Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.

BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination. METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 109 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed.

Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation.

Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFκB promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

Amphipathic α-helices in apolipoproteins are crucial to the formation of infectious hepatitis C virus particles.

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures.

Several autophagy proteins contain an LC3-interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B-positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP-ALFY-LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR-binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.

[Recent progress in adenovirus vectors: focusing on VA-deleted AdV].

First-generation adenovirus vectors (FG-AdVs) are widely used because transduction efficiency of the vectors is very high. However, severe immune responses especially to the liver have been a serious problem of this vector. We succeeded to identify a viral protein that cause the immune responses and reported ''low-inflammatory AdVs'' that mostly solve this problem. However, to develop the ultimate form of this vector, it is necessary to remove virus-associated RNA (VA RNA) genes from the AdV vector genome. VA RNAs are transcribed by polymerase III; they are not essential for viral growth but have important roles to make appropriate circumstances for this virus. Large amount of VA RNAs are required in the late phase to support viral growth. Hence it is difficult to establish 293 cell lines that can support replication of AdVs lacking VA RNA genes (VA-deleted AdVs) supplying sufficient amount of VA RNA in trans. Recently we have developed a method for efficient production of VA-deleted AdVs and succeeded to obtain a high titer of VA-deleted AdVs. Then we construct VA-deleted AdVs expressing shRNA that knockdown the replication of hepatitis C virus (HCV). In fact, VA-deleted AdVs expressing these shRNAs suppressed HCV replication more effectively than conventional FG-AdV. Therefore, we showed that VA RNAs expressed from FG-AdVs probably compete with shRNA in the maturation pathway and reduce the effect of shRNAs. We think that VA-deleted AdV may substitute for current FG-AdVs and become a standard AdV.

BabA-mediated adherence is a potentiator of the Helicobacter pylori type IV secretion system activity.

Chronic infection of Helicobacter pylori in the stomach mucosa with translocation of the bacterial cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV secretion system (TFSS) into host epithelial cells are major risk factors for gastritis, gastric ulcers, and cancer. The blood group antigen-binding adhesin BabA mediates the adherence of H. pylori to ABO/Lewis b (Le(b)) blood group antigens in the gastric pit region of the human stomach mucosa. Here, we show both in vitro and in vivo that BabA-mediated binding of H. pylori to Le(b) on the epithelial surface augments TFSS-dependent H. pylori pathogenicity by triggering the production of proinflammatory cytokines and precancer-related factors. We successfully generated Le(b)-positive cell lineages by transfecting Le(b)-negative cells with several glycosyltransferase genes. Using these established cell lines, we found increased mRNA levels of proinflammatory cytokines (CCL5 and IL-8) as well as precancer-related factors (CDX2 and MUC2) after the infection of Le(b)-positive cells with WT H. pylori but not with babA or TFSS deletion mutants. This increased mRNA expression was abrogated when Le(b)-negative cells were infected with WT H. pylori. Thus, H. pylori can exploit BabA-Le(b) binding to trigger TFSS-dependent host cell signaling to induce the transcription of genes that enhance inflammation, development of intestinal metaplasia, and associated precancerous transformations.

Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration.

Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.

Comparison of efficiency between FLPe and Cre for recombinase-mediated cassette exchange in vitro and in adenovirus vector production.

Cre and FLP recombinases mediate not only specific deletions and insertions, but also the recombinase-mediated cassette exchange (RMCE) reaction, which is used in cell biotechnology including ES cells and mouse genetics. However, comparison of efficiencies for Cre and FLP in RMCE has not been made. We here examined the detailed process of RMCE with Cre and FLP in vitro using mutant loxP 2272 and three mutant FRTs (FRT G, FRT H, and FRT F3) and then quantitatively compared the RMCE reactions in vitro. Interestingly, in the in vitro reactions, the RMCE efficiency of Cre reached a plateau level of approximately 5% and did not proceed further, whereas that of FLPe reached approximately 12-13%, showing that FLPe reached a higher level of efficiency than Cre possibly when they were supplied at a very high concentration. Moreover, we quantitatively compared the production efficiency of E1-deleted adenovirus vector using the RMCE method with Cre or FLP. The results showed that FLPe was again found more efficient than Cre in RMCE reaction. Thus, although Cre is considered more active than, or similar to, FLPe, it may not be necessarily true for RMCE reaction. Possible reasons explaining these results are discussed.

Influence of loxP insertion upstream of the cis-acting packaging domain on adenovirus packaging efficiency.

