Effect of Genome Size on AAV Vector Packaging

TLDR

AAV vector genomes have traditionally been limited to ~5 kb, but recent studies suggest that larger genomes can be packaged intact. The authors tested plasmid‑encoded AAV vectors ranging from 4.7 to 8.7 kb using AAV2, 5, and 8 capsids to assess packaging. Southern blotting showed that packaged genomes never exceeded 5.2 kb, and although larger plasmids produced heterogeneous, 5′‑truncated genomes that still expressed a reporter at high MOI, the expression efficiency was markedly lower, suggesting recombination restores intact genomes in transduced cells.

Abstract

Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5' end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination.