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. 2012 Dec 18;21(2):318–323. doi: 10.1038/mt.2012.258

Minimizing the Inhibitory Effect of Neutralizing Antibody for Efficient Gene Expression in the Liver With Adeno-associated Virus 8 Vectors

Jun Mimuro 1,*, Hiroaki Mizukami 2, Shuji Hishikawa 3, Tomokazu Ikemoto 4, Akira Ishiwata 1, Asuka Sakata 1, Tsukasa Ohmori 1, Seiji Madoiwa 1, Fumiko Ono 5, Keiya Ozawa 2, Yoichi Sakata 1
PMCID: PMC3594013  PMID: 23247100

Abstract

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10–10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59–12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06–9.0 ± 2.37%) in the presence of NAbs (14–56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

Introduction

Gene and cell therapies are expected to be the next generation of therapies for a variety of inherited diseases. Hemophilia is thought to be an ideal target disease for these approaches as it is caused by a genetic abnormality in the factor VIII gene for hemophilia A, or the factor IX (FIX) gene for hemophilia B.1,2,3,4,5,6,7 The current strategy of hemophilia gene therapy involves inducing expression of the normal coagulation factor gene or transplanting cells expressing the respective coagulation factor. The liver is normally the primary target of gene transfer for coagulation factors since the majority of these coagulation factors are synthesized in the liver with appropriate post-translation modifications before secretion into the circulatory system.

Substantial effort has been applied to express coagulation factor genes using various vector types. Among the viral vectors, recombinant adeno-associated virus (AAV) vectors are preferred for therapeutic gene transfer in vivo because they reside in the episome and rarely integrate into genomes. However, retrovirus vectors including lentivirus vectors require integration into the host cell genome.6,7 In addition, AAV vectors can transfer genes to nondividing cells and allow long-term expression of transgenes in these cells.

Clinical trials for hemophilia gene therapy have recently been conducted using various types of vectors.4,5,6,7,8,9,10,11 These trials were designed based upon data obtained from mouse models of hemophilia and hemophiliac dogs and proved to be more efficient in these models than for humans. Species differences between humans and these other animal models might partially account for the results observed. Therefore, gene transfer studies in non-\human primates may well predict the efficacy of gene transfer in humans. Indeed, FIX gene transfer studies using a new type of vector have been conducted in rhesus macaques.12,13 The results from these studies provided the basis for recent hemophilia B gene therapy clinical trials employing an AAV8 vector.13,14,15,16 Gene transfer in mice using AAV vectors results in excellent transduction efficiency. This is especially so for AAV8 vector-mediated gene transfer in the mouse liver;12,13,14,17 however, the efficacy of AAV8 vectors is modest in macaques.13

There are also difficulties associated with FIX gene expression when using AAV8 vectors in nonhuman primates. Growing evidence suggests that the presence of neutralizing antibodies (NAbs) against AAV8, due to previous natural infection by wild-type AAV, significantly inhibits transduction in the macaque liver. It is likely that antibodies against one serotype of AAV cross-react with other AAV serotypes.18 A hemophilia B gene therapy clinical study using an AAV8 vector was successfully conducted in hemophilia B patients negative for pre-existing antibodies against AAV8.15 Because of the high prevalence of AAV infection in humans,18 evading NAbs against this virus is an important hurdle to overcome before AAV8 vectors can be routinely and effectively employed for therapies.

The aim of our study was to develop an administration method of AAV8 vectors that assisted in minimizing the inhibitory effect of NAbs against AAV in macaques that were already seropositive for AAV8 antibodies.

