Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

Mol Ther. 2017 Jun 7;25(6):1363-1374. doi: 10.1016/j.ymthe.2017.03.028. Epub 2017 Apr 24.


Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal repeats (mTRs), sequences that were previously thought to be required for functional self-complementary AAV vectors. The use of shDNAs as artificial mTRs opens the door to engineering a new generation of AAV vectors with improved potency, genetic stability, and safety for both preclinical studies and human gene therapy.

Keywords: adeno-associated virus; genome homogeneity; replication template switching; self-complementary AAV; short hairpin DNA; short hairpin RNA; viral genome truncation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • DNA Replication
  • DNA, Viral*
  • Dependovirus / genetics*
  • Gene Expression
  • Gene Order
  • Gene Transfer Techniques
  • Genes, Reporter
  • Genetic Vectors / genetics*
  • Genome, Viral*
  • Humans
  • Inverted Repeat Sequences*
  • Male
  • Mice
  • Models, Biological
  • Nucleic Acid Conformation
  • Plasmids / genetics
  • RNA, Small Interfering
  • Sequence Analysis, DNA
  • Sequence Deletion
  • Transduction, Genetic


  • DNA, Viral
  • RNA, Small Interfering