Deletion of the B-B\u2019 and C-C\u2019 regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression

Qingzhang Zhou,Wenhong Tian,Xiaobing Wu,Chunguo Liu,Zhonghui Lian,Xiaoyan Dong

doi:10.1038/s41598-017-04054-4

Qingzhang Zhou, Wenhong Tian + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-04054-4

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Abstract

Inverted terminal repeats (ITRs) of the adeno-associated virus (AAV) are essential for rescue, replication, packaging, and integration of the viral genome. While ITR mutations have been identified in previous reports, we designed a new truncated ITR lacking the B-B’ and C-C’ regions named as ITRΔBC and investigated its effects on viral genome replication, packaging, and expression of recombinant AAV (rAAV). The packaging ability was compared between ITRΔBC rAAV and wild-type (wt) ITR rAAV. Our results showed the productivity of ITRΔBC rAAV was reduced 4-fold, which is consistent with the 8-fold decrease in the replication of viral genomic DNA of ITRΔBC rAAV compared with wt ITR rAAV. Surprisingly, transgene expression was significantly higher for ITRΔBC rAAV. A preliminary exploration of the underlying mechanisms was carried out by inhibiting and degrading the ataxia telangiectasia mutated (ATM) protein and the Mre11 complex (MRN), respectively, since the rAAV expression was inhibited by the ATM and/or MRN through cis interaction or binding with wt ITRs. We demonstrated that the inhibitory effects were weakened on ITRΔBC rAAV expression. This study suggests deletion in ITR can affect the transgene expression of AAV, which provides a new way to improve the AAV expression through ITRs modification.

Highlights

Adeno-associated virus (AAV) is a nonpathogenic member of the Parvoviridae family[1] that has received much attention as a promising vector for gene therapy and has been assessed in clinical trials[2]
Inverted terminal repeats (ITRs) is the unique cis element involved in recombinant AAV (rAAV) packaging
To directly assess whether deletion of the B-B’ and C-C’ regions in the two ITRs affects productivity, pAAV2biΔBC was used in rAAV packaging

Summary

Introduction

Adeno-associated virus (AAV) is a nonpathogenic member of the Parvoviridae family[1] that has received much attention as a promising vector for gene therapy and has been assessed in clinical trials[2]. We found the B-B’ region in the upstream ITRs was lost in the pAAV2neo, which was a rAAV vector plasmid in our laboratory, this deletion did not impact the packaging function of viral vector. The ability of Rep[68] to bind wt hairpin ITRs or mutant ITRs in which the B-B’ and C-C’ regions were deleted was identical. Effects of mutant ITRs on AAV have been limited It is unclear whether removal of the B-B’ and C-C’ regions from ITRs impairs the packaging and transgene expression of rAAV. To address these issues, we designed a rAAV vector plasmid harboring two mutant ITRs that lacked the B-B’ and C-C’ regions, and examined the effects of ITR truncation on packaging and expression of rAAV

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Results

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