Introduction

The liver is an important target for gene therapy because (i) the fenestrated endothelium permits exposure to an intravenously delivered vector, (ii) hepatocytes are well suited for the secretion of therapeutic proteins into the circulation for systemic delivery, and (iii) it is the affected organ in many genetic disorders. Indeed, hepatocytes are primarily involved in more than 5,000 identified metabolic pathways,¹ and, not surprisingly, numerous inherited metabolic diseases originate from defects in hepatocyte metabolism. These disorders can potentially be corrected with liver-directed gene therapy. Helper-dependent adenoviral vectors (HDAds), devoid of all viral coding sequences, show tremendous potential for liver-directed gene therapy because they can mediate long-term transgene expression without chronic toxicity, leading to sustained phenotypic correction of several disease models.² For example, in rodents, a single intravenous injection of an HDAd can result in life-long phenotypic correction of a genetic disease.^{3, 4} Similarly, in large animals (dogs and baboons), hepatic transduction by a single injection of an HDAd results in long-term transgene expression for at least 1-2 years of observation.^{5, 6, 7} In both small and large animals, hepatic transduction by HDAd is not associated with any chronic toxicity due to the absence of viral gene expression.^{2, 3, 4, 5, 6, 7} However, efficient hepatocyte transduction by systemic intravascular delivery requires high vector doses. Numerous studies have shown a non-linear dose response, with low doses yielding very low to undetectable levels of transgene expression, but with higher doses resulting in disproportionately high levels of transgene expression in both mice^{8, 9, 10} and non-human primates.^{11, 12, 13} Unfortunately, the high doses required for efficient hepatic transduction result in dose-dependent activation of the innate immune response characterized by increases in pro-inflammatory cytokines and resulting in acute toxicity with potentially severe and lethal consequences in non-human primates and humans.^{12, 13, 14, 15, 16, 17, 18, 19} Although the mechanism of adenoviral vector-mediated innate immune activation is yet to be fully elucidated, the severity is clearly dose-dependent.^{12, 13, 19, 20} This is the major obstacle currently hindering clinical usefulness of HDAds for liver-directed gene therapy. Therefore, strategies to improve hepatic transduction efficiency with low vector doses would greatly increase the therapeutic index of HDAds.

In this regard, we have previously shown that rapid, large-volume tail vein injection (called hydrodynamic injection, a technique first demonstrated in mice using plasmid DNA [pDNA],^{21, 22} in which 100 ml/kg is injected within 7 seconds) of HDAds into mice results in significantly higher-efficiency hepatic transduction with lower vector doses than conventional injection.²³ This result is accompanied by reduced systemic vector dissemination and reduced pro-inflammatory cytokines and therefore represents a potential method for increasing the therapeutic index of HDAds significantly.²³ It is believed that the high intra-hepatic pressures achieved with hydrodynamic injection enlarge the liver fenestrations (100 nm), enhancing extravasation of the HDAd virion (90-100 nm) and thereby increasing hepatocyte-vector contact.²³ Unfortunately, this method of vector delivery is not applicable to larger animals, because the rapid, systemic injection of a large volume would be too dangerous, especially for human clinical application. However, the tremendous potential of the method led us to seek to develop a safe and effective method that mimics hydrodynamic injection in large animals but does not require rapid, large-volume injection.

Results

HDAds need not be injected hydrodynamically for high hepatic transduction

As a first step toward developing a method of mimicking hydrodynamic HDAd injection into large animals, we investigated whether an HDAd must necessarily be injected rapidly in a large volume to achieve the higher-efficiency hepatic transduction with low vector doses. To accomplish this, we injected 1 10¹² vector particles (vp)/kg HD28E4LacZ, an HDAd containing a cytomegalovirus-LacZ (CMV-LacZ) expression cassette, into the tail vein of mice under four conditions (Table 1): (1) conventionally, (2) hydrodynamically, (3) conventionally 10 minutes after hydrodynamic injection of Ringer's solution, and (4) conventionally 30 minutes after hydrodynamic injection of Ringer's solution. Forty-eight hours after injection, the livers were removed for analysis. As expected, compared with conventional injection of the HDAd (condition 1, Figure 1a), hydrodynamic injection of the HDAd (condition 2, Figure 1b) resulted in qualitatively higher-efficiency hepatic transduction as determined by X-gal histochemistry. Surprisingly, conventional injection of the HDAd either 10 minutes (condition 3, Figure 1c) or 30 minutes (condition 4, Figure 1d) after hydrodynamic injection of Ringer's solution resulted in high-level hepatic transduction similar to that following hydrodynamic injection (condition 2, Figure 1b).

Figure 1.

