Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors

Abstract

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

Neuronal calcineurin transcriptional targets parallel changes observed in Alzheimer disease brain

“…Previous literature on the transcriptional impact of AAV transduction is relatively sparse, but a handful of studies have been reported, showing in particular that AAV serotype 2 could elicit an ER stress response and trigger UPR signaling pathways in HeLA cells, a phenomenon less prominent after single‐stranded AAV2 transduction (Balakrishnan et al . ). Other literature reports immune pathways activated by AAV (McCaffrey et al .…”

“…Approximately 400 genes are differentially expressed between GFP and PBS controls using the same cut-offs; several of which of are ribosomal genes. Previous literature on the transcriptional impact of AAV transduction is relatively sparse, but a handful of studies have been reported, showing in particular that AAV serotype 2 could elicit an ER stress response and trigger UPR signaling pathways in HeLA cells, a phenomenon less prominent after single-stranded AAV2 transduction (Balakrishnan et al 2013). Other literature reports immune pathways activated by AAV (McCaffrey et al 2008;Zhu et al 2009), although the AAV host responses likely vary upon the serotype, the injection route and the transduced cells.…”

Cellular unfolded protein response against viruses used in gene therapy

“…Another way to repress UPR could be by the use of silencing (si) RNAs against specific components of the UPR pathway. Our study had previously shown a modest increase in transgene expression from AAV vectors when the PERK and IRE-1α pathways were inhibited by specific siRNA in vitro (Balakrishnan et al, 2013). shRNAs against UPR components can also be potentially tested in vivo.…”

Identification of signaling pathways modulated by RHBDD2 in breast cancer cells: a link to the unfolded protein response

“…This situation was also evident for the chaperone CRT, but in this case, the peak of response is observed at 4 h (b), as well as observed for PERK, ATF4, and CHOP, which also showed a greater response in silenced cells compared with the scramble (c). Asterisk: Statistically significant differences (p <0.01) transcription of several stress genes including BiP and CHOP (CCAAT /enhancer -binding protein homologous protein ) (Yoshida et al 2001;Liu and Kaufman 2003;Chakrabarti et al 2011;Balakrishnan et al 2013).…”