Results

The AAV8 vector carrying the macaque FIX T262A gene located downstream of the liver-specific chimeric promoter consisted of an enhancer element of hepatic control region (HCR) of the ApoE/C-I gene and the 5′ flanking region of the α1-antitrypsin (HAAT) gene (AAV8-HCRHAAT-macFIXT262A). This vector was used to express mutant macaque FIX containing a single amino acid substitution of Thr to Ala at the position 262 (macaque FIX T262A) in the following experiments. Macaque FIX T262A but not wild-type macaque FIX could be bound to human FIX-specific monoclonal antibody 3A6, thereby macaque FIX T262A expressed in macaques with AAV8-HCRHAAT-macFIXT262A could be precisely quantified by an enzyme immunoassay with 3A6.17 The amino acid sequence of macaque FIX is highly homologous to the human FIX amino acid sequence. Twelve amino acid residues of human FIX are different at corresponding positions of macaque FIX, while only one amino acid of macaque FIX T262A is different from wild-type macaque FIX. Expression of macFIX T262A in a macaque would mimic a situation where normal human FIX is expressed in a hemophilia B patient with a missense mutation in the FIX gene.

Results corresponding to the expression of macaque FIX T262A following injection of AAV8HCRHAATmacFIXT262A can be seen in Table 1. When AAV8HCRHAATmacFIXT262A (5 × 1012 vector genome copies (vg)/kg) was injected into the saphenous veins of three AAV8 NAb-negative macaques (#28, #30, #31), expression of macFIX T262A in the therapeutic range (>5% of normal FIX concentration) was achieved. However, injection of the same vector (1 × 1012–1 × 1013 vg/kg) into the mesenteric vein branches of AAV8 NAb-positive macaques (#14, #17, #24; inhibitory titers: 14–56×) resulted in subtherapeutic levels (<0.2%) of macFIX T262A expression. The amount of vector DNA in the liver of AAV8 NAb-positive macaques was ~1% of that seen in AAV8 NAb-negative macaques (Table 1). These data suggest that low titers of NAbs against AAV8 significantly inhibit transduction even when the vector is injected into the mesenteric vein branches. In addition, only short period of time may be required for NAbs in the blood to neutralize the AAV8 vector since the blood of the mesenteric vein rapidly goes to the liver through the portal vein after gathering with the blood from other viscera.

Table 1. Expression of macaque T262A in nonhuman primates with AAV8-HCRHAAT-macFIXT262A.

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Evading AAV8 NAbs could be achieved by ensuring the AAV8 vector and NAbs do not come into physical contact with each other in the blood. Blood enters the liver from the hepatic artery and portal vein. The hepatic artery accounts for ~20–30% of blood flow, while the portal vein supplies the remaining blood flow to hepatocytes.19,20 Blood from the portal vein and hepatic artery are eventually mixed in the sinusoids of the liver; however, the blood from the portal vein mainly supplies hepatocytes. Therefore, direct injection of AAV8 vectors into the portal vein branch was investigated to determine whether saline flushing to remove blood from the portal vein just before injection of the vector would diminish the inhibitory effects of anti-AAV8 NAbs. Three macaques (#26, #27, #29; inhibitory titers: 14–28×) were directly injected with vector (5 × 1012 vg/kg) into the left portal vein after flushing saline to remove blood (Supplementary Table S1). Expression of transgene-derived FIX (macaque FIX T262A) increased to therapeutic levels with the AAV8 vector carrying the macaque FIX T262A gene and persisted for greater than 1 year in the three macaques (Figure 1). Average FIX and vector genome levels in macaque liver tissues are presented in Table 2. Compared with the results of vector injection to the mesenteric vein of NAb-positive macaques #14, #17, and #24 (Table 1), the levels of macaque FIX T262A in the circulation of the macaques #26, #27, and #29 that received vector injection directly to the left portal vein with flushing to remove blood, were increased to therapeutic levels with significant amounts of vector genome detected (Table 2).

Figure 1.

Figure 1

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Expression of FIX T262A in macaques after direct vector injection into portal veins. Macaques (n = 3) were subjected to direct injection of AAV8 vector into the portal vein. Concentrations of FIX T262A in macaque plasma samples (macaque #26, open triangles; #27, open circles; #29, closed squares) were measured by ELISA. AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay; FIX, factor IX.

Table 2. Expression of macaque T262A in nonhuman primates with direct, and balloon catheter-guided vector (AAV8-HCRHAAT-macFIXT262A) injection into the portal vein.