Helper-dependent adenoviral vectors (HDAds) need not be injected hydrodynamically for high hepatic transduction. X-gal histochemistry of mouse livers following tail vein injection of HD28E4LacZ under the following conditions: (a) condition 1: conventional injection of 1 10¹² vector particles (vp)/kg HDAd; (b) condition 2: hydrodynamic injection of 1 10¹² vp/kg HDAd; (c) condition 3: hydrodynamic injection of Ringer's solution followed, 10 minutes later, by conventional injection of 1 10¹² vp/kg HDAd; (d) condition 4: hydrodynamic injection of Ringer's solution followed, 30 minutes later, by conventional injection of 1 10¹² vp/kg HDAd. (e) -galactosidase activity in the liver of mice following tail vein injection of HDAd under conditions 1, 2, 3, and 4. Serum (f) asparate aminotransferase (AST) and (g) alanine aminotransferase (ALT) in mice 6 and 48 hours after tail vein injection of HDAd under conditions 1 and 2, and after condition 5, hydrodynamic injection of Ringer's solution without vector. Mean SD is shown.

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Table 1 - Injection conditions in mice.

Full table

To measure the amount of transgene expression, protein was extracted from the livers and the amount of -galactosidase activity was determined. The result for hydrodynamic HDAd injection (condition 2) was 2.6-fold higher (P = 0.03) than that for conventional injection (condition 1) (Figure 1e). Furthermore, conventional HDAd injection either 10 minutes (condition 3) or 30 minutes (condition 4) after hydrodynamic injection of Ringer's solution resulted in levels of -galactosidase activity comparable to hydrodynamic injection (condition 2) (P = 0.14 for condition 3 versus condition 2 and P = 0.11 for condition 4 versus condition 2). Moreover, conventional HDAd injection either 10 minutes (condition 3) or 30 minutes (condition 4) after hydrodynamic injection of Ringer's solution resulted in levels of -galactosidase activity that were 3.6-fold (P = 0.006) and 1.7-fold higher (P = 0.01), respectively, than the level for conventional HDAd injection (condition 1) (Figure 1e). Together, these results indicate that HDAds need not be injected rapidly in a large volume to reap the benefits of the hydrodynamic effect of increased hepatic transduction, and we have taken advantage of this important observation in developing an approach to mimic hydrodynamic injection in non-human primates (see below). It is important to note that, unlike HDAds, pDNA must be injected hydrodynamically (i.e., rapidly in a large volume) to achieve hepatic transfection.²⁴

To determine hepatotoxicity, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 6 and 48 hours after injection of the vector. For conventional HDAd injection (condition 1), a mild AST increase to 182 9 U/l was observed at 6 hours, followed by a further increase to 675 235 U/l at 48 hours (Figure 1f). Similarly, a mild ALT increase to 62 6 U/l was observed at 6 hours, followed by a further increase to 153 72 U/l at 48 hours (Figure 1g). For hydrodynamic HDAd injection, a more dramatic AST increase to 13,729 5,488 U/l was observed at 6 hours, followed by a decline to 803 522 U/l at 48 hours (Figure 1f). Likewise, a dramatic ALT increase to 16,857 5,102 U/l was observed at 6 hours, followed by a decline to 703 287 U/l at 48 hours (Figure 1g). As a control, mice were subjected to injection condition 5, hydrodynamic injection of Ringer's solution without vector (Table 1), and exhibited a similarly dramatic AST increase to 9,206 7,415 U/l at 6 hours, followed by a decline to 371 102 U/l at 48 hours (Figure 1f); ALT rose to 10,828 7,623 U/l at 6 hours but declined to 195 94 U/l at 48 hours (Figure 1g). Together, these results show that systemic hydrodynamic injection, with or without vector, causes acute and extreme, albeit transient, hepatotoxicity in mice.

Duration of the hydrodynamic effect on hepatic transduction

We next determined the duration of the hydrodynamic effect. Mice were subjected to four additional injection conditions (Table 1): conventional injection of 5 10¹¹ vp/kg HD28E4LacZ (condition 6) and conventional injection of 5 10¹¹ vp/kg HD28E4LacZ 30 minutes (condition 7), 6 hours (condition 8), or 24 hours (condition 9) after hydrodynamic injection of Ringer's solution. Forty-eight hours after injection of the HDAd, X-gal histochemistry revealed that conventional HDAd injection (condition 6, Figure 2a) resulted in lower hepatic transduction than conventional HDAd injection 30 minutes (condition 7, Figure 2b) or 6 hours (condition 8, Figure 2c) after hydrodynamic injection of Ringer's solution. However, conventional HDAd injection 24 hours after hydrodynamic injection of Ringer's solution (condition 9, Figure 2d) resulted in low hepatic transduction comparable to conventional HDAd injection (condition 6, Figure 2a).

Figure 2.

Duration of the hydrodynamic effect on hepatic transduction. X-gal histochemistry of mouse livers following tail vein injection of HD28E4LacZ under the following conditions: (a) condition 6: conventional injection of 5 10¹¹ vector particles (vp)/kg of helper-dependent adenoviral vector (HDAd); (b) condition 7: hydrodynamic injection of Ringer's solution followed, 30 minutes later, by conventional injection of 5 10¹¹ vp/kg HDAd; (c) condition 8: hydrodynamic injection of Ringer's solution followed, 6 hours later, by conventional injection of 5 10¹¹ vp/kg HDAd; (d) condition 9: hydrodynamic injection of Ringer's solution followed, 24 hours later, by conventional injection of 5 10¹¹ vp/kg HDAd. (e) -galactosidase activity in the liver of mice after tail vein injection of HDAd under conditions 6, 7, 8, and 9.