First-generation AdV enables efficient gene transduction, although its immunogenicity is an important problem in vivo. Helper-dependent AdV (HD-AdV) is one possible solution to this problem. The construction of HD-AdV requires a helper virus, in which the viral packaging domain is flanked by two inserted loxP to hamper its packaging in Cre-expressing 293 cells. Here, we constructed 19L viruses containing loxP at 191 nt from the left end of the genome upstream of the packaging domain, 15L viruses bearing loxP at 143 nt, and a control ΔL virus lacking loxP at these positions. The 19L position is used worldwide, and the 15L position has been reported to result in a lower titer than that of 19L. When the titers were compared for six pairs of 19L and 15L AdV, the 19L AdV produced titers similar to, or sometimes lower than, the 15L and ΔL AdV, unlike the results of previous reports. We next chose one pair of 15L and 19L AdV that produced titers similar to that of ΔL and a competitor AdV lacking loxP for use in a competition assay. When a small amount of the competitor AdV was co-infected, both the 15L and the 19L AdV, but not ΔL, gradually became minority components during subsequent viral passages. Therefore, the loxP insertions at 143 nt and 191 nt decreased the viral packaging efficiency.

Keratinocyte growth factor gene transduction ameliorates pulmonary fibrosis induced by bleomycin in mice.

Pulmonary fibrosis has high rates of mortality and morbidity, but there is no established therapy at present. We demonstrate here that bleomycin-induced pulmonary fibrosis in mice is ameliorated by intratracheal administration of keratinocyte growth factor (KGF)-expressing adenovirus vector. Progressive pulmonary fibrosis was created by continuous subcutaneous administration of 120 mg/kg of bleomycin subcutaneously using an osmotic pump twice from Day 1 to 7 and Day 29 to 35. The mice initially exhibited subpleural fibrosis and then exhibited advanced fibrosis in the parenchyma of the lungs. These histopathological changes were accompanied by reduced lung compliance (0.041 ± 0.011 versus 0.097 ± 0.004; P < 0.001), reduced messenger expression of surfactant proteins, and reduced KGF messenger expression in the lungs at 4 weeks compared with naive group. Intratracheal instillation of Ad-KGF at 1 week after the first administration of bleomycin increased KGF mRNA expression in the lungs compared with the fibrosis-induced mice that received saline alone. The phenotype was associated with alveolar epithelial cell proliferation, increased pulmonary compliance (0.062 ± 0.005 versus 0.041 ± 0.011; P = 0.023), and decreased mortality (survival rate on Day 56: 68.8% versus 0%; P = 0.002), compared with mice receiving only the saline vehicle. These observations suggest the therapeutic utility of a KGF-expressing adenoviral vector for pulmonary fibrosis.

Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

Acetophenone 4-nitrophenylhydrazone inhibits Hepatitis B virus replication by modulating capsid assembly.

Hepatitis B virus (HBV) is the causative agent of chronic liver disease and is correlated with the development of subsequent hepatic cirrhosis and hepatocellular carcinoma. Current antiviral therapy using nucleos(t)ide analogs is effective in suppressing viral replication and interrupting disease progression, but HBV is rarely cured completely. Thus, there remains an unmet need for the development of novel anti-HBV drugs. Here, we report the identification of N-(4-Nitrophenyl)-1-phenylethanone hydrazone (ANPH) as a novel structural class of selective inhibitors targeting the replication of the HBV genome using adenovirus vector-mediated HBV genome transduction. ANPH inhibited viral genome replication in HepG2.2.15 cells by inducing the formation of empty capsids devoid of pregenomic RNA without affecting its transcription and translation. Biochemical assays using a truncated core protein consisting of the assembly domain showed that ANPH accelerates the formation of morphologically intact capsids. Taken together, we propose that ANPH might provide a new structural scaffold to design a new anti-HBV drug in medicinal chemistry as well as chemical probes for HBV core protein functions in the future.

Alpha-catenin is essential in intestinal adenoma formation.

A loss-of-function mutation in the APC gene initiates colorectal carcinogenesis. Although the molecular mechanism of tumor initiation is complex, several modifier genes have been identified using mouse models, including the ApcMin mouse. Among the familial adenomatous polyposis mouse lines carrying a truncation mutation at codon 580 in Apc (Apc580D), one line (line19-Apc(580D/+)) showed a remarkably reduced incidence of intestinal adenomas (<5% compared with other lines). Extensive genetic analysis identified a deletion in the alpha-catenin (Ctnna1) gene as the cause of this suppression. Notably, the suppression only occurred when the Ctnna1 deletion was in cis-configuration with the Apc580D mutation. In all adenomas generated in line19-Apc(580D/+), somatic recombination between the Apc and Ctnna1 loci retained the wild-type Ctnna1 allele. These data strongly indicate that simultaneous inactivation of alpha-catenin and Apc during tumor initiation suppresses adenoma formation in line19-Apc(580D/+), suggesting that alpha-catenin plays an essential role in the initiation of intestinal adenomas. Although accumulating evidence obtained from human colon tumors with invasive or metastatic potential has established a tumor-suppressive role for alpha-catenin in late-stage tumorigenesis, the role of alpha-catenin in the initiation of intestinal tumorigenesis is not well documented, especially compared with that of beta-catenin. A mouse model used in this study focused on the early stage of tumor initiation and clearly indicated an essential role for alpha-catenin. Thus, alpha-catenin has dual roles in intestinal tumorigenesis, a supporting role in tumor initiation, and a suppressive role in tumor progression.

An animal model of preclinical diagnosis of pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease, which is usually diagnosed in an advanced stage. Animal PDA models which reflect the human condition are clearly necessary to develop early diagnostic tools and explore new therapeutic approaches. We have established transgenic rats carrying a mutated H- or K-ras gene (Hras250 and Kras327) controlled by Cre/loxP activation. These animals develop PDA which are histopathologically similar to that in humans. We utilized this model to identify biomarkers to detect early PDA. We report here that serum levels of Erc/Mesothelin are significantly higher in rats bearing PDA than in controls. Importantly, the levels are significantly elevated in rats before grossly visible carcinomas develop. Even in rats with very small microscopic ductal carcinoma lesions, elevated serum Erc/Mesothelin can be detected. We believe this is the first report of a pancreas tumor animal model in which pre-symptomatic lesions can be diagnosed.

Autophagy regulates lipid metabolism through selective turnover of NCoR1.

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in ß-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates ß-oxidation and ketone bodies production.

Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice.

At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, treatment using recombinant KGF protein is limited by its short effective duration. Thus, we investigated the effectiveness of intratracheal KGF gene transduction using adenoviral vector in ALI. We constructed an adenoviral vector expressing mouse KGF (mKGF), and 1.0 x 10(9 ) plaque-forming units of mKGF cDNA-expressing (Ad-KGF) and control (Ad-1w1) adenoviral vector was intratracheally instilled, using a MicroSprayer, into anesthetized BALB/c mice. Three days later, the mice were exposed to >90% oxygen for 72 hr, and the effect of KGF on hyperoxia-induced lung injury was examined. In the Ad-KGF group, KGF was strongly expressed in the airway epithelial cells, while peribronchiolar and alveolar inflammation caused by adenoviral vector instillation was minimal. The KGF overexpression not only induced proliferation of surfactant protein C-positive cuboidal cells, especially in the terminal bronchiolar and alveolar walls, but also prevented lung injury including intraalveolar exudation/hemorrhage, albumin permeability increase, and pulmonary edema. The arterial oxygen tension and the survival rate were significantly higher in the KGF-transfected group. These findings suggest that KGF gene transduction into the airway epithelium is a promising potential treatment for ALI.

Expression of pIX gene induced by transgene promoter: possible cause of host immune response in first-generation adenoviral vectors.

First-generation (FG) adenoviral vectors (AdVs) have been widely used not only for gene therapy but also for basic studies. Because vectors of this type lack the E1A gene that is essential for the expression of other viral genes, their expression levels in target cells have been considered low. However, we found that the viral pIX gene, located immediately downstream of the inserted expression unit of the transgene, was significantly coexpressed with the transgene in cells infected with FG AdV. Whereas CAG and SRalpha promoters activated the pIX promoter considerably through their enhancer effects, the EF1alpha promoter hardly did. Moreover, when the expression unit was inserted in the rightward orientation, not only the pIX protein but also a fusion protein consisting of the N-terminal part of transgene product and pIX were sometimes coexpressed with the transgene product through an aberrant splicing mechanism. In in vivo experiments, a LacZ-expressing AdV bearing the CAG promoter caused an elevation of alanine aminotransferase, but an AdV bearing the EF1alpha promoter produced no detectable levels. Whereas the FG AdV expressing human growth hormone under the control of the CAG promoter maintained a high hormone level for less than 1 month, the FG AdV under the control of the EF1alpha promoter maintained a high level for at least 6 months. These results suggest that pIX coexpression may be one of the main causes of AdV-induced immune responses, and that the EF1alpha promoter is probably valuable for the long-term expression of FG AdV. Thus, the in vivo utility of FG AdV should be reevaluated.