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Blood chemistry analysis and liver biopsies were conducted following administration of the vector to determine whether there were any adverse effects induced by the injection. Moderate increases in liver enzymes, such as transaminases, were observed just after injection of the vector (Supplementary Figure S1). However, no significant pathological changes were seen in liver biopsy samples taken on days 14, 28 or 48 (data not shown). We did not observe an increase in the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) -positive hepatocytes in the liver biopsy specimens (data not shown).

The direct injection of the AAV8 vector into the left portal vein branch with saline flushing to remove blood from the portal vein just before injection of the vector was effective to minimize the inhibitory effects of anti-AAV8 NAbs. Therefore, we explored the possibility of utilizing a balloon catheter to perform the vector injection into the portal vein branch with saline flushing to remove blood, taking the concern about the safety of the procedures into consideration. Using a microballoon catheter, we injected the vector into the left portal vein of three anti-AAV8 antibody-positive macaques (#37, # 38, #42; inhibitory titers: 14–56×) (Table 2, Supplementary Table S2). Fluorography in macaque #37 representing angiography of the portal vein branch is shown in Figure 2 and Supplementary Video S1. Increase of FIX T262A to therapeutic levels was achieved in the three macaques (#37, # 38, #42), and macaque FIX T262A expression in the circulation persisted (Figure 3, Table 2). The two portal vein vector delivery methods were successful in expressing macaque FIX T262A with the AAV8 vector in NAb-positive macaques (Table 2). The data suggest that the gene transfer efficiency using the catheter-guided vector injection method is similar to that of the direct vector injection into the portal vein branch with flushing.

Figure 2.

Figure 2

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Fluorography in macaque #37. A balloon catheter was inserted into the portal vein of macaque #37 and contrast medium injected before vector administration. The left portal vein branches can be visualized. The arrow (white) indicates the tip of the catheter. See Supplementary Video S1 which also recorded inflation of the balloon before the vector administration and deflation of the balloon after the administration.

Figure 3.

Figure 3

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Expression of FIX T262A in macaques following balloon catheter-guided vector injection into portal veins. Three macaques (n = 3) were subjected to balloon catheter-guided vector injection into the portal vein. Concentrations of FIX T262A in macaque plasma samples (#37, closed circles; #38, open squares; #42, closed triangles) were measured by ELISA. ELISA, enzyme-linked immunosorbent assay; FIX, factor IX.

Blood chemistry analysis and liver biopsies were conducted following injection of the vector. Increases in the levels of liver enzymes just after injection of the vector were not observed, suggesting that the ischemic effect of the temporary occlusion of the left portal vein branch was minimum compared with that of the direct vector injection procedure. Moderate increases in transaminases were observed following the vector injection, but did not persist (Supplementary Figure S1). Although the cause of the changes in the liver enzymes was not elucidated, no animals showed pathological changes, including histology of liver biopsy samples (data not shown).

Vector injection rates of the four different vector injection procedures are listed in Table 3 for comparison. The vector injection rates of the portal vein-directed strategies were similar to those of bolus vector injection into the saphenous vein and the mesenteric vein. Thus, the effect of vector injection speed on the transduction efficiency of the vector was thought to be minimal.

Table 3. Vector injection rate.

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Discussion

There are many features that make recombinant AAV vectors attractive for transferring therapeutic genes into target organs, and many vectors have been tried for the treatment of various diseases.6,7,11,15,21,22,23 However, lines of evidence suggest that NAbs against AAV interfere with AAV vector-mediated gene transfer by intravascular vector delivery.7,23,24,25,26 A clinical gene therapy trial for hemophilia B using a self-complementary AAV8 vector carrying the FIX gene has been conducted and reported to be successful.15 However, even the self-complementary AAV8 vector failed to express FIX in a subject with a relatively high anti-AAV8 antibody titer compared with other subjects with no or lower antibody titers.15

According to the previous reports on the prevalence of NAbs against various AAV serotypes in normal subjects, the seropositivity against AAV8 is 15–30%, which is lower than that against AAV2 (50–60%), although the technical details of the NAb assay is different.27,28 These reports have also demonstrated that the antibody titer against AAV8 is generally lower than for AAV2. Our data suggest that a low titer of NAbs against AAV8 can interfere with transduction even if the vector is injected into the mesenteric vein. Therefore, the use of another serotype vector such as AAV5 vector could be the next approach for this type of gene therapy because of the divergence in capsid sequence of AAV5 from other AAV serotypes.13,16 Although the prevalence of NAbs against AAV5 is much lower than those against AAV1 and AAV2, and the prevalence of NAbs against AAV5 is comparable to or even lower than that against AAV8 in humans,25,27 it is possible that subjects of gene therapy may contain cross-reactive NAbs against various AAV serotypes.