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To measure the amount of transgene expression, total protein was extracted from the livers and -galactosidase activity was determined (Figure 2e). As expected, conventional HDAd injection either 30 minutes (condition 7) or 6 hours (condition 8) after hydrodynamic injection of Ringer's solution resulted in fourfold (P = 0.001) and twofold (P = 0.047) higher activities than conventional HDAd injection (condition 6), respectively (Figure 2e). However, conventional HDAd injection 24 hours after hydrodynamic injection of Ringer's solution (condition 9) resulted in a twofold decrease (P = 0.041) in activity compared with conventional HDAd injection (condition 6) (Figure 2e). Together, these results indicate that the hydrodynamic effect is transient, decreasing with time and lasting <24 hours.

Pseudo-hydrodynamic injection of helper-dependent adenoviral vectors into baboons results in high hepatic transduction

On the basis of the results in mice, we developed a method to mimic hydrodynamic injection in larger animals without rapid, large-volume injection. In this method, hepatic venous outflow is occluded for 30 minutes using two balloon occlusion catheters percutaneously placed in the inferior vena cava (IVC), above and below the hepatic veins (Figure 3). Because blood entering the liver from the hepatic artery and portal vein remains unobstructed, an increase in intra-hepatic pressure is expected. We hypothesize that this may mimic the high pressures achieved by systemic hydrodynamic injection, resulting in endothelial fenestration enlargement and thereby increasing the efficiency of hepatocyte transduction following a simple peripheral intravenous injection of an HDAd without the need for rapid, large-volume systemic injection.

Figure 3.

Pseudo-hydrodynamic injection of helper-dependent adenoviral vector (HDAd) into non-human primates. Two balloon occlusion catheters are percutaneously positioned in the inferior vena cava (IVC) under fluoroscopic guidance. Inflation of the balloons with contrast medium results in hepatic venous outflow occlusion from the hepatic veins (HV) while blood inflow from the portal vein (PV) and hepatic artery (HA) increases the intra-hepatic pressure. Following 30 minutes of occlusion, the balloon occlusion catheters are removed from the animal and the HDAd is administered by systemic peripheral intravenous injection.

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Two baboons, 14721 and 11754, were subjected to this procedure. Because occlusion of the IVC obstructs blood return to the heart, a decrease in systemic blood pressure was anticipated. To minimize this, 20 ml/kg saline was intravenously infused before IVC occlusion, and phenylephrine was titrated intravenously during the 30 minutes of occlusion. In addition, 250 ml of saline was injected retrograde at 2.5 ml/s into the liver of 11754 via one of the balloon occlusion catheters during the 30 minutes of occlusion in an attempt to further increase intra-hepatic pressure. This was not done for 14721. When the balloons were inflated, systemic blood pressure dropped 50% and intra-hepatic pressure rose from 5 mm Hg to 20-30 mm Hg in both animals. Both systemic and intra-hepatic pressures remained at these altered levels during the occlusion and immediately returned to normal when the balloons were deflated. Following deflation and removal of the balloons, 1 10¹¹ vp/kg HD28E4LacZ diluted in 5 ml saline was slowly injected through a forelimb peripheral vein at 1 ml/min. Otherwise the procedure was uneventful and well tolerated. Following an uneventful recovery, the animals were necropsied 96 hours after vector injection.

Acute laboratory abnormalities were noted (Figure 4). For 14721, AST peaked at 6.6 the upper limit of the normal range (ULN) at 24 hours. ALT peaked at 2.9 ULN at 48 hours. Lactate dehydrogenase peaked at 2.8 ULN at 24 hours. Interleukin-6 rose from 9.6 pg/ml at baseline to 280 pg/ml at 1 hour. Interleukin-12 rose from a baseline of 54.5 pg/ml to 91.3 pg/ml at 1 hour. For 11754, AST and ALT peaked at 2.5 ULN and 4 ULN, respectively, at 24 hours. Lactate dehydrogenase peaked at 2.5 ULN at 24 hours. Interleukin-6 rose from 2.9 pg/ml at baseline to 412 pg/ml at 3.5 hours. Interleukin-12 rose from undetectable to 32.5 pg/ml at 3.5 hours. Platelet counts remained within the normal range in both animals. For both animals, all laboratory abnormalities were transient, returning toward the normal range by 72 or 96 hours (Figure 4). The relatively mild increases in AST and ALT observed in these two baboons following pseudo-hydrodynamic injection (Figure 4) are in sharp contrast with the extreme elevations observed in mice following systemic hydrodynamic injection (Figure 1f and g), illustrating the clinical utility of our approach.

Figure 4.

Laboratory findings from baboons following pseudo-hydrodynamic injection of 1 10¹¹ vector particles/kg of helper-dependent adenoviral vector (HDAd). Baboons 14721 (9 years, 28.48 kg) (solid squares) and 11754 (10 years, 36.29 kg) (solid circles) were given HD28E4LacZ. Baboons 14200 (6 years, 28.4 kg) (open circles) and 14245 (6 years, 32.9 kg) (open squares) were given HD21.7E4PEPCK-bAFP-WL. The white area indicates the normal range. ALT, alanine aminotransferase; AST, aspartate aminotransferase; bAFP, baboon -fetoprotein; IL-6, interleukin-6; IL-12, interleukin-12; LDH, lactate dehydrogenase.

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X-gal histochemistry of the livers from both baboons revealed that hepatic transduction was lower in 14721 (Figure 5a) than in 11754 (Figure 5b). The higher efficiency observed in 11754 may have been due to the 250 ml of saline injected retrograde into the liver before vector administration, which may have increased the intra-hepatic pressure and thus accentuated the hydrodynamic effect, although normal variations between animals or slight unperceived variations in the procedures cannot be ruled out. Regardless, the hepatic transduction in both animals was significantly higher than was obtained with a fivefold higher dose (5 10¹¹ vp/kg) given by simple intraportal injection to a rhesus monkey without hepatic inflow occlusion, which resulted in no detectable hepatic transduction (Figure 5c).¹² In fact, the levels of hepatic transduction achieved in 14721 and 11754 were comparable to, if not higher than, that obtained with a 10-fold higher systemic dose (1 10¹² vp/kg) in a rhesus monkey (Figure 5d)¹² or a baboon (Figure 5e).¹³ As hepatic transduction efficiency of first-generation adenoviral vectors or HDAds is comparable in baboons and rhesus monkeys (Supplementary Figure S1), these comparisons indicate that pseudo-hydrodynamic injection of HDAds can dramatically enhance hepatic transduction. Hematoxylin and eosin histology revealed no histological abnormalities in the liver of 14721 (Figure 6a) and rare foci of mild periportal inflammation and steatosis in the liver of 11754 (Figure 6b). These results indicate that pseudo-hydrodynamic injection did not result in significant liver damage, at least at 96 hours after injection.

Figure 5.

X-gal histochemistry of liver sections from non-human primates. (a) and (b) Pseudo-hydrodynamic injection of 1 10¹¹ vector particles (vp)/kg of a helper-dependent adenoviral vector (HDAd) expressing -galactosidase or conventional systemic intravascular injection (intra-portal or peripheral intravenous) of (c) 5 10¹¹ vp/kg or (d and e) 1 10¹² vp/kg of a first-generation, E1-deleted adenoviral vector expressing -galactosidase. c and d are from Nunes et al.¹² and e is from Morral et al.¹³

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Figure 6.

Hematoxylin and eosin histology of liver from baboons. (a) Baboon 14721 showing no abnormalities and (b) baboon 11754 showing rare foci of mild periportal inflammation and steatosis 96 hours after injection. Liver samples from (c) baboon 14200 and (d) baboon 14245 were taken 28 days after vector administration. The liver in c shows no abnormalities and the liver in d shows a focus of lymphocyte infiltrate.

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Pseudo-hydrodynamic injection of helper-dependent adenoviral vectors into baboons results in long-term transgene expression without chronic toxicity

Two important characteristics of HDAds are their ability to mediate long-term transgene expression after transduction of hepatocytes and the absence of chronic toxicity.^{2, 3, 4, 5, 6, 7} Therefore, we next determined whether pseudo-hydrodynamic injection of an HDAd compromised the duration of transgene expression or resulted in chronic toxicity. Pseudo-hydrodynamic injection of 1 10¹¹ vp/kg HD21.7E4PEPCK-bAFP-WL, an HDAd containing the baboon -fetoprotein (AFP) reporter gene under the control of a liver-restricted expression cassette,⁶ was performed on two baboons, 14200 and 14245. The baboons were given 20 ml/kg saline before occlusion, and phenylephrine was titrated intravenously during the occlusion. During the 30 minutes of occlusion, 360 ml of saline was injected retrograde into the liver through one of the balloon occlusion catheters in an attempt to further increase the intra-hepatic pressure. An increase in intra-hepatic pressure from a baseline of 5 mm Hg to a transient peak of 35 mm Hg in 14200 and >50 mm Hg in 14245 was achieved. Balloon inflation resulted in an 50% reduction in systemic blood pressure. Otherwise, the procedure was uneventful and well tolerated. Both animals returned to their pre-injection state 1 hour after injection. The animals were monitored weekly for the first month and every month thereafter to determine the duration of transgene expression and to look for evidence of chronic toxicity.

For 14200, AST peaked at 3.5 ULN at 24 hours, ALT peaked at 1.7 ULN at 24 hours, and lactate dehydrogenase peaked at 1.4 ULN at 6 hours (Figure 4). Interleukin-6 rose from 2.2 pg/ml at baseline to 79 pg/ml at 1 hour. Interleukin-12 rose from 7.4 pg/ml at baseline to 72.2 pg/ml at 1 hour. For 14245, AST peaked at 3.1 ULN at 24 hours, ALT peaked at 2.1 ULN at 24 hours, and lactate dehydrogenase peaked at 1.6 ULN at 24 hours (Figure 4). Interleukin-6 rose from 2.4 pg/ml at baseline to 396 pg/ml at 3 hours. Interleukin-12 rose from undetectable at baseline to 37.9 pg/ml at 1 hour. In both animals, platelet counts remained within the normal range. After laboratory parameters returned to baseline, no further abnormalities were observed for at least 413 days (data not shown), indicating an absence of chronic toxicity. Needle biopsies from the liver were obtained from both animals 28 days after vector injection for hematoxylin and eosin histology. No histological abnormalities were noted in 14200 (Figure 6c), but rare foci of lymphocytes were noted for 14245 (Figure 6d). Together, these results indicate that pseudo-hydrodynamic injection of HDAds does not cause chronic hepatotoxicity.

To determine the duration of transgene expression, baboon AFP was measured in the serum at various times after injection. High levels of serum baboon AFP were achieved that remained stable for the duration of the observation period of at least 413 days in both animals (Figure 7), indicating that pseudo-hydrodynamic injection of HDAds does not compromise the duration of HDAd-mediated transgene expression.

Figure 7.

Duration of transgene expression after pseudo-hydrodynamic injection of 1 10¹¹ vector particles/kg of HD21.7E4PEPCK-bAFP-WL into baboons. Serum levels of baboon -fetoprotein (bAFP) from baboons 14200 (open circles) and 14245 (open squares) are shown.¹³

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Discussion

We have shown in mice that HDAds need not be injected hydrodynamically to reap the benefit of increased hepatic transduction caused by the hydrodynamic effect provided the vector is delivered conventionally within 6 hours of the initiation of the hydrodynamic effect. This is in contrast to the case of pDNA, which must be delivered hydrodynamically to achieve hepatic transfection.²⁴ Although we do not know the reason for this difference, it has been shown that hydrodynamic injection causes both enlargement of the hepatic fenestrations and transient formation of membrane pores.²⁵ Whereas both may be required for pDNA to enter hepatocytes, only enlargement of the hepatic fenestrations may be required for increased hepatic transduction by HDAds²⁶ because the virus, unlike pDNA, can enter cells through receptor-mediated endocytosis,²⁷ thus doing away with the requirement for membrane pores. For this reason, our pseudo-hydrodynamic injection method would not work for pDNA. We also have shown that the hydrodynamic effect responsible for increased hepatic transduction by HDAds is transient, being most pronounced within the first 30 minutes of initiation of the hydrodynamic effect and then decreasing until it is no longer evident by 24 hours.

We have previously shown in mice that hydrodynamic injection could substantially increase the therapeutic index of HDAds,²³ but, unfortunately, this method of vector delivery cannot be used for humans. Therefore, on the basis of our results in mice in this study, we have developed a method of mimicking hydrodynamic injection in baboons that does not require the vector to be injected rapidly in a large volume. In this method, an increase in intra-hepatic pressure was achieved by transiently occluding the hepatic venous outflow using balloon occlusion catheters to simulate the high intra-hepatic pressures achieved by systemic hydrodynamic injection in the mouse. Following transient occlusion, an HDAd was delivered by systemic peripheral intravenous injection. This pseudo-hydrodynamic injection technique proved successful in achieving higher hepatic transduction than conventional systemic intravenous injection. This percutaneous approach was minimally invasive and well tolerated, resulted in relatively mild, transient acute toxicity and no chronic toxicity, and did not compromise long-term transgene expression from the transduced hepatocytes. Whether the improved hepatic transduction efficiency afforded by pseudo-hydrodynamic injection is biologically significant cannot be determined using our current animal model. However, in general, we believe that any increase would be beneficial because adenoviral vector-mediated acute toxicity is exquisitely dose dependent. As our technique cannot be performed in small animals, careful assessment in a large-animal model of a genetic disease (such as hemophilia in dogs) will be required to address this important question.

Localized hydrodynamic injection of pDNA into the liver of small and large animals has been performed previously.^{28, 29} However, unlike our approach, these experiments required the rapid injection of a relatively large volume of pDNA directly into the liver. Furthermore, unlike our percutaneous approach, the method of Zhang et al.²⁸ necessitates an invasive laparotomy to achieve isolation of the hepatic circulation. Finally, the duration of transgene expression and long-term toxicity were not investigated in these studies.

We have previously shown that comparable high-efficiency hepatic transduction could be achieved using the same vector at the same dose in baboons by delivering the vector preferentially into the surgically isolated liver.⁶ However, compared with the surgical method, which required an invasive laparotomy and mobilization of the liver to control the hepatic circulation, the pseudo-hydrodynamic method described here is significantly less invasive, requiring only percutaneous positioning of balloon occlusion catheters in the IVC, and, consequently, it was much better tolerated by the animals. Moreover, in our previous study,⁶ we showed long-term, stable expression of baboon AFP from HD21.7E4PEPCK-bAFP-WL for at least 476 and 665 days in baboons following hepatic transduction. In the present study, we have demonstrated that this important characteristic of HDAds is not compromised by pseudo-hydrodynamic injection, which yielded stable expression for at least 413 days. Morral et al.⁷ also observed long-term transgene expression in baboons following systemic injection of an HDAd. However, although expression was detectable for 1-2 years in that study, it slowly declined over the observation period to 10% of peak levels. This difference in expression stability may be attributable to the differences in the age of the animals at the time of vector administration; whereas we used adult baboons (>28 kg), Morral et al.⁷ used juvenile baboons (3 kg), and they speculated that growth of their animals may have, at least in part, been responsible for the decline in expression. Differences in the HDAd such as vector backbone sequences, cis-acting elements, and transgene may also be contributing factors.^{30, 31, 32, 33} Regardless, our present study, in conjunction with our previous study,⁶ confirms that HDAds can mediate stable, long-term transgene expression in non-human primates.

One undesirable side effect of pseudo-hydrodynamic injection is the transient hypotension caused by obstruction of the venous return to the heart that was observed in all four baboons. This potential complication has also been reported in humans during IVC occlusion for regional chemotherapy,³³ although at a reduced incidence compared with our baboons: only 3 of 10 patients experienced hypotension. The lack of hypotension in seven patients may have been due to the presence of a collateral venous system returning blood from the abdomen to the chest (which would not affect the achievement of high intra-hepatic pressure), and this may be more prevalent in humans than in baboons. However, it is important to note that the hypotension was completely and immediately reversible when the balloons were deflated and that the baboons appear to have suffered no ill effects. Whether this potential transient hypotension is clinically acceptable will be dictated by the risk/benefit ratio.

In summary, we have developed a novel method for delivering HDAds preferentially into the liver in non-human primates. This method results in higher-efficiency hepatic transduction and stable, long-term transgene levels using lower vector doses compared with conventional intravascular systemic delivery. Although acute elevations in liver enzymes and serum cytokines were observed, they were all relatively mild and transient. This approach may pave the way toward clinical liver-directed gene therapy using HDAds for a wide variety of genetic and acquired diseases.

Materials and Methods

Helper-dependent adenoviral vectors. HD28E4LacZ contains an MCMV-LacZ expression cassette and has been described in detail previously.³⁴ HD27.1E4PEPCK-bAFP-WL contains a liver-restricted baboon -fetoprotein expression cassette and has been described in detail previously.⁶ Both HDAds were produced in 116 cells³⁴ with the helper virus AdNG163 (ref. 35) as described in detail elsewhere.³⁴ Helper virus contamination levels were determined as described elsewhere³⁴ and were found to be <0.05% for both vector preparations. DNA analyses of HDAd genomic structure was confirmed for both vectors as described elsewhere.³⁴ All vector preparations were tested using Multi-test Limulus Amebocyte Lysate (Pyrogent; Biowhittaker, Walkersville, MD) for the presence of endotoxin and were found to be below the limit of detection (endotoxin <0.5 EU/ml).

Hydrodynamic injection in mice and analyses. Nine- to twelve-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were used for all the experiments. All procedures involving mice were approved by the Institutional Animal Care and use committee at Baylor College of Medicine, Houston. Hydrodynamic and conventional injections were performed as previously described.²³ Briefly, hydrodynamic injection into the tail vein was performed with 100 ml/kg Ringer's solution within 7 seconds, and conventional injection was performed with 200 l. Unless otherwise stated, all treatment groups consisted of three mice. Blood and livers were collected for analysis as previously described.²³ X-gal histochemistry and -galactosidase activity measurements were performed as described previously.¹⁹ Statistical analyses were performed using the t-test.

Non-human primates studies. Four adult male baboons (Papio sp.) were used in this study. Blood was collected from all animals as described previously⁶ before vector injection for blood cell counts, blood chemistries, and serum cytokines to establish baseline levels. Serum levels of neutralizing anti-Ad5 antibodies were determined before injection as described elsewhere³⁶ and were undetectable for all animals. Serum levels of neutralizing anti-Ad5 antibodies rose to a titer of 1,000 for baboons 14200 and 14245 at 56 days after injection. Blood was collected 1, 3, 6, 24, 72 and 96 hours after injection of HD28E4LacZ from baboons 14721 and 11754. For baboons 14200 and 14245 injected with HD27.1E4PEPCK-bAFP-WL, blood was collected 1, 3, 6, 24, and 72 hours after vector injection then weekly for the first month and monthly thereafter for blood cell counts, blood chemistries, and serum cytokines and to determine serum baboon AFP levels. Serum interleukin-6 concentrations were determined by Specialty Laboratories (Santa Monica, CA). Interleukin-12 was measured by ELISA according to the manufacturer's instructions (Biosource, Camarillo, CA). Serum baboon AFP was measured as previously reported.⁶ Needle biopsies of the liver were taken from baboons 14721 and 11754 for hematoxylin and eosin histology 28 days after vector injection.

Pseudo-hydrodynamic injection. An 11 French (F) sheath was placed in the right and left femoral veins using a standard sterile percutaneous technique. A 20-gauge arterial catheter was placed in the femoral artery for continuous blood pressure monitoring during the procedure. A Meditech occlusion balloon catheter (Boston Scientific, Watertown, MA) was advanced into the right femoral vein sheath and positioned in the IVC at the IVC-right atrial junction. A second Meditech occlusion balloon catheter was then advanced through the left femoral vein sheath and positioned in the IVC inferior to the first balloon. The balloons were then inflated with saline-contrast solution. Positioning of the balloons was confirmed by injection of a small amount of contrast solution into the inferior balloon catheter to demonstrate complete occlusion of hepatic venous outflow and monitored with intermittent fluoroscopy. As indicated in the Results section, saline was injected through the inferior balloon catheter into the IVC in the space between the two balloons to further increase the intra-hepatic pressure in baboons 11754, 14200, and 14245. The hepatic venous pressure was measured in real time via a transducer attached to the inferior balloon catheter. After 30 minutes of occlusion, the balloons were deflated, the catheters were removed from the femoral venous sheaths, and the HDAd vector at a dose of 1 10¹¹ vp/kg in 5 ml saline was injected into a peripheral vein of the forelimb at a rate of 1 ml/min.

