Transduction Properties of an Adenovirus Vector Containing Sequences Complementary to a Liver-Specific microRNA, miR-122a, in the 3′-Untranslated Region of the E4 Gene in Human Hepatocytes from Chimeric Mice with Humanized Liver

Abstract

Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3′-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3′-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3′-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3′-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.

INTRODUCTION

Replication-incompetent adenovirus (Ad) vectors have attracted much attention as gene delivery vehicles in not only gene therapy studies but also basic researches due to their various advantages, which include efficient transduction, a relatively large capacity for transgenes, and high titer production. The E1A gene, which is crucial for the Ad replication cycle, is genetically deleted from the Ad vector genome in a replication-incompetent Ad vector. Theoretically, the deletion of the E1A gene makes it impossible to transcribe other Ad genes in the Ad vector genome. However, other Ad genes in the Ad vector genome are slightly transcribed following transduction, resulting in Ad vector-induced cytotoxicity and immune responses against Ad proteins.^1–4) In particular, Ad vector-induced hepatotoxicity has been reported in clinical and nonclinical studies, because Ad vectors efficiently accumulate in the liver following systemic administration.^1,2,5–7) In order to overcome these problems, next-generation Ad vectors, including Ad vectors with the E2A and/or the E4 deletion in addition to the E1/E3 deletion, and helper-dependent Ad vectors, have been developed so far, but these Ad vectors are often difficult to produce at high titers.^8–11)

We previously developed a novel Ad vector containing four copies of sequences perfectly complementary to miR-122a, which is a hepatocyte-specific microRNA (miRNA), in the 3′-untranslated region (UTR) of the E4 gene in order to suppress the leaky expression of Ad genes from the Ad vector genome in the hepatocytes.^4,12–14) This novel Ad vector mediated significant reductions in the Ad gene expression and cytotoxicity in the human hepatocellular carcinoma cell line Huh-7 cells, primary mouse hepatocytes, and mouse liver, leading to efficient transgene expression; however, it remained to be elucidated whether this Ad vector with miR-122a-targeted sequences in the 3′-UTR of the E4 gene also showed superior transgene expression profiles without apparent cytotoxicity in human hepatocytes. Hepatocytes are multi-functional cells, and play highly crucial roles in various functions of the liver, including protein synthesis, transformation of carbohydrates, detoxification, modification, and excretion of exogenous and endogenous substances, and synthesis of cholesterol and lipids.¹⁵⁾ Hepatocytes are an important target for gene therapy of various diseases, including hemophilia, ornithine transcarbamylase deficiency, and viral hepatitis.^13,16–19) In addition, primary human hepatocytes are often used in studies of drug metabolism and toxicology.²⁰⁾ Therefore, methods for the safe and efficient delivery and expression of foreign genes in human hepatocytes are urgently needed not only for gene therapy studies but also gene function analyses.

In this study, we examined the transgene expression and cytotoxicity profiles of Ad vectors containing miR-122a-targeted sequences in the 3′-UTR of the E4 gene in primary human hepatocytes isolated from humanized-liver mice. Insertion of miR-122a-targeted sequences in the 3′-UTR of the E4 gene improved the transgene expression and cytotoxicity profiles of Ad vectors in the primary human hepatocytes, indicating that an Ad vector containing miR-122a-targeted sequences in the 3′-UTR of the E4 gene efficiently delivers transgenes to primary human hepatocytes without apparent toxicity.

