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.

Figure 1. Activation of the cellular unfolded protein response (UPR) pathways against AAV2 vectors in vitro.

HeLa cells were infected with self-complementary (sc) or single-stranded (ss) AAV2 vectors at an MOI of 5,000 vgs/cell. At various time-points (2/6/12/24/48 h) after infection, total RNA was isolated and the transcript levels of the UPR pathway genes were measured by real-time PCR. Dithiothreitol (DTT, 2 mM) was used as a positive control of UPR activation. A. The fold variation in UPR target genes (BiP, PERK, IRE1α and ATF6) expression in cells infected with single-stranded AAV2 at different time points. B. The fold variation in UPR target genes (BiP, PERK, IRE1α and ATF6) expression in cells infected with self-complementary AAV2 vectors relative to mock infected cells is shown. *p<0.05 Vs mock infected cells.

Figure 2. Western blot analysis of UPR activation.

HeLa cells were mock-infected or infected in triplicates with 5,000 vgs/cell of AAV2-EGFP vectors. The protein lysates were harvested at 2, 6, 12, 24 and 48 hours post infection for western blot analysis. The protein levels of PERK, phosphorylated-elF2α, IRE1α and BiP at different time points after single stranded AAV2 (A–E) or self complementary AAV2 (F–J) infection. β-actin was used as a loading control. The band intensities of all the test and control conditions was calculated by two independent densitometric scans using ImageJ software (NIH ImageJ, http://rsb.info.nih.gov/nih-image/) The data is mean +/− S.E from two independent experiments done with protein lysates pooled from triplicate conditions of mock- or AAV infection.

Figure 3. Self-complementary AAV2 infection activates PERK1 and IRE1α pathway and its downstream targets.

A. Total RNA from HeLa cells mock-infected or infected with of 5,000 vgs/cell of scAAV2-CB-EGFP vectors was used to profile the expression of downstream targets of IRE1α and PERK target genes such as ATF4 or CHOP by real-time PCR analysis at 2, 6, 12, 24 and 48 hours post infection. *p<0.05 Vs mock infected cells. B. Qualitative reverse-transcription PCR amplification of XBP1 (283 bp) and spliced variant sXBP1 (257 bp) at various time points, 2 h (lane 1), 6 h (lane 2), Molecular weight ladder (lane 3), 12 h (lane 4), 24 h (lane 5) and 48 h (lane 6) analyzed. Dithiothreitol (DTT, lane 7) was used as a positive control of UPR activation.

Figure 4. Alternate serotypes AAV 1 and AAV6 induce cellular unfolded protein response.

A. HeLa cells were infected with 5,000 vgs/cell of scAAV1- EGFP or scAAV6-EGFP vectors under identical conditions. Twelve hours post infection, the differential gene expression of UPR targets were assessed between mock-infected or AAV infected cells. Expression level of PERK, ATF6, IRE1 and CHOP from cells treated with AAV1 and AAV6. DTT (Dithiothreitol) was used as a positive control of UPR activation. *p<0.05 Vs mock infected cells.

Figure 5. Comparative analysis of AAV mediated transduction efficiency in HeLa cells after siRNA mediated knock down of PERK or IRE1α pathways.

A. Transgene expression was measured in HeLa cells 48 hrs post-infection with self-complementary AAV2-EGFP or AAV6-EFGP vectors either in the presence or absence of specific siRNA or scrambled siRNA control. B. Quantitative analyses of the data from (A) by fluorescence microscopy. Images from five visual fields were analyzed quantitatively by ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel²) per visual field (mean ± SD) and normalized to 1 for the control. Error bars represent standard error and the graph is a representative data set of at least three independent experiments. *p<0.05 Vs scrambled siRNA treated cells C. Western blot analysis of HeLa cellular extracts following mock (PBS)-infection or infection with AAV vectors, either in the presence or absence of PERK or IRE1α siRNA or scrambled siRNA control. β-actin was used as a loading control.

Figure 6. Self-complementary AAV2 mediated hepatic gene transfer in BALB/c mice activates UPR signalling.

Groups of mice (n = 4) were injected with 1×10¹¹ vg of scAAV2 vgs/animal intravenously with or without prior treatment with the UPR inhibitor, metformin (Met) (250 mg/kg body weight). Animals which received tunicamycin (1 µg/g) were used as positive controls for UPR activation. Twenty four hours after vector injection, the animals were euthanized and hepatic mRNA was assayed for the levels of PERK (A) or IRE1α (B) genes by real time PCR. *p<0.05 Vs AAV2 vector administered mice. Tunicamycin (Tm) injected animals were used a positive control for UPR activation. Mice treated with metformin alone were used as mock control.

Figure 7. Comparative gene expression profiling of AAV vector-induced inflammatory and immune response markers in the presence or absence UPR inhibitor during hepatic gene transfer in vivo.

Hepatic gene expression of various inflammatory cytokines in the scAAV2 injected BALB/c mice was measured 24 hours post vector administration. Genes which are significantly different between (2 fold, p<0.05) between mice that received AAV2 and metformin compared to the vector administered group alone, are shown.

Figure 8. Pharmacological inhibition of UPR increases self-complementary AAV2 mediated transgene expression in vivo.

C57/BL6 mice were either mock injected or injected with scAAV2 alone or with metformin and scAAV2 vectors at a dose of 1×10¹¹ vgs per mouse. Four weeks later, mice were euthanized and the liver lobes were studied for EGFP expression by fluorescence microscopy. All images were taken at an identical exposure of 576 milliseconds, gain of 1.5 and an intensity of 2. A. Representative images from each of the groups. B. Images from five visual fields per group were analyzed quantitatively using image-J software. *p<0.05 Vs scAAV2 treated mice.