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. 2012 Jul 24;20(10):1863–1870. doi: 10.1038/mt.2012.143

Balloon Catheter Delivery of Helper-dependent Adenoviral Vector Results in Sustained, Therapeutic hFIX Expression in Rhesus Macaques

Nicola Brunetti-Pierri 1,7, Aimee Liou 2, Priti Patel 2,7, Donna Palmer 1, Nathan Grove 1, Milton Finegold 3, Pasquale Piccolo 4, Elizabeth Donnachie 5, Karen Rice 6, Arthur Beaudet 1, Charles Mullins 2, Philip Ng 1,*
PMCID: PMC3464633  PMID: 22828499

Abstract

Hemophilia B is an excellent candidate for gene therapy because low levels of factor IX (FIX) (≥1%) result in clinically significant improvement of the bleeding diathesis. Helper-dependent adenoviral (HDAd) vectors can mediate long-term transgene expression without chronic toxicity. To determine the potential for HDAd-mediated liver-directed hemophilia B gene therapy, we administered an HDAd expressing hFIX into rhesus macaques through a novel and minimally invasive balloon occlusion catheter-based method that permits preferential, high-efficiency hepatocyte transduction with low, subtoxic vector doses. Animals given 1 × 1012 and 1 × 1011 virus particle (vp)/kg achieved therapeutic hFIX levels for the entire observation period (up to 1,029 days). At 3 × 1010 and 1 × 1010 vp/kg, only subtherapeutic hFIX levels were achieved which were not sustained long-term. Balloon occlusion administration of HDAd was well tolerated with negligible toxicity. Five of six animals developed inhibitors to hFIX. These results provide important information in assessing the clinical utility of HDAd for hemophilia B gene therapy.

Introduction

Hemophilia B is an attractive target for gene therapy because a very low level of factor IX (FIX) is therapeutic (≥1% of normal activity or ≥50 ng/ml). Consequently, the therapeutic vector dose can be potentially low and thus attractive from a safety perspective. Helper-dependent adenoviral vectors (HDAds) are devoid of all viral genes and thus can provide long-term transgene expression in the absence of chronic toxicity.1 We have demonstrated that HDAds can transduce the liver of FIX-deficient mice2 and dogs3 to provide long-term expression of the vector-encoded FIX following systemic intravenous administration. However, efficient hepatocyte transduction by adenoviral (Ad)-based vectors following systemic intravascular injection requires high vector doses because of a nonlinear dose response which has been well documented in both mice4,5 and nonhuman primates.6,7,8 Unfortunately, these high doses resulted in dose-dependent activation of the innate immune response resulting in acute toxicity with potentially severe and lethal consequences in nonhuman primates and humans.7,8,9,10,11 While the mechanism of Ad-mediated innate immune activation remains to be fully elucidated, the severity is clearly dose-dependent.7,8,9,10,11,12 To address this, we have recently developed a novel, minimally invasive balloon occlusion catheter-based method for delivering HDAd preferentially into the liver of baboons so that efficient hepatocyte transduction can be achieved using low, subtoxic vector doses.13 This route of administration was very well tolerated and up to an 80-fold hepatocyte transduction enhancement can be achieved in baboons using clinically relevant low vector doses with minimal-to-negligible toxicity.13 However, that study used a non-therapeutic secreted reporter protein and thus the therapeutic potential could not be determined. Therefore, in the present study, we evaluated the therapeutic potential of delivering HDAd expressing hFIX using this novel route of administration in the rhesus macaque as a preclinical model for hemophilia B gene therapy.

Results

In the present study, we evaluated the therapeutic potential of an HDAd expressing hFIX from a liver-specific promoter (HDΔ21.7E4gE-hFIX) delivered at various doses by a balloon occlusion method13 directly into the livers of six rhesus macaques as a preclinical model for hemophilia B gene therapy.