MATERIALS AND METHODS

Ad Vectors

An AHA promoter (a liver-specific apolipoprotein E enhancer-hepatocyte control region-human α1-antitrypsin promoter)-driven mouse secreted alkaline phosphatase (mSEAP) expression cassette-containing conventional Ad vector, Ad-AHASEAP and an Ad vector with miR-122a-targeted sequences in the 3′-UTR of the E4 gene, Ad-E4-122aT-AHASEAP, were previously produced.⁴⁾ The CA promoter (a fusion promoter composed of chicken β-actin promoter and cytomegalovirus (CMV) enhancer)-driven mSEAP expression cassette-containing conventional Ad vector, Ad-CASEAP, and the Ad vector with miR-122a-targeted sequences in the 3′-UTR of the E4 gene, Ad-E4-122aT-CASEAP, were prepared by an improved in vitro ligation method.^21,22) Briefly, the fragment containing the mSEAP gene, which were recovered from pCpG-mSEAP (InvivoGen, SanDiego, CA, U.S.A.), was inserted under the CA promoter in pHMCA6,²³⁾ producing pHMCA6-mSEAP. I-CeuI/PI-SceI-digested pHMCA6-mSEAP was ligated with I-CeuI/PI-SceI-digested pAdHM4²¹⁾ and pAdHM4-E4-122aT,⁴⁾ producing pAdHM4-CAmSEAP and pAdHM4-E4-122aT-CAmSEAP, respectively. pAdHM4-CAmSEAP and pAdHM4-E4-122aT-CAmSEAP were digested with PacI, followed by transfection in HEK293 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Carlsbad, CA, U.S.A.), producing Ad-CASEAP and Ad-E4-122aT-CASEAP, respectively. The CMV promoter-driven enhanced green fluorescent protein (GFP) expression cassette-containing conventional Ad vector, Ad-GFP, was previously produced.²⁴⁾ The CMV promoter-driven GFP expression cassette-containing Ad vector with miR-122a-targeted sequences in the 3′-UTR of the E4 gene, Ad-E4-122aT-CMVGFP, were similarly produced using pAdHM4-E4-122aT, and pHMCMV-GFP1.²⁴⁾ The Ad vectors were amplified and purified as previously described.⁴⁾ The virus particles (VPs) were determined using a spectrophotometric method.²⁵⁾ The infectious units (IFU) were determined using an Adeno-X Rapid Titer Kit (Clontech Laboratories, Mountain View, CA, U.S.A.). The Ad vectors used in this study are shown in Fig. 1.

Fig. 1. Schematic Diagrams of the Replication-Incompetent Ad Vectors Used in This Study

A GFP or mSEAP expression cassette was inserted into the E1-deleted region in Ad-GFP, Ad-E4-122aT-CMVGFP, Ad-AHASEAP, Ad-E4-122aT-AHASEAP, Ad-CASEAP, and Ad-E4-122aT-CASEAP. CMV, the cytomegalovirus promoter; GFP, enhanced green fluorescent protein; AHA, a synthetic promoter composed of the apolipoprotein E enhancer, the hepatocyte control region, and the human α1-antitrypsin promoter; CA, a fusion promoter composed of the chicken β-actin promoter and the CMV enhancer; ITR, inverted terminal repeat; pA, bovine growth hormone (BGH) gene polyadenylation signal.

Cells

HEK293 cells (a human transformed embryonic kidney cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM L-glucose, 100 µg/mL streptomycin, and 100 U/mL penicillin. Primary human hepatocytes which were recovered from chimeric mice with humanized liver (PXB-mice^®) (PXB-cells; PhoenixBio, Hiroshima, Japan), were seeded at a density of 7 × 10⁴ cells/well on type I collagen-coated 96-well plates using maintenance medium as previously described.²⁶⁾ Transduction with Ad vectors was performed 3 d after cell seeding as described below. Contamination of mouse-derived cells in the primary human hepatocyte culture was less than several percent.²⁶⁾

Ad Vector-Mediated Transduction in Primary Human Hepatocytes

For evaluation of Ad vector-mediated GFP expression in the primary human hepatocytes, cells were transduced with Ad vectors at a multiplicity of infection (MOI) of 30. GFP expression was examined using a Keyence Biozero BZ-9000 fluorescence microscope (KEYENCE, Osaka, Japan) 2 d after transduction. For evaluation of Ad vector-mediated SEAP expression in the primary human hepatocytes, cells were transduced with Ad vectors at an MOI of 50. The culture supernatants were recovered on the indicated day points following transduction with Ad vectors. The culture supernatants were replaced with fresh media every other day. mSEAP expression levels were determined using a Great EscAPe SEAP Chemiluminescence Kit, version 2.0 (TaKaRa Bio, Otsu, Japan). Cell morphologies were observed under the microscope on the indicated day points. Cell viabilities were assessed 14 d after transduction by water-soluble tetrazolium (WST)-8 assay using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan).

