Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression

Abstract

Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated virus (AAV) genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat (ITR) sequences. These cis-elements, primarily from AAV serotype 2, are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution; however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1-4, 6, and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression both in vitro and in vivo. However, we observed three classes of AAV ITR expression in vitro. Class I ITRs (AAV2 and 3) generated the highest level, whereas class II (AAV 4) had intermediate levels, and class III (AAV1 and 6) had the lowest levels. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. In vivo, all classes had promoter activity. Next-generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the Rep binding element. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influence on transgene expression cassettes independent of promoter selection.

Viral vector platforms within the gene therapy landscape

“…However, new evidence suggests that the ITRs of different serotypes can confer differential promoter activity. 168 Furthermore, there is evidence that the ITRs can produce transcripts on the minus strand, independent of a classical promoter on the reverse strand. 169 , 170 The result of such action is the formation of double-stranded RNAs, which can trigger the host innate immune system.…”

Adeno-Associated Virus Vector Mobilization, Risk Versus Reality

“…This may be attributed to the equal efficiency of wtAAV and rAAV genome encapsidation, 75 yet the improved efficiency of the rAAV vector yield may be a result of when the rep and cap genes are supplied by the wtAAV genome, which is assumed to have the optimal rep/cap expression ratios during production in the laboratory setting. 56 Although still unknown, this result may be related to the following: (1) inherent ITR transcriptional activity that may fine-tune expression of known or currently undescribed AAV ORFs 76 and/or perhaps (2) the ability of the wtAAV helper plasmid to replicate, an activity shown to increase rAAV production. 56 Since the rAAV vector production via cotransfection in the laboratory mimics the production of wtAAV and rAAV with regard to replication substrates, these observations strongly suggest the likelihood of wtAAV and rAAV particle production at similar efficiencies after cotransduction (Fig.…”

Wild type AAV, recombinant AAV, and Adenovirus super infection impact on AAV vector mobilization

“…This may be attributed to the equal efficiency of wtAAV and rAAV genome encapsidation 67 , yet the improved efficiency of rAAV vector yield may be a result of when the rep and cap genes are supplied by the wtAAV genome, which is assumed to have the optimal rep/cap expression ratios during production in the laboratory setting 68 . Although still unknown, this result may be related to: 1) inherent ITR transcriptional activity that may fine-tune expression of known or currently undescribed AAV ORFs 69 and or perhaps 2) the ability of the wtAAV helper plasmid to replicate, an activity shown to increase rAAV production 68 . Since the rAAV vector production via co-transfection in the laboratory mimics the production of wtAAV and rAAV with regard to replication substrates, these observations strongly suggest the likelihood of wtAAV and rAAV particle production at similar efficiencies post co-transduction (Fig.…”