Rhesus 17664 and 17650

We administered, by the balloon occlusion method, 1 × 1012 and 1 × 1011 virus particle (vp)/kg HDΔ21.7E4gE-hFIX (lot no. 040908) into rhesus 17664 and 17650, respectively (Table 1). The percutaneous catheterization procedure, vector administration, and recovery were well tolerated and uneventful. Pressure was applied to the inguinal areas for ~30 minutes following removal of catheters until bleeding stopped. However, the next day, 17664 was found with an unexpected complication of extravasation in the left inguinal region, where percutaneous access was performed. Rhesus 17664 presented with an extremely swollen left leg and reluctance to use the limb. With extensive hydrotherapy and massage, this complication resolved and 17664 remained in a healthy, active state with no clinical manifestations of toxicity for the duration of observation (1,029 days). Extravasation in the left inguinal area also was found the next day in 17650 but with more severe clinical presentation and disuse of the left leg. Rhesus 17650 was given the same clinical intervention but did not resolve completely and was permanently impaired. Extensive clinical care was provided and his condition began to improve. Blood was collected weekly from the two animals to measure hFIX levels, total anti-hFIX antibodies, inhibitors (neutralizing anti-hFIX antibodies), aspartate transaminase (AST) and alanine transaminase (ALT). Unfortunately, over the next few months, the post-procedural complication worsened for 17650 with muscle wasting, fused ankle, ulcerated left foot and inflexible, curled toes. Although appetite and weight remained normal, the veterinarian recommended euthanasia due to poor quality of life. Rhesus 17650 was euthanized 99 days after vector administration.

Table 1. Summary of rhesus macaques, procedures, and outcomes.

graphic file with name mt2012143t1.jpg

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Rhesus 17664, injected with 1 × 1012 vp/kg, achieved an average hFIX level of 1,608 ng/ml during the first 28 days post-vector, after which hFIX levels declined and stabilized at a therapeutic average of 391 ng/ml until necropsy on day 1,029 (Figure 1 and Supplementary Table S1). Hepatotoxicity was assessed by measuring serum AST and ALT which remained within the normal range for the entire observation period with one exception; a moderate rise in ALT (3.2× upper limit of normal (ULN)) at day 7 (Figure 2 and Supplementary Table S1). Fibrinogen levels, prothrombin time (PT), and partial thromboplastin time (PTT) were within the normal range at the time of necropsy. Total anti-hFIX antibodies were first detected 49 days post-vector and persisted until day 343 at a titer of 1:100, and then fell to undetectable levels for the remainder of the observation period (Figure 3 and Supplementary Table S1). Inhibitors were undetectable before vector injection (day 0), but were present at low levels on days 43 (0.55 Bethesda units (BU)/ml) and 338 (2.35 BU/ml), and became undetectable on day 1,029 (Figure 3 and Supplementary Table S1). Before necropsy on day 1,029, ultrasound examination of the liver revealed no abnormalities and physical examination showed that the animal was in good clinical condition with adequate amounts of fat, normal muscle bulk, and hydration. Thorough visual examination during necropsy revealed no significant gross abnormalities in any organ/tissue. Careful examination of ~5 mm slices made through the entire liver revealed no macroscopic abnormalities. A wide variety of organs (see Materials and Methods) were examined by hematoxylin and eosin (H&E) histology with no significant findings.

Figure 1.

Figure 1

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Plasma hFIX levels in rhesus macaques. Vector was administrated on day 0. Normal; >30% of normal. Mild; >5–30% of normal. Moderate; ≥1–5% of normal. Severe; <1% of normal. Normal hFIX level is ~5,000 ng/ml.

Figure 2.

Figure 2

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Serum ALT and AST levels in rhesus macaques. Normal range for ALT is 5–61 U/l. Normal range for AST is 12–63 U/l. Grade 1 (mild) = 1.1–2.5× upper limit of normal (ULN). Grade 2 (moderate) = 2.6–5.0× ULN. Grade 3 (severe) = 5.1–10× ULN. Grade 4 (potentially life-threatening) >10× ULN. ALT, alanine transaminase; AST, aspartate transaminase.

Figure 3.

Figure 3

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Levels of total anti-hFIX antibodies and inhibitors in rhesus macaques. Vector was administered on day 0. BU, Bethesda units.