Real-Time RT-PCR Analysis

Total RNA was recovered 7 d after transduction from the primary human hepatocytes using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions on the indicated day points following Ad vector transduction. cDNA was synthesized using a Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific). Real-time RT-PCR analysis was performed using a StepOnePlus System (Thermo Fisher Scientific) and THUNDERBIRD SYBR qPCR Mix reagents (TOYOBO, Osaka, Japan). The values were normalized by the mRNA levels of a housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers are available on request.

Statistical Analysis

Statistical significance was analyzed by the one-way ANOVA followed by Bonferroni multiple comparison test.

RESULTS

Transgene Expression Levels of Ad-E4-122aT-AHASEAP and Ad-E4-122aT-CASEAP in Primary Human Hepatocytes

In order to examine whether suppression of the leaky expression of Ad genes from the Ad vector genome improved the transduction properties of Ad vectors in primary human hepatocytes, Ad vectors containing the miR-122a-targeted sequences in the 3′-UTR of the E4 gene were added to the primary human hepatocytes at a MOI of 30. A previous study demonstrated that transduction efficiencies of an Ad vector in primary human hepatocytes reached a plateau at a MOI of 25.²⁷⁾ Both the conventional Ad vector, Ad-GFP, and improved Ad vector, Ad-E4-122aT-CMVGFP, produced almost 100% GFP-positive cells at 48 h after transduction, indicating that the primary human hepatocytes were highly susceptible to Ad vector-mediated transduction (Fig. 2a). Next, we performed a time-course study of transgene expression in the primary human hepatocytes using mSEAP-expressing Ad vectors (Fig. 2b). In order to obtain higher transgene expression, we used the CA promoter instead of the CMV promoter because the CA promoter was stronger than the CMV promoter.^28,29) We also tested the AHA promoter, which is a hepatocyte-specific promoter.³⁰⁾ mSEAP production levels by Ad-E4-122aT-CASEAP and Ad-CASEAP were higher than those by Ad-AHASEAP and Ad-E4-122aT-AHASEAP by about 1.4-fold on day 2, and by about 6-fold on day 4, indicating that the CA promoter mediated more efficient transcription than the AHA promoter in primary human hepatocytes. Ad-E4-122aT-CASEAP produced 1.8-fold higher levels of mSEAP than Ad-CASEAP on day 7. Ad-E4-122aT-AHASEAP mediated 1.6-fold higher levels of mSEAP expression than Ad-AHASEAP on day 7. There were no statistically significant differences in the mSEAP expression in the culture supernatants between Ad-E4-122aT-AHASEAP-treated and Ad-AHASEAP-treated cells, or between Ad-E4-122aT-CASEAP-treated and Ad-CASEAP-treated cells on days 2 and 4. These results indicated that insertion of miR-122a-targeted sequences in the 3′-UTR of the E4 gene significantly improved the transgene expression profiles of an Ad vector in primary human hepatocytes.

Fig. 2. Transgene Expression Profiles of Ad Vectors in the Primary Human Hepatocytes

(a) GFP expression in the primary human hepatocytes following transduction with Ad-GFP and Ad-E4-122aT-CMVGFP. The primary human hepatocytes were transduced with Ad vectors at an MOI of 30. GFP expression was observed 2 d after transduction. The scale bar indicates 200 µm. (b) mSEAP expression levels in the culture supernatants of primary human hepatocytes following Ad vector transduction. Primary human hepatocytes were transduced with Ad vectors at an MOI of 50. The culture supernatants were recovered on days 2, 4, and 7 after transduction, followed by measurement of mSEAP levels in the culture supernatants. mSEAP levels in the culture supernatants of mock-transduced cells were less than 0.01 µg/mL. The data are expressed as the means ± standard deviation (S.D.) (n = 4). * p < 0.05. (Color figure can be accessed in the online version.)

