Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System

Abstract

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.

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

“…First, the relative proportion of packaged capsids increased to 40% from 10%, and second, the packaging of non-specific sequences was reduced by 10-fold. [56] The incorporation of RBE in the original One-Bac1.0 was responsible for high-frequency co-packaging of rep/cap sequences in AAV capsids, confirming its role as a packaging signal. [50] Excision of the RBE component in the One-Bac2.0/AAV5 cell line resulted in more than 3-and 4-log reductions in the frequency of rep and cap sequence copackaging, respectively, with final rep/cap packaging levels reaching as low as 0.001% and 0.02%, respectively.…”

“…The inverted terminal repeat sequence of the AAV genome is the only element from the wild-type AAV that is maintained in a recombinant vector and is present in cis in a transgene expression cassette. A recent report by Savy et al [56] showed that the unmodified AAV2 wildtype ITR (wtITR) sequence promotes enhanced and specific encapsidation of the rAAV8 vector sequence in contrast to truncated ITRs, which is found in traditional packaging plasmids such as pSUB201 and its variants. The latter was found to be responsible for non-specific packaging.…”

“…The second question is related to the efficiency of vector genome packaging in insect cells. In general, insect cells have been reported to produce more AAV empty capsids [43,56,93] compared to mammalian cell platforms, [94][95][96] which counteracts any success achieved with genomic and functional particles of wtAAV in the presence of helper virus functions. [97] Moreover, in contrast to well-defined and wellcharacterized helper genes of adenovirus or HSV, [98,99] the baculovirus helper elements have not yet been fully identified, even though their helper function has been known for almost two decades.…”

“…A recent report by Savy et al. [ 56 ] showed that the unmodified AAV2 wild‐type ITR (wtITR) sequence promotes enhanced and specific encapsidation of the rAAV8 vector sequence in contrast to truncated ITRs, which is found in traditional packaging plasmids such as pSUB201 and its variants. The latter was found to be responsible for non‐specific packaging.…”

“…First, the relative proportion of packaged capsids increased to 40% from 10%, and second, the packaging of non‐specific sequences was reduced by 10‐fold. [ 56 ]…”