Another approach for evading NAbs against AAV could be the use of chemically or genetically modified AAV variants. Such variants could include AAV vector mutants with amino acid substitutions, or chimeric AAV vectors made by serotype shuffling.23 Approaches that enable evasion of NAb inhibitory effects are necessary if researchers and clinicians wish to effectively apply AAV vectors for gene therapy because of NAb cross-reactivity.

An alternative approach for overcoming the inhibitory effect of NAb against AAVs is to develop a vector injection method. In the current study, two portal vein vector delivery strategies were employed that ensured that the AAV8 vector and NAbs do not come into physical contact with each other in the blood. These strategies were investigated using macaques whether the strategies could efficiently transduce hepatocytes with the AAV8 vector in the presence of NAbs. The first approach was the direct injection of AAV8 vectors into the portal vein branch after flushing with saline to remove blood. This strategy proved to be successful for the vector expressing FIX T262A in anti-AAV8 antibody-positive macaques. Since there are safety concerns about the direct vector injection method, injection of the vector into the portal vein using a balloon catheter was investigated. The catheter-guided vector injection may be less invasive than the direct vector injection into the portal vein branch because exfoliation of hepatic hilum is not required. In addition, fine surgical skills, such as manipulation of the hepatic hilum and suturing the venotomy site of portal vein after the direct vector injection without causing stenosis, are required for the direct vector injection method into the left portal vein. Obviously, catheterization from the mesenteric vein branch is required for the balloon catheter-guided vector injection method but insertion of a catheter into the portal vein from a branch of the mesenteric vein is not difficult for a cardiologist and a radiologist familiar with angiography. In addition, suturing the venotomy site of the mesenteric vein branch is easier and safer than suturing the venotomy site of portal vein, and the ischemic effect of this procedure was expected to be less than that of the direct vector injection into the portal vein branch. Taken together, these studies suggested that both the direct vector injection into the left portal vein and the balloon catheter-guided vector injection into the left portal vein were similarly effective for hepatocyte transduction with the AAV8 vector in the presence of low titer NAbs but the balloon catheter-guided vector injection method into the left portal vein was thought to be safer than the direct vector injection into the left portal vein.

Considering that the antibody titer against AAV8 was generally lower than that against AAV2 and that NAbs at low titers could interfere with the AAV8 vector-mediated gene transfer to the liver significantly, we selected macaques with low NAb titers for the portal vein vector delivery strategies. However, the impact of the presence of high titer NAbs on the efficacy of these methods was not studied. Thus, the extent of AAV8 NAb titer, for that this approach is effective, needs to be investigated in the future.

In conclusion, we have provided the basis for an alternative approach for gene transfer to the liver that minimizes the deleterious effects of anti-AAV NAbs. Our result might expand the potential of the AAV vector-mediated gene delivery for medical application.

