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.

The ability to produce large quantities of recombinant Adeno-Associated Virus (rAAV) vectors is an important factor for the development of gene therapy-based medicine. The baculovirus/insect cell expression system is one of the major systems for large scale rAAV production. So far, most technological developments concerned the optimization of the AAV rep and cap genes in order to be expressed correctly in a heterologous system. However, the effect of the baculovirus infection on the production of rAAV has not been examined in detail. In this study we show that the baculoviral cathepsin (v-CATH) protease is active on several (but not all) rAAV serotypes, leading to a partial degradation of VP1/VP2 proteins. Subsequently, we identified the principal v-CATH cleavage site in the rAAV8 capsid proteins and demonstrated that the cleavage is highly specific. The proteolytic degradation of VP1/VP2 AAV capsid proteins reduces the infectivity of rAAV vectors but can be prevented by the use of a baculovirus vector with a deletion of the chiA/v-cath locus or by addition of the E64 protease inhibitor during production. Moreover, the codon optimization of AAV cap performed for several serotypes and originally aimed at the removal of potential alternative initiation codons, resulted in incorporation of additional forms of truncated VP1 into the rAAV capsids.

Recombinant adeno-associated virus (rAAV) has become the most widely used vector in the gene therapy field with hundreds of clinical trials ongoing and already several products on the market. AAV's physicochemical stability, and the various natural and engineered serotypes allow for targeting a broad range of cell types and tissue by diverse routes of administration. Progressing from early clinical studies to eventual market approval, many critical quality attributes have to be defined and reproducibly quantified, such as AAV stability, purity, aggregates, empty/full particles ratio, and rAAV genome titration. Droplet digital PCR (ddPCR) is becoming the tool of choice to perform absolute quantification of rAAV genomes. In the present study, we have identified critical parameters that could impact AAV titration and characterization accuracy, such as Poisson distribution confidence interval, primers/probe position, and potential aggregates. Our work presents how ddPCR can help to better characterize AAV vectors on the single particle level and highlights challenges that we are facing today in terms of AAV titration.