Leaky Expression of Ad Genes from Ad Vectors in Primary Human Hepatocytes

Next, in order to examine whether insertion of miR-122a-targeted sequences in the 3′-UTR of the E4 gene suppressed the leaky expression of Ad genes from the Ad vector genome, Ad gene expression levels in the primary human hepatocytes were measured by real-time RT-PCR analysis following Ad vector transduction. Ad gene expression levels in the Ad-E4-122aT-AHASEAP-treated and Ad-E4-122aT-CASEAP-treated primary human hepatocytes were substantially lower than those in the Ad-AHASEAP-treated and Ad-CASEAP-treated cells, although there were no statistically significant differences in the Ad gene expression levels between the Ad-E4-122aT-AHASEAP-treated and the Ad-AHASEAP-treated cells (Fig. 3). The mRNA levels of the E2A, E4, and hexon genes following transduction with Ad-E4-122aT-CASEAP were approximately 91-fold, 11-fold, and 1200-fold lower than those following transduction with Ad-CASEAP, respectively. The mRNA levels of the E2A, E4, and hexon genes following transduction with Ad-E4-122aT-AHASEAP were approximately 10-fold, 3-fold, and 22-fold lower than those following transduction with Ad-AHASEAP, respectively. These results indicate that insertion of miR-122a-targeted sequences in the 3′-UTR of the E4 gene largely reduced the leaky expression of Ad genes from the Ad vector genome in primary human hepatocytes. In addition, Ad-CASEAP exhibited approximately 3.4-fold and 4.8-fold higher levels of the E2A and E4 gene expression in the primary human hepatocytes compared to Ad-AHASEAP, respectively. These data suggested that the transcriptional activities of the promoters driving the transgene expression in the Ad vector genome would affect the leaky expression levels of Ad genes.

Fig. 3. Leaky Expression Levels of Ad Genes from the Ad Vector Genome in Primary Human Hepatocytes Following Transduction

Primary human hepatocytes were transduced with Ad vectors at an MOI of 50. Total RNA was recovered from the cells 7 d after transduction, followed by real-time RT-PCR analysis. The data are expressed as the means ± S.D. (n = 4). N.S., not significant. ** p < 0.01, *** p < 0.005.

Ad Vector-Induced Cytotoxicity in Primary Human Hepatocytes

Next, in order to examine whether leaky expression of Ad genes from the Ad vector genome resulted in Ad vector-mediated cytotoxicity in primary human hepatocytes, the morphologies of the primary human hepatocytes were observed following Ad vector transduction at an MOI of 100. No apparent morphological changes in the primary human hepatocytes were not found on day 4 following transduction with any of the types of Ad vectors (Fig. 4a); however, Ad-CASEAP significantly induced a severe damage on the primary human hepatocytes on days 6 and 10. Ad-E4-122aT-CASEAP slightly induced cytotoxicities in the primary human hepatocytes on day 10. No cytotoxicity was evident following transduction with Ad-AHASEAP or Ad-E4-122aT-AHASEAP. Severe cytotoxicity was not induced by any of the Ad vectors transduced at MOIs of 25 or 50 (data not shown). High levels of cytotoxicity were also found in Ad-CASEAP-treated cells in WST-8 assay (Fig. 4b). These results suggested that leaky expression of Ad genes from the Ad vector genome partly contributed to the Ad vector-mediated cytotoxicity in primary human hepatocytes, and that suppression of the leaky expression of Ad genes by miR-122a-mediated post-transcriptional gene silencing mitigated the Ad vector-mediated cytotoxicity.