References

1. Nguyen, TH and Ferry, N (2004). Liver gene therapy: advances and hurdles. Gene Ther 11: S76–S84. | Article | PubMed | ChemPort |
2. Palmer, D and Ng, P (2005). Helper-dependent adenoviral vectors for gene therapy. Hum Gene Ther 16: 1–16. | Article | PubMed | ChemPort |
3. Kim, IH, Jozkowicz, A, Piedra, PA, Oka, K and Chan, L (2001). Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector. Proc Natl Acad Sci USA 98: 13282–12387. | Article | PubMed | ChemPort |
4. Toietta, G, Mane, VP, Norona, WS, Finegold, MJ, Ng, P, McDonagh, AF et al. (2005). Lifelong elimination of hyperbilirubinemia in the gunn rat with a single injection of helper-dependent adenoviral vector. Proc Natl Acad Sci USA 102: 3930–3935. | Article | PubMed | ChemPort |
5. Brunetti-Pierri, N, Nichols, TC, McCorquodale, S, Merricks, E, Palmer, DJ, Beaudet, AL et al. (2005). Sustained phenotypic correction of canine hemophilia B following systemic administration of helper dependent adenoviral vectors. Hum Gene Ther 16: 811–820. | Article | PubMed | ChemPort |
6. Brunetti-Pierri, N, Ng, T, Iannitti, DA, Palmer, DJ, Beaudet, AL, Finegold, MJ et al. (2006). Improved hepatic transduction, reduced systemic vector dissemination, and long-term transgene expression by delivering helper-dependent adenoviral vectors into the surgically isolated liver of nonhuman primates. Hum Gene Ther 17: 391–404. | Article | PubMed | ChemPort |
7. Morral, N, O'Neal, W, Rice, K, Leland, M, Kaplan, J, Piedra, PA et al. (1999). Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver directed gene transfer in baboons. Proc Natl Acad Sci USA 96: 12816–12821. | Article | PubMed | ChemPort |
8. Morral, N, O'Neal, W, Rice, K, Leland, M, Kaplan, J, Piedra, PA et al. (1998). High doses of a helper-dependent adenoviral vector yield supraphysiological levels of 1-antitrypsin with negligible toxicity. Hum Gene Ther 9: 2709–2716. | PubMed | ISI | ChemPort |
9. Tao, N, Gao, GP, Parr, M, Johnston, J, Baradet, T, Wilson, JM et al. (2001). Sequestration of adenoviral vector by kupffer cells leads to a nonlinear dose response of transduction in liver. Mol Ther 3: 28–35. | Article | PubMed | ISI | ChemPort |
10. Schiedner, G, Hertel, S, Johnston, M, Dries, V, Van Rooijen, N and Kochanek, S (2003). Selective depletion or blockade of kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors. Mol Ther 7: 35–43. | Article | PubMed | ChemPort |
11. Sullivan, DE, Dash, S, Du, H, Hiramatsu, N, Aydin, F, Kolls, J et al. (1997). Liver-directed gene transfer in non-human primates. Hum Gene Ther 8: 1195–1206. | PubMed | ISI | ChemPort |
12. Nunes, FA, Furth, EE, Wilson, JM and Raper, SE (1999). Gene transfer into the liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration. Hum Gene Ther 10: 2515–2526. | Article | PubMed | ChemPort |
13. Morral, N, O'Neal, WK, Rice, K, Leland, MM, Piedra, PA, Aguilar-Cordova, E et al. (2002). Lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons. Hum Gene Ther 13: 143–154. | Article | PubMed | ISI | ChemPort |
14. Muruve, DA (2004). The innate immune response to adenovirus vectors. Hum Gene Ther 15: 1157–1166. | Article | PubMed | ISI | ChemPort |
15. Muruve, DA, Cotter, MJ, Zaiss, AK, White, LR, Liu, Q, Chan, T et al. (2004). Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo. J Virol 78: 5966–5972. | Article | PubMed | ISI | ChemPort |
16. Schnell, MA, Zhang, Y, Tazelaar, J, Gao, GP, Yu, QC, Qian, R et al. (2001). Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors. Mol Ther 3: 708–722. | Article | PubMed | ISI | ChemPort |
17. Raper, SE, Chirmule, N, Lee, FS, Wivel, NA, Bagg, A, Gao, GP et al. (2003). Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80: 148–158. | Article | PubMed | ISI | ChemPort |
18. Muruve, DA, Barnes, MJ, Stillman, IE and Libermann, TA (1999). Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum Gene Ther 10: 965–976. | Article | PubMed | ISI | ChemPort |
19. Brunetti-Pierri, N, Palmer, DJ, Beaudet, AL, Carey, D, Finegold, M and Ng, P (2004). Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into non human primates. Hum Gene Ther 15: 35–46. | Article | PubMed | ISI | ChemPort |
20. Zhang, Y, Chirmule, N, Gao, GP, Qian, R, Croyle, M, Joshi, B et al. (2001). Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages. Mol Ther 3: 697–707. | Article | PubMed | ChemPort |
21. Liu, F, Song, YK and Liu, D (1999). Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6: 1258–1266. | Article | PubMed | ISI | ChemPort |
22. Zhang, G, Budker, V and Wolff, JA (1999). High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 10: 1735–1737. | Article | PubMed | ISI | ChemPort |
23. Brunetti-Pierri, N, Palmer, DJ, Mane, V, Finegold, M, Beaudet, AL and Ng, P (2005). Increased hepatic transduction with reduced systemic dissemination and proinflammatory cytokines following hydrodynamic injection of helper-dependent adenoviral vectors. Mol Ther 12: 99–106. | Article | PubMed | ChemPort |
24. Andrianaivo, F, Lecocq, M, Wattiaux-De Coninck, S, Wattiaux, R and Jadot, M (2004). Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection. J Gene Med 6: 877–883. | Article | PubMed | ISI | ChemPort |
25. Zhang, G, Gao, X, Song, YK, Vollmer, R, Stolz, DB, Gasiorowski, JZ et al. (2004). Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther 11: 675–682. | Article | PubMed | ChemPort |
26. Lievens, J, Snoeys, J, Vekemans, K, Van Linthout, S, de Zanger, R, Collen, D et al. (2004). The size of sinusoidal fenestrae is a critical determinant of hepatocyte transduction after adenoviral gene transfer. Gene Ther 11: 1523–1531. | Article | PubMed | ChemPort |
27. Meier, O and Greber, UF (2003). Adenovirus endocytosis. J Gene Med 5: 451–462. | Article | PubMed | ChemPort |
28. Zhang, G, Vargo, D, Budker, B, Armstrong, N, Knechtle, S and Wolff, JA (1997). Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers. Hum Gene Ther 8: 1763–1772. | PubMed | ChemPort |
29. Eastman, SJ, Baskin, KM, Hodges, BL, Chu, Q, Gates, A, Dreusicke, R et al. (2002). Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA. Hum Gene Ther 13: 2065–2077. | Article | PubMed | ChemPort |
30. Parks, RJ, Bramson, JL, Wan, Y, Addison, CL and Graham, FL (1999). Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors. J Virol 73: 8027–8034. | PubMed | ChemPort |
31. Sandig, V, Youil, R, Bett, AJ, Franlin, LL, Oshima, M, Maione, D et al. (1999). Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc Natl Acad Sci USA 97: 1002–1007. | Article |
32. Schiedner, G, Hertel, S, Johnston, M, Biermann, V, Dries, V and Kochanek, S (2002). Variables affecting in vivo performance of high-capacity adenovirus vectors. J Virol 76: 1600–1609. | Article | PubMed | ChemPort |
33. Berkenstadt, H, Ben-Ari, G and Perel, A (1998). Hemodynamic changes during a new procedure for regional chemotherapy involving occlusion of the thoracic aorta and inferior vena cava. J Clin Anesth 10: 636–640. | Article | PubMed | ChemPort |
34. Palmer, DJ and Ng, P (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8: 846–852. | Article | PubMed | ISI | ChemPort |
35. Palmer, DJ and Ng, P (2004). Physical and infectious titers of helper-dependent adenoviral vectors: a method of direct comparison to the adenovirus reference material. Mol Ther 10: 792–798. | Article | PubMed | ChemPort |
36. Croyle, MA, Chirmule, N, Zhang, Y and Wilson, JM (2001). "Stealth" adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol 75: 4792–4801. | Article | PubMed | ISI | ChemPort |
37. Varnavski, AN, Zhang, Y, Schnell, M, Tazelaar, J, Louboutin, JP, Yu, QC et al. (2002). Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity. J Virol 76: 5711–5719. | Article | PubMed | ISI | ChemPort |

Acknowledgements

This work was supported by the National Institutes of Health P50 HL59314 (A.L.B.) and R01 DK069369 (P.N.) and the Texas Affiliate of the American Heart Association 0465102Y (P.N.). The financial support of Telethon-Italy (Fellowship GFP04008) to N.B.-P. is gratefully acknowledged. We thank the Morphology Core Laboratory of the Gulf Coast Digestive Disease Center and Angela Major and Dorene M. Rudman for the enzyme histochemistry.