Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector

Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14294-9. doi: 10.1073/pnas.1102883108. Epub 2011 Aug 15.

Abstract

Adeno-associated virus (AAV) is a single-stranded parvovirus retaining the unique capacity for site-specific integration into a transcriptionally silent region of the human genome, a characteristic requiring the functional properties of the Rep 78/68 polypeptide in conjunction with AAV terminal repeat integrating elements. Previous strategies designed to assemble these genetic elements into adenoviral (Ad) backbones have been limited by the general intolerability of AAV Rep sequences, prompting us to computationally reengineer the Rep gene by using synonymous codon pair recoding. Rep mutants generated by using de novo genome synthesis maintained the polypeptide sequence and endonuclease properties of Rep 78, while dramatically enhancing Ad replication and viral titer yields, characteristics indistinguishable from adenovirus lacking coexpressed Rep. Parallel approaches using domain swaps encompassing WT and recoded genomic segments, coupled with iterative computational algorithms, collectively established that 3' cis-acting Rep genetic elements (and not the Rep 78 polypeptide) retain dominant-acting sequences inhibiting Ad replication. These data provide insights into the molecular relationships of AAV Rep and Ad replication, while expanding the applicability of synonymous codon pair reengineering as a strategy to effect phenotypic endpoints.

Publication types

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

MeSH terms

  • Base Sequence
  • Codon / genetics
  • Computational Biology / methods*
  • Dependovirus / genetics*
  • Dependovirus / physiology
  • Endonucleases / metabolism
  • Genes, Viral / genetics
  • Genetic Vectors / genetics*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mutation / genetics
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism
  • Virus Replication / physiology

Substances

  • Codon
  • Viral Proteins
  • Endonucleases