Materials and Methods

AAV vector production. Construction of pAAV2-HCRHAAT-macFIX T262A and production of AAV8 carrying the macaque FIX T262A gene (AAV8HCRHAATmacFIXT262A) has been previously described.17 Briefly, DNA fragments harboring the macFIXT262A gene located downstream of the chimeric promoter consisted of an enhancer element of HCR of the human ApoE/C-I gene and the 5′ flanking region of the human HAAT gene (HCRHAAT promoter), and the SV40 polyadenylation signal sequence flanked by AAV2 inverted terminal repeats in pAAV2-HCRHAAT-macFIX T262A. The genes were packaged by triple plasmid transfection of human embryonic kidney 293 cells (Avigen, San Diego, CA) to generate AAV8-HCRHAAT-macFIXT262A, with the chimeric packaging plasmid (AAV2 rep/AAV8 cap), and the adenovirus helper plasmid pHelper (Stratagene, La Jolla, CA), as previously described.17 The chimeric packaging plasmid for AAV8 capsid pseudotyping29 was constructed by inserting the synthetic AAV8 Cap gene (Takara Bio, Otsu, Shiga, Japan) downstream of the AAV2 Rep gene of pHelp19. For virus vector purification, the DNase (Benzonase; Merck Japan, Tokyo, Japan)-treated viral particles containing samples were subjected to two rounds of cesium chloride-density gradient ultracentrifugation in HEPES-buffered saline (pH 7.4) supplemented with 25 mmol/l EDTA at 21 °C, as previously described.17 Titration of recombinant AAV vectors was carried out by quantitative PCR using a real-time PCR system (StepOnePlus; Applied Biosystems Japan, Tokyo, Japan).17 AAV8HCRHAATmacFIXT262A was previously shown to express macaque FIXT262A in mice efficiently.17 Human FIX could be expressed in macaques and detected, however, macaques developed antibody against human FIX under certain experimental conditions. Only one amino acid residue at position 262 was humanized in macaque FIX T262A for detection with the human FIX-specific monoclonal antibody.

Animals. Cynomolgus macaques were bred and maintained at the Tsukuba Primate Research Center (Ibaraki, Japan). The animal experiments using macaques were performed at the Tsukuba Primate Research Center according to the guidelines of the Institutional Animal Care and Concern Committees at Jichi Medical University and the Tsukuba Primate Research Center. The use of macaques in animal experiments was approved by the Animal Care and Concern Committees. All surgical procedures were carried out under anesthesia, with vital signs and electrocardiogram monitoring conducted in accordance with the stipulated guidelines. Male macaques with low NAb titers (<56×) were used in this study.

Vector injection from peripheral and mesenteric vein. Injection of AAV8 vector to a saphenous vein (peripheral vein) was performed under intramuscular anesthesia. Injection of the AAV8 vector into a terminal branch of the superior mesenteric vein was carried out with laparotomy under anesthesia with isoflurane and electrocardiogram monitoring.

Direct portal vein vector injection with saline flushing. Direct injection of the vector solution into the left portal vein was carried out after induction of general anesthesia with isoflurane and sterilization. A right subcostal incision (5 cm) was made through the skin and the subcutaneous tissue. The abdominal cavity was explored and the soft tissue of hepatic hilum was exfoliated surgically, then the main portal vein and its right and left branches were exposed. The main portal vein was cannulated with a plastic cannula type 20G needle (Surflo; Terumo, Tokyo, Japan), which was advanced into the left portal vein branch. The left and right portal vein branches were then clamped with vascular forceps. After flushing the left portal vein with saline, the vector solution was injected, and then a second saline solution, for flushing, was injected. Volumes of solutions used in the experiments were determined by taking a standard liver volume, a hepatic vascular bed volume, and effects of solutions on the systemic circulation into consideration.19,30 A standard liver volume of a macaque was estimated with the formula (standard liver volume = 706.2 × body surface area + 2.4)31 and the vascular bed volume of the liver was estimated to 25–30% of the standard liver volume.19 A hepatic vascular bed volume can increase to 60% of the liver volume upon infusion of solutions and this may function as a reservoir and reduce the effects of the solutions on the systemic circulation.20,32,33 The forceps were then removed immediately and the venotomy site was closed with an 8-0 prolene suture.

Catheter-guided vector injection to the portal vein with saline flushing. Balloon catheter-guided injection of the vector into the left portal vein of AAV8 antibody-positive macaques was carried out after the induction of general anesthesia. A 5-cm right paramedian incision was made through the skin and subcutaneous tissue. The abdominal cavity was carefully entered, with a part of the ileum identified and brought out through the incision. A peripheral branch of the superior mesenteric vein was cannulated with a plastic cannula type 20G needle (Surflo; Terumo). A temporary occlusion microcatheter (Iiguman 3.3F; Fuji System, Tokyo, Japan) was advanced into the left portal vein using a guide-wire (run through 0.014 (0.36 mm); Terumo) under a fluoroscope. The positions of the catheter and the balloon were confirmed by imaging with contrast medium. Blood flow in the left portal vein was occluded with a silicone balloon catheter and 40 ml of saline, followed by the AAV8 vector solution, and another 20 ml of saline was injected sequentially through the microcatheter. Volumes of solutions used in the experiments were determined as above with taking the result of the experiment of direct vector injection to the left portal vein branch into consideration. Following deflation of the balloon, the microcatheter was withdrawn and the peripheral venotomy ligated. The abdominal wall was then closed in layers.

