Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors

Marcos Tejero, Ozgun F. Duzenli, Colin Caine, Hisae Kuoch, George Aslanidi

Research output: Contribution to journalArticlepeer-review


The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.

Original languageEnglish (US)
Article number1866
Issue number10
StatePublished - Oct 2023

Bibliographical note

Publisher Copyright:
© 2023 by the authors.


  • Rep gene
  • adeno-associated virus
  • genome encapsulation
  • upstream production

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.


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