Studies of efficacy and liver toxicity related to adeno-associated virus-mediated RNA interference

Abstract

Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.

FIG. 1.

In vitro inhibition of HBV gene expression by H1- or U6-driven shRNAs. (a) Schematic representation of the shRNA-encoding pAAVEMBL plasmids containing the H1 or U6 promoter. The predicted structures and sequences of the three HBV-specific shRNAs and the control GL2 shRNA are depicted, with the sense strand shown in bold. (b) Inhibition of HBsAg expression by shRNAs. Huh-7 cells were transfected with pHBV1.3 alone or together with the indicated amounts of shRNA-expressing pAAVEMBL-H1 or pAAVEMBL-U6 plasmids for 72 hr, and then HBsAg levels in the culture supernatant were analyzed by enzyme-linked immunosorbent assay. The data are presented as a percentage of that produced by cells transfected with pHBV1.3 alone (mock transfection, mean±SD by three wells per condition). (c) Small RNAs from cells transfected with 1 μg of the different pAAVEMBL-H1 or pAAVEMBL-U6 plasmids transfected were detected by Northern blot using radiolabeled probes identical in sequence to the antisense strand of the corresponding HBV or luciferase shRNAs. 5.8S rRNA was stained with ethidium bromide as a loading control. HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; ITR, inverted terminal repeat; SD, standard deviation; shRNA, short hairpin RNA.

FIG. 2.

In vivo HBV DNA inhibition by AAV8-H1- or AAV8-U6-encoded shRNAs. Groups of ICR/HBV mice (n=7) were injected intravenously with 10¹² vg per mouse of AAV8-H1 or AAV8-U6 vectors encoding HBV-specific shRNAs (HBV-S1, sAg19, or sAg25) or control GL2 shRNA, and then serum samples were collected weekly for analysis of HBV DNA levels and the results displayed as a percentage of the pretreatment titer for each group (mean±SD). The experiment was repeated twice with similar results.

FIG. 3.

Expression of shRNA and the copy number of AAV vectors in the liver. ICR/HBV mice were injected intravenously with 10¹² vg per mouse of the different AAV8-H1 or AAV8-U6 vectors as in Figure 2 and then were euthanized after 1 or 6 weeks, two mice at each time point. (a) Northern blot analysis of shRNA and miR-122. Total liver RNA was analyzed by Northern blot using radiolabeled probes specific for the antisense strand of each shRNA, endogenous miR-122, or 5S rRNA. (b) Southern blot analysis of AAV vectors. Total liver DNA was digested with HindIII and XbaI and hybridized with a radiolabeled DNA probe against the nonencoded green fluorescent protein sequence present in all AAV8 vectors. Each lane represents an individual mouse sample. The figure shows one representative set of data from two independent experiments. (c-e) The density of the signals in each lane was quantitated using ImageQuant software. The amount of shRNAs (c) or miR-122 (e) in each group is presented as the ratio of the density of the band to that of 5S rRNA. (d) The AAV genome copy number per cell in each group was calculated using the reference standard generated from the pAAVEMBL plasmid. Data are the summarized results of four mice from two independent experiments and are presented as the mean±SD. AAV, adeno-associated virus.

FIG. 4.

Liver injury induced by AAV8-H1- and AAV8-U6-encoded shRNAs in ICR/HBV mice. Groups of ICR/HBV mice (n=8) were injected intravenously with 10¹² vg per mouse of different AAV8-H1 or AAV8-U6 vectors as in Figure 2, and serum samples collected at the indicated times for measurement of ALT activity (a) and albumin levels (b). The data (mean±SD) are for one representative result from two independent experiments. The dashed lines represent the mean value for ALT activity (a) or albumin levels (b) in untreated ICR/HBV mice (n=6). ALT, alanine aminotransferase.

FIG. 5.

Liver injury induced by AAV8-U6/sAg19 in different mouse strains and analysis of intrahepatic leukocytes and their contribution to liver injury caused by toxic shRNA. (a) ICR mice, C57BL/6 mice, FVB mice, or Nod-scid/IL2Rγ^−/− mice (n=5–7 per group) were injected intravenously with 10¹² vg of AAV8-U6/sAg19 and serum samples collected at the indicated times for analysis of ALT activity (mean±SD). (b and c) Groups of C57BL/6 mice (n=6) were injected with saline, AAV8-H1/sAg19, or AAV8-U6/sAg19, and liver leukocytes were isolated on day 4 postinjection. The number of total intrahepatic leukocytes (b) and different cell subsets (c) per liver from 3 mice per group are shown (mean±SD). , Saline; □, AAV8-H1/sAg19; ■, AAV8-U6/sAg19. The number of cells per liver for individual cell types was calculated by multiplying the percentage of the individual cell type by the total number of isolated liver leukocytes per liver. The experiment was performed twice with similar results. (d) Groups of wild-type C57BL/6 mice treated with anti-asialo-GM1 antiserum, anti-Gr-1 monoclonal antibody, clodronate liposome or GdCl3, CD1d^−/− mice, I-Aβ^−/− mice, or CD8^−/− mice (n=5–8) were injected with AAV8-U6/sAg19 on day 0, and then serum samples were collected at day 4 for analysis of ALT activity (mean±SD). Wild-type C57BL/6 mice treated with control rabbit antiserum, control rat immunoglobulin G, or saline were included as controls.

FIG. 6.

Analysis of hepatic cytokine profiles in C57BL/6 mice treated with shRNA-encoding AAV vectors. Total liver protein (a–c) or RNA (d) from C57BL/6 mice (n=3 per group) collected 4 days after injection of 10¹² vg of AAV8-H1/sAg19 or AAV8-U6/sAg19 was assayed by the mouse cytokine array (a–c) or real-time reverse transcriptase polymerase chain reaction (d). (a) List of antibodies on the cytokine array membrane. Each antibody is represented by duplicate spots. (b) A representative blot for two independent experiments. (c) Average net optical intensity for each pair of cytokine spots. (d) Cytokine and chemokine mRNAs in AAV8-vector-treated animals measured by real-time reverse transcrptase polymerase chain reaction. mRNAs are expressed as a fold-change compared with the control (mean±SD).