.