Analysis of macaque FIX T262A expression in macaques. Macaque FIX T262A was bound to 3A6, a human FIX-specific monoclonal antibody for analyses. An enzyme-linked immunosorbent assay (ELISA) for the detection of macaque FIX T262A was carried out using 3A6, as previously described.17,34

NAb assay. An assay for the detection of anti-AAV8 NAbs was performed as previously reported, with some modifications.35,36 Briefly, 5 × 104 2V6.11 cells/well were seeded in the wells of 96-well culture plates. Ponasterone A was added to the culture media the day before transduction to induce expression of the E4 gene. On the day of transduction, 10 µl of serum (undiluted, or subject to serial twofold dilutions) was incubated with the vector (AAV8-CMV-LacZ, 5 × 107 vg/10 µl) at 37 °C for 1 hour, and this mixture was added to a culture well. Sucrose was added to the culture medium such that the final concentration was 125 mmol/l. The culture medium was removed after a 48-hour incubation, and β-galactosidase activity quantified with a β-Gal assay kit (Invitrogen, Carlsbad, CA). Briefly, o-nitrophenyl-β-D-galactopyranoside was added to cell lysates, incubated for 30 minutes, and color change quantified with a microplate reader (Benchmark Plus; Bio-Rad, Hercules, CA). If β-galactosidase activity was inhibited with a test sample that contained more than 50% of control fetal bovine serum, it was judged as positive for neutralizing capacity. The inhibitory titer of the serum sample was expressed as the highest final dilution in the culture medium that showed inhibitory activity.

Quantitation of AAV8 vector DNA in macaque tissue. Quantitation of AAV8 vector DNA in macaque tissues was performed using quantitative PCR assays using a StepOnePlus instrument (Applied Biosystems Japan). DNA was isolated from macaque tissues using a DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) and subjected to PCR using primers 5′-GAT AAC TGG GGT GAC CTT GG-3′ and 5′-GCC TGG TGA TTC TGC CAT GA-3′, and Cybergreen reagent (Applied Biosystems Japan).

SUPPLEMENTARY MATERIAL Figure S1. Changes in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in macaques. Table S1. Direct vector injection into the portal vein of macaques. Table S2. Balloon catheter-guided vector injection into macaques. Video S1.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (20591155, 21591249, and 21790920) and the Support Program for Strategic Research Infrastructure from the Japanese Ministry of Education, Culture, Sports, Science and Technology; and Health, Labor, and Science Research Grants for Research on HIV/AIDS and Research on Intractable Diseases from the Japanese Ministry of Health, Labor, and Welfare. This work was done in Shimotsuke, Tochigi and Tsukuba, Ibaraki, Japan. The authors declared no conflict of interest.

Supplementary Material

Figure S1.

Changes in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in macaques.

Click here for additional data file. (120.6KB, pdf)
Table S1.

Direct vector injection into the portal vein of macaques.

Click here for additional data file. (126.1KB, pdf)
Table S2.

Balloon catheter-guided vector injection into macaques.

Click here for additional data file. (40.3KB, pdf)
Video S1.
Click here for additional data file. (3.5MB, mov)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Changes in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in macaques.

Click here for additional data file. (120.6KB, pdf)
Table S1.

Direct vector injection into the portal vein of macaques.

Click here for additional data file. (126.1KB, pdf)
Table S2.

Balloon catheter-guided vector injection into macaques.

Click here for additional data file. (40.3KB, pdf)
Video S1.
Click here for additional data file. (3.5MB, mov)

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