A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors

Abstract

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.

Keywords: ITRs; nanopore sequencing; packaging; potency; productivity; rAAV.

Figure 1

(a) WT AAV2 ITR in the flip and flop configurations. In this study, the ITR with flop configuration was utilized. Rep binding element (RBE), the second Rep binding element (RBE’), and the terminal resolution site (TRS) are highlighted in black boxes. (b) Highlighted sequences for different deletions in ITRs. B loop deletion: orange and red base pairs. C loop deletion: purple and red base pairs. A region deletion: base pairs highlighted in yellow. D sequence deletion: base pairs highlighted in green. (c) Deletions were generated in either one or two of the ITRs. In total, 10 different constructs were created.

Figure 2

Mutations were identified in the ITRs during the plasmid construction phase. Initially, a plasmid containing intact 145 bp ITRs was utilized. However, following digestion and subsequent construct generation, approximately 40% of the colonies exhibited mutations within the ITRs. Notably, these ITR mutations were observed exclusively in one of the ITRs. Upon colony selection, the sequence of the ITRs was preserved throughout the plasmid expansion process.

Figure 3

The impact of deletions within the ITRs on crude harvest titer and percentage of non-empty capsids (%). (a) Vector genome titration in the crude harvest using ddPCR. (b) Measurement of the DNA-filled capsid percentage (non-empty capsids) using SEC HPLC. The error bars represent standard deviation of the mean (n = 2).

Figure 4

The assessment of AAV genome replication in HEK293 cells with and without Rep. The crude harvest samples were not treated with Benzonase, and the genome copy number was determined by ddPCR. The error bars represent standard deviation of the mean (n = 2).

Figure 5

The coverage of nanopore sequencing reads for 10 design constructs (SPE samples). The GOI carrying plasmid was linearized as reference and reads were mapped. Key features are annotated, with the two triangles indicating the ITR regions; the promoter; intron, hFIX, and Poly A (turquoise triangle) are within the two ITRs. AmpR and Ori are in the plasmid backbone region.

Figure 6

The coverage of nanopore sequencing reads for 10 design constructs (post-CsCl samples). The GOI carrying plasmid was linearized as a reference and reads were mapped. Key features were annotated; the two triangles indicate the ITR regions, the promoter, intron, hFIX, and Poly A (turquoise triangle) are within the two ITRs. AmpR and Ori are in the plasmid backbone region.

Figure 7

The attribution of Nanopore sequencing reads for 10 designed AAV vectors (SPE samples). The possible sources of packaged DNA are listed: the GOI sequence, the GOI plasmid backbone sequence, the host cell DNA, the Rep-Cap plasmid, and the helper plasmid. The sequencing reads that could not be mapped to any of the above categories were grouped into the unaligned category.

Figure 8

The attribution of Nanopore sequencing reads for 10 designed AAV vectors (post-CsCl samples).

Figure 9

In vitro potency of AAV-DJ-hFIX vectors. (a) The expression of hFIX at a series of MOIs ranging from 1000 to 25,600 vg/cell in Huh7 cells was quantified by ELISA and is shown as ng/mL per 1 × 10⁵ cells. (b) Purified vectors with deletions in both ITRs were compared to those with wild-type ITRs.