Fig. 4. Cytotoxicities Associated with Ad Vector Transduction in Primary Human Hepatocytes

(a) Morphologies of the primary human hepatocytes following Ad vector transduction. Primary human hepatocytes were transduced with Ad vectors at an MOI of 100. Cell morphologies were observed on days 4, 6, and 10 after transduction. The representative images from two independent experiments are shown. Scale bars indicate 100 µm. (b) Cell viabilities following Ad vector transduction. Primary human hepatocytes were transduced as described above. Cell viabilities were assessed on day 14 by WST-8 assay. The data is the averages of duplicate samples (n = 2).

DISCUSSION

In this study, we examined the transduction properties of an Ad vector possessing sequences perfectly complementary to a liver-specific miRNA, miR-122a, in the 3′-UTR of the E4 gene in primary human hepatocytes. This study demonstrated that while a conventional Ad vector containing the CA promoter, Ad-CASEAP, induced severe cytotoxicity in the primary human hepatocytes at 10 d after transduction at an MOI of 100, insertion of miR-122a-targeted sequences in the 3′-UTR of the E4 gene improved not only the Ad vector-mediated cytotoxicity but also the transgene expression profiles of an Ad vector in the primary human hepatocytes by suppressing the leaky expression of Ad genes. These findings indicated that an Ad vector with miR-122a-targeted sequences in the 3′-UTR of the E4 gene is an efficient gene delivery vehicle for primary human hepatocytes. Development of an efficient gene delivery vehicle to primary human hepatocytes has been eagerly anticipated because human hepatocytes are an important research target for both gene therapy studies and basic studies. An efficient gene delivery vehicle to primary human hepatocytes in vitro makes it possible to genetically manipulate primary human hepatocytes by over-expressing foreign genes and knocking down/out target genes. In a previous work, we successfully achieved genome editing in the primary human hepatocytes by efficient delivery of a CRISPR-Cas9 system using an Ad vector with miR-122a-targeted sequences in the 3′-UTR of the E4 gene.³¹⁾

Insertion of miR-122a-targeted sequences in the 3′-UTR of the E4 gene resulted in the reduction in the mRNA levels of not only the E4 gene but also the E2A and hexon genes in the primary hepatocytes. This is probably because the E4 gene products have various functions, including enhancement of the export of virus mRNA from the nucleus and late viral protein production.³²⁾ The E4 gene products are involved in the expression of the E2A and hexon genes.

The hexon mRNA levels were comparable in Ad-AHASEAP-treated and Ad-CASEAP-treated cells, however, the hexon mRNA levels were approximately 8-fold higher than the E2A and E4 mRNA levels following transduction with Ad-AHASEAP and Ad-CASEAP (data not shown). The mechanism of leaky expression of the hexon gene would be somewhat different from that of the E2A and E4 genes.

This study suggests that transgene expression levels in the primary human hepatocytes were correlated with Ad vector-mediated cytotoxicity levels. Significant differences in the mSEAP levels were not found on days 2 and 4 between conventional Ad vectors and Ad-E4-122aT vectors containing the same transgene expression cassette (Fig. 2b). Apparent morphological changes were not observed on day 4 (Fig. 4a). On the other hand, apparent cytotoxicities were found on day 6 in Ad-CASEAP-treated cells. Significant differences in the mSEAP levels were found on day 7 between Ad-AHASEAP and Ad-E4-122aT-AHASEAP, and between Ad-CASEAP and Ad-E4-122aT-CASEAP. These data indicate that suppression of Ad vector-mediated cytotoxicity is important for improvement of transgene expression profiles.

The mechanism of Ad protein-induced cytotoxicity via a non-adaptive immune response is highly complex and remains to be clarified. Among the Ad proteins, the E4 gene products have been demonstrated to contribute to Ad vector-induced cytotoxicity.^{1,10,33–35)} Moreover, among the several E4 gene products, the E4 open reading frame (ORF) 4 was shown to induce apoptosis via caspase activation in a cell line-specific manner.³⁶⁾ In addition, the E4 ORF4 preferentially induced apoptosis in transformed cells, not non-transformed cells.³⁷⁾ In the present study, we did not find significant up-regulation of apoptosis-related genes, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Bcl-2-associated X protein (Bax), following transduction with Ad vectors in the primary human hepatocytes in this study (data not shown). Other mechanisms different from the classical apoptosis pathway and/or the other E4 ORF products are likely be involved in Ad vector-mediated in vitro cytotoxicity in the primary human hepatocytes.

As described above, leaky expression of Ad genes from the Ad vector genome is involved in Ad vector-induced cytotoxicity. Ad-CASEAP mediated higher levels of the E2A and E4 gene expression in the primary human hepatocytes than Ad-AHASEAP, and thereby led to more severe morphological changes in the primary human hepatocytes than Ad-AHASEAP. These data indicate that we should be mindful of the risk of Ad vector-mediated cytotoxicity when using a strong promoter for transgene expression. Ad-E4-122aT-CASEAP slightly induced the cytotoxicity on day 10, by contrast, we did not find apparent morphological changes in Ad-AHASEAP-treated cells on day 10 (Fig. 3). We did not find statistically significant differences in the leaky expression levels of the Ad genes in Ad-AHASEAP-treated or Ad-E4-122aT-CASEAP-treated cells (Fig. 4). Differences in the leaky expression levels of other Ad genes except for the E2A, E4, and hexon genes might explain the differences in the cytotoxicity levels between Ad-AHASEAP-treated and Ad-E4-122aT-CASEAP-treated cells. For example, the pIX mRNA levels in Ad-E4-122aT-CASEAP-treated cells were approximately 30-fold higher than those in Ad-AHASEAP-treated cells (data not shown). Furthermore, high levels of mSEAP expression might be involved in the cytotoxicity in Ad-E4-122aT-CASEAP-treated cells. Approximately 10-fold higher levels of mSEAP expression were found in Ad-E4-122aT-CASEAP-treated cells than in Ad-AHASEAP-treated cells (Fig. 1b).

mSEAP production levels in Ad-E4-122aT-AHASEAP-treated cells were 1.5-fold higher than those in Ad-AHASEAP-treated cells on day 7. Although we did not find the apparent differences in the cell morphology or survival between Ad-AHASEAP and Ad-E4-122aT-AHASEAP, mRNA levels of hepatocyte-specific genes, including the CYP3A4 and albumin genes, in Ad-E4-122aT-AHASEAP-treated cells were significantly higher than those in Ad-AHASEAP-treated cells (data not shown). These data indicated that Ad-E4-122aT-AHASEAP-induced cytotoxicity levels were lower than those by Ad-AHASEAP. Lower levels of cytotoxicity in Ad-E4-122aT-AHASEAP-treated cells than those in Ad-AHASEAP-treated cells resulted in the higher levels of mSEAP production by Ad-E4-122aT-AHASEAP than by Ad-AHASEAP on day 10.

In summary, we demonstrated that an Ad vector containing miR-122a-targeted sequences in the 3′-UTR of the E4 gene showed superior transgene expression profiles and safety profiles in the primary human hepatocytes. This Ad vector is an efficient gene delivery vehicle for primary human hepatocytes in not only gene therapy studies but also gene function analysis.

Acknowledgments

We thank Dr. Mark A Kay (Stanford University, Stanford, CA, U.S.A.) and Dr. Jun-ichi Miyazaki (Osaka University, Osaka, Japan) for kindly providing the AHA promoter and the CA promoter, respectively. This study was supported by Grants-in-Aid for Young Scientists (A) and for Scientific Research (A) and from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and by Grants from the Japan Agency for Medical Research and Development (AMED) under Grant Number 21fk0310109s0205. T. Tsukamoto is a Research Fellow of the Japan Society for the Promotion of Science.

Conflict of Interest

YI and CT are paid employees of PhoenixBio Co., Ltd. PXB-mice and PXB-cells are products of PhoenixBio Co., Ltd.

REFERENCES

• 1) Gao GP, Yang Y, Wilson JM. Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J. Virol., 70, 8934–8943 (1996).
• 2) Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. U.S.A., 91, 4407–4411 (1994).
• 3) Engelhardt JF, Ye X, Doranz B, Wilson JM. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. U.S.A., 91, 6196–6200 (1994).
• 4) Shimizu K, Sakurai F, Tomita K, Nagamoto Y, Nakamura S, Katayama K, Tachibana M, Kawabata K, Mizuguchi H. Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome. Mol. Ther. Methods Clin. Dev., 1, 14035 (2014).
• 5) Gallo-Penn AM, Shirley PS, Andrews JL, Tinlin S, Webster S, Cameron C, Hough C, Notley C, Lillicrap D, Kaleko M, Connelly S. Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs. Blood, 97, 107–113 (2001).
• 6) Reddy PS, Sakhuja K, Ganesh S, Yang L, Kayda D, Brann T, Pattison S, Golightly D, Idamakanti N, Pinkstaff A, Kaloss M, Barjot C, Chamberlain JS, Kaleko M, Connelly S. Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector. Mol. Ther., 5, 63–73 (2002).
• 7) Lin X, Huang H, Li S, Li H, Li Y, Cao Y, Zhang D, Xia Y, Guo Y, Huang W, Jiang W. A phase I clinical trial of an adenovirus-mediated endostatin gene (E10A) in patients with solid tumors. Cancer Biol. Ther., 6, 648–653 (2007).
• 8) Lusky M, Christ M, Rittner K, Dieterle A, Dreyer D, Mourot B, Schultz H, Stoeckel F, Pavirani A, Mehtali M. In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted. J. Virol., 72, 2022–2032 (1998).
• 9) Zhou H, O’Neal W, Morral N, Beaudet AL. Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted. J. Virol., 70, 7030–7038 (1996).
• 10) Andrews JL, Kadan MJ, Gorziglia MI, Kaleko M, Connelly S. Generation and characterization of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII. Mol. Ther., 3, 329–336 (2001).
• 11) Kochanek S, Clemens PR, Mitani K, Chen HH, Chan S, Caskey CT. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc. Natl. Acad. Sci. U.S.A., 93, 5731–5736 (1996).
• 12) Iizuka S, Sakurai F, Shimizu K, Ohashi K, Nakamura S, Tachibana M, Mizuguchi H. Evaluation of transduction properties of an adenovirus vector in neonatal mice. Biomed. Res. Int., 2015, 685374 (2015).
• 13) Iizuka S, Sakurai F, Tachibana M, Ohashi K, Mizuguchi H. Neonatal gene therapy for hemophilia B by a novel adenovirus vector showing reduced leaky expression of viral genes. Mol. Ther. Methods Clin. Dev., 6, 183–193 (2017).
• 14) Shimizu K, Sakurai F, Iizuka S, Ono R, Tsukamoto T, Nishimae F, Nakamura SI, Nishinaka T, Terada T, Fujio Y, Mizuguchi H. Adenovirus vector-induced IL-6 promotes leaky adenoviral gene expression, leading to acute hepatotoxicity. J. Immunol., 206, 410–421 (2021).
• 15) Schulze RJ, Schott MB, Casey CA, Tuma PL, McNiven MA. The cell biology of the hepatocyte: a membrane trafficking machine. J. Cell Biol., 218, 2096–2112 (2019).
• 16) Aravalli RN, Belcher JD, Steer CJ. Liver-targeted gene therapy: approaches and challenges. Liver Transpl., 21, 718–737 (2015).
• 17) Perrin GQ, Herzog RW, Markusic DM. Update on clinical gene therapy for hemophilia. Blood, 133, 407–414 (2019).
• 18) Mian A, McCormack WM Jr, Mane V, Kleppe S, Ng P, Finegold M, O’Brien WE, Rodgers JR, Beaudet AL, Lee B. Long-term correction of ornithine transcarbamylase deficiency by WPRE-mediated overexpression using a helper-dependent adenovirus. Mol. Ther., 10, 492–499 (2004).
