Adeno-associated virus (AAV) inverted terminal repeats (ITRs) are key elements of AAV. These guanine-cytosine-rich structures are involved in the replication and encapsidation of the AAV genome, along with its integration in and excision from the host genome. These sequences are the only AAV-derived DNA sequences conserved in recombinant AAV (rAAV), as they allow its replication, encapsidation, and long-term maintenance and expression in target cells. Due to the original vector design, plasmids containing the gene of interest flanked by ITRs and used for rAAV production often present incomplete, truncated, or imperfect ITR sequences. For example, pSUB201 and its derivatives harbor a truncated (14 nt missing on the external part of the ITR), flop-orientated ITR plus 46 bp of non-ITR viral DNA at each end of the rAAV genome. It has been shown that rAAV genomes can be replicated, even with incomplete, truncated, or imperfect ITR sequences, leading to the production of rAAV vectors in transfection experiments. Nonetheless, it was hypothesized that unmodified wild-type (WT) ITR sequences could lead to a higher yield of rAAV, with less non-rAAV encapsidated DNA originating from the production cells and/or baculovirus shuttle vector genomes. This work studied the impact of imperfect ITRs on the level of encapsidated rAAV genomes and baculovirus-derived DNA sequences using the baculovirus/Sf9 cells production system. Replacement of truncated ITRs with WT and additional wtAAV2 sequences has an impact on the two major features of rAAV production: (1) a rise from 10% to 40% of full capsids obtained, and (2) up to a 10-fold reduction in non-rAAV encapsidated DNA. Furthermore, this study considered the impact on these major parameters of additional ITR elements and ITRs coupled with various regulatory elements of different origins. Implementation of the use of complete ITRs in the frame of the baculovirus-based rAAV expression system is one step that will be required to optimize the quality of rAAV-based gene therapy drugs.

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