Rhesus 17650, injected with 1 × 1011 vp/kg, achieved an average hFIX level of 122 ng/ml during the first 34 days post-vector, after which hFIX levels declined but remained therapeutic for 83 days (average 61 ng/ml) before declining to subtherapeutic levels at day 97 (Figure 1 and Supplementary Table S1). At day 99, 17650 was euthanized for the reasons cited above. AST and ALT remained within the normal range for the entire observation period with one exception; a moderate rise in ALT (3.3× ULN) and AST (1.2× ULN) at day 7 (Figure 2 and Supplementary Table S1). Low titer anti-hFIX antibodies (1:10) were first detected 48 days post-vector and persisted at this low level for the duration of the observation period (Figure 3 and Supplementary Table S1). Inhibitors were undetectable at days 0 and 41 but were present at a low level on day 97 (1.14 BU/ml) (Figure 3 and Supplementary Table S1).

Rhesus 17633 and 18407

Hemophilia B patients regularly receive intravenous exogenous hFIX, many of whom are on a prophylactic regimen. Therefore, we sought to determine whether pre-exposure to hFIX affected our gene therapy in the rhesus macaque. Thus, 100 units/kg recombinant hFIX (BeneFIX) was infused once a week into rhesus 17633 and 18407 for 6–8 consecutive weeks before HDAd administration. hFIX in blood was detected 30 minutes after each infusion, but became undetectable 7 days postinfusion due to its short half-life (Figure 1 and Supplementary Table S1). One week after the last BeneFIX infusion, 1 × 1012 and 1 × 1011 vp/kg of HDΔ21.7E4gE-hFIX was administered into 17633 and 18407, respectively, using the balloon occlusion method. A different preparation of the same vector, HDΔ21.7E4gE-hFIX (lot no. 102308), was used for these two animals. The percutaneous catheterization procedure, vector administration, and recovery were well tolerated and uneventful. To avoid the post-procedural complication of extravasation at the site of percutaneous access, pressure was applied to the inguinal areas for ~1 hour following catheter removal, to ensure stable clot formation. This mitigated the post-procedural complication experienced by the previous two animals. Both animals remained in a healthy, active state with no clinical manifestations of toxicity for the duration of the observation period of 833 and 725 days, respectively.

Rhesus 17633, injected with 1 × 1012 vp/kg, achieved an average hFIX level of 3,874 ng/ml during the first 182 days post-vector, after which hFIX levels declined and stabilized at a therapeutic average of 1,289 ng/ml until necropsy on day 833 (Figure 1 and Supplementary Table S1). AST and ALT remained within the normal range for the entire observation period with one exception; a mild rise in ALT (1.4× ULN) at day 7 (Figure 2 and Supplementary Table S1). Fibrinogen levels, PT, and PTT were within the normal range at necropsy (data not shown). Total anti-hFIX antibodies were first detected 28 days post-vector, rising to a peak of 1:100 by day 211, then declining to undetectable levels at day 343 and remained undetectable until the end of the observation period (Figure 3 and Supplementary Table S1). Inhibitors were undetectable on day −42 (before the first BeneFIX infusion) but were present on days 189 (7.6 BU/ml) and 833 (3.9 BU/ml) (Figure 3 and Supplementary Table S1).

Rhesus 18407, injected with 1 × 1011 vp/kg, achieved a therapeutic average hFIX level of 986 ng/ml during the first 208 days post-vector, after which hFIX levels declined and stabilized at a therapeutic average of 591 ng/ml until necropsy on day 725 (Figure 1 and Supplementary Table S1). AST and ALT remained within the normal range for the entire observation period (Figure 2 and Supplementary Table S1). Fibrinogen levels, PT, and PTT were within the normal range at necropsy. No anti-hFIX antibodies were detected and inhibitors were not detected on days −56 (before the first BeneFIX), 214, and 725 (Figure 3 and Supplementary Table S1).

Although injected with the same doses of HDΔ21.7E4gE-hFIX, 17664 and 17650 achieved lower hFIX levels compared with 17633 and 18407 (Figure 1). However, as mentioned above, two different preparations of HDΔ21.7E4gE-hFIX were used; 17664 and 17650 received lot no. 040908 while 17633 and 18407 received lot no. 102308. Therefore, it was likely that the infectivity of lot no. 040908 was lower than lot no. 102308. To investigate this, one group of C57BL/6 mice was injected with 1 × 1012 vp/kg of lot no. 040908 while another group was injected with 1 × 1012 vp/kg of lot no. 102308 and plasma hFIX levels were determined 7 and 34 days postinjection. Consistent with our hypothesis, lot no. 040908 yielded lower levels of hFIX than lot no. 102308 in the injected mice (Figure 4).

Figure 4.

Figure 4

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Plasma hFIX levels in mice. Mice injected with two different preparations (lot no. 040908 or lot no. 102308) of HDΔ21.7E4gE-hFIX at 1 × 1012 virus particle/kg. N = 5 per group. Mean ± SD shown.

Before necropsy, both animals were in good clinical condition with normal amounts of fat, muscle bulk, and hydration. Their livers were examined by ultrasound which revealed no abnormalities. Gross examination at necropsy revealed some fibrous adhesions of the right caudal lung to the pleura and diaphragm, and the cardiac lobes were both ~30% atelectatic and red for 17633. Although we do not know the cause of this abnormality, it is unlikely to be related to the HDAd or balloon procedure because of the length of time between vector administration and necropsy (833 days) and because this was not observed in the other treated animals. This abnormality may have spontaneously arisen from an episode of pneumonia which is the sixth most common natural pathology at the nonhuman primate facility where our experiments were conducted.14 No significant gross abnormalities were noted in 18407. Careful examination of thin slices (~5 mm) made through the entire liver in these two animals revealed no macroscopic abnormalities. A wide variety of organs were examined by H&E histology which revealed no significant findings except for some areas of acute necrosis in the liver of 17633 (Supplementary Figure S1a) and von Meyenburg complexes in the liver of 18407 (Supplementary Figure S1b), both of which were isolated rare findings which we do not attribute to the gene therapy and consider clinically insignificant.

Rhesus 19152 and 19313

We next investigated the effect of even lower vector doses. Thus, 3 × 1010 and 1 × 1010 vp/kg HDΔ21.7E4gE-hFIX (lot no. 102308) were administered to 19152 and 19313, respectively, by the balloon occlusion method. These two animals were infused with BeneFIX as described above before vector administration. The percutaneous catheterization procedure, vector administration, and recovery for both animals were well tolerated and uneventful. The post-procedural complication of extravasation at the site of percutaneous access was again avoided by prolonged pressure application of the inguinal area following catheter removal. Both animals remained in a healthy, active state with no clinical manifestations of toxicity for the duration of the observation period of 378 and 456 days, respectively.

For 19152, 3 × 1010 vp/kg yielded a subtherapeutic average of 24 ng/ml hFIX for 126 days and then declined to undetectable levels (Figure 1 and Supplementary Table S1). AST and ALT remained within the normal range for the entire observation period with one exception; a mild elevation of ALT (1.4× ULN) and AST (2.5× ULN) was observed on day 112 (Figure 2 and Supplementary Table S1). Total anti-hFIX antibodies were first detected at a titer of 1:10–1:100 during the period of BeneFIX infusions before vector administration. Following vector administration, total anti-hFIX antibodies remained detectable (1:10–1:1,000) for the duration of the observation period (Figure 3 and Supplementary Table S1). Inhibitors were undetectable on day −42 before the first BeneFIX infusion but were detected on day 0, before vector injection, at a titer of 7.34 BU/ml. Inhibitors remained detectable on days 217 (3.18 BU/ml) and 378 (3.14 BU/ml)(Figure 3 and Supplementary Table S1). Fibrinogen levels, PT, and PTT were within the normal range at day 322 and at the end of the observation period indicating that the endogenous rhesus FIX was unaffected by the humoral response to hFIX.

For 19313, 1 × 1010 vp/kg yielded 4.6 ng/ml hFIX on day 20 (Figure 1 and Supplementary Table S1) but was undetectable at all subsequent times. AST and ALT remained within the normal range for the entire observation period (Figure 2 and Supplementary Table S1). Total anti-hFIX antibodies were first detected 27 days after vector administration at 1:10 and rose to 1:100 by day 62 and remained at that titer for the duration of the observation period (Figure 3 and Supplementary Table S1). Inhibitors were undetectable at day −42 (before the first BeneFIX infusion) but was detectable at days 27 (16.22 BU/ml), 174 (17.34 BU/ml), and 456 (10.69 BU/ml) (Figure 3 and Supplementary Table S1). Fibrinogen levels, PT, and PTT were within the normal range at day 456 indicating that the endogenous rhesus FIX was unaffected by the humoral response to hFIX.

Vector biodistribtuion was determined for rhesus 17664, 17633, and 18407. As expected, vector genome was found primarily in the liver but a significant amount was also found in the spleen of rhesus 17633 (Figure 5). With the exception of the left lateral lobe of 17664, the amount of vector DNA found in the livers correlate well with hFIX levels. Detection of higher number of vector genome copies in three of the four liver lobes in 17633 compared with 17664 is consistent with higher infectivity of lot no. 102308 compared with lot no. 040808.

Figure 5.

Figure 5

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Vector biodistribution in rhesus macaques. Vector genome copies were determined by real-time PCR in different organs in rhesus 17664, 17633, and 18407.

Discussion

In this study, we have evaluated the utility of balloon occlusion catheter-based delivery of HDAd expressing hFIX from a liver-specific promoter in the rhesus macaque. We found that 1 × 1012 and 1 × 1011 vp/kg resulted in therapeutic hFIX levels for the duration of the observation period of up to 1,029 days. Inhibitors were detectable in three of these four animals receiving these vector doses but were not sufficient to extinguish hFIX. At 3 × 1010 vp/kg, hFIX was detectable for 126 days but at subtherapeutic levels which then decline to undetectable levels. In this animal, inhibitors were first detected after BeneFIX infusion and before vector administration, but were not high enough to extinguish hFIX, at least not for the first 126 days. Eventual loss of hFIX may be due to a combination of persistent inhibitors and spontaneous loss of transduced hepatocytes due to physiologic hepatocyte turnover. At 1 × 1010 vp/kg, hFIX was undetectable for the entire observation period except for a low, subtherapeutic amount on day 20. High titer inhibitors were detected in this animal which persisted and likely contributed to loss of hFIX. Thus, the minimum therapeutic HDΔ21.7E4gE-hFIX dose appears to be >3 × 1010 vp/kg when delivered by the balloon occlusion method, at least in this animal model. However, additional animals will need to be evaluated to confirm this finding. It may be possible to further reduce the therapeutic dose by improving hFIX expression level using strategies proven to enhance transgene expression (stronger promoters, appropriate enhancers and cis-acting elements, and codon optimization). However, 1 × 1011 vp/kg, which appears to yield sustained therapeutic hFIX in the rhesus, may be clinically relevant because an intravascular dose of 2 × 1011 vp/kg of an Ad-based vector was relatively well tolerated in an OTC (ornithine transcarbamylase) gene therapy trial.9 That trial also revealed that an intravascular dose of 6 × 1011 vp/kg resulted in fatal toxicity in one of two patients.10

Development of inhibitors to FIX is a major concern and we found inhibitor development in five of the six animals. This frequency is higher than in hemophilia B patients (1.5–3%), but is perhaps not surprising given that the hFIX is foreign to the rhesus macaque, despite >97% amino acid identity with the endogenous rhesus FIX.15 In support of this, at least one of the four animals (19152) developed inhibitors following exposure to BeneFIX alone, a frequency that appears to be substantially higher than the frequency of 1.5–3% observed for hemophilia B patients. Therefore, the frequency or magnitude of inhibitