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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. [ABSTRACT FROM AUTHOR]

DNA methylation is a major epigenetic event that affects not only cellular gene expression but that also has the potential to influence bacterial and viral DNA in their host-dependent functions. Adeno-associated virus (AAV) genome contains a high degree of CpG sequences capable of methylation in its terminal repeat sequences, which are the sole elements retained in AAV-based vectors used in gene therapy. The present study determined the influence of methylation status of the host cell on wild type (wt) AAV integration and recombinant (r) AAV transgene expression in HeLa cells. Results of the study indicated that hypo-methylation significantly enhanced both wtAAV chromosomal integration and transgene expression of rAAV. A direct influence of methylation on AAV integration was further confirmed by methylating the AAVS1 integration sites prior to viral infection with DNA trans-complementation assay. These results signify the importance of epigenetic status of target cells as one of the key factors in long-term transgene expression in AAV gene therapy. [ABSTRACT FROM AUTHOR]