• 19) Gebbing M, Bergmann T, Schulz E, Ehrhardt A. Gene therapeutic approaches to inhibit hepatitis B virus replication. World J. Hepatol., 7, 150–164 (2015).
• 20) Li AP. Human hepatocytes: isolation, cryopreservation and applications in drug development. Chem. Biol. Interact., 168, 16–29 (2007).
• 21) Mizuguchi H, Kay MA. Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum. Gene Ther., 9, 2577–2583 (1998).
• 22) Mizuguchi H, Kay MA. A simple method for constructing E1- and E1/E4-deleted recombinant adenoviral vectors. Hum. Gene Ther., 10, 2013–2017 (1999).
• 23) Nagamoto Y, Takayama K, Ohashi K, Okamoto R, Sakurai F, Tachibana M, Kawabata K, Mizuguchi H. Transplantation of a human iPSC-derived hepatocyte sheet increases survival in mice with acute liver failure. J. Hepatol., 64, 1068–1075 (2016).
• 24) Okada N, Saito T, Masunaga Y, Tsukada Y, Nakagawa S, Mizuguchi H, Mori K, Okada Y, Fujita T, Hayakawa T, Mayumi T, Yamamoto A. Efficient antigen gene transduction using Arg-Gly-Asp fiber-mutant adenovirus vectors can potentiate antitumor vaccine efficacy and maturation of murine dendritic cells. Cancer Res., 61, 7913–7919 (2001).
• 25) Maizel JV Jr, White DO, Scharff MD. The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology, 36, 115–125 (1968).
• 26) Yamasaki C, Ishida Y, Yanagi A, Yoshizane Y, Kojima Y, Ogawa Y, Kageyama Y, Iwasaki Y, Ishida S, Chayama K, Tateno C. Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: novel, long-term, functional two-dimensional in vitro tool for developing new drugs. PLOS ONE, 15, e0237809 (2020).
• 27) Castell JV, Hernandez D, Gomez-Foix AM, Guillen I, Donato T, Gomez-Lechon MJ. Adenovirus-mediated gene transfer into human hepatocytes: analysis of the biochemical functionality of transduced cells. Gene Ther., 4, 455–464 (1997).
• 28) Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene, 272, 149–156 (2001).
• 29) Sakurai F, Kawabata K, Yamaguchi T, Hayakawa T, Mizuguchi H. Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities. Gene Ther., 12, 1424–1433 (2005).
• 30) Miao CH, Ohashi K, Patijn GA, Meuse L, Ye X, Thompson AR, Kay MA. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol. Ther., 1, 522–532 (2000).
• 31) Tsukamoto T, Sakai E, Nishimae F, Sakurai F, Mizuguchi H. Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells. J. Biotechnol., 304, 1–9 (2019).
• 32) Leppard KN. E4 gene function in adenovirus, adenovirus vector and adeno-associated virus infections. J. Gen. Virol., 78, 2131–2138 (1997).
• 33) Christ M, Louis B, Stoeckel F, Dieterle A, Grave L, Dreyer D, Kintz J, Ali Hadji D, Lusky M, Mehtali M. Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products. Hum. Gene Ther., 11, 415–427 (2000).
• 34) Gorziglia MI, Lapcevich C, Roy S, Kang Q, Kadan M, Wu V, Pechan P, Kaleko M. Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3. J. Virol., 73, 6048–6055 (1999).
• 35) Täuber B, Dobner T. Molecular regulation and biological function of adenovirus early genes: the E4 ORFs. Gene, 278, 1–23 (2001).
• 36) Livne A, Shtrichman R, Kleinberger T. Caspase activation by adenovirus e4orf4 protein is cell line specific and is mediated by the death receptor pathway. J. Virol., 75, 789–798 (2001).
• 37) Shtrichman R, Sharf R, Barr H, Dobner T, Kleinberger T. Induction of apoptosis by adenovirus E4orf4 protein is specific to transformed cells and requires an interaction with protein phosphatase 2A. Proc. Natl. Acad. Sci. U.S.A., 96, 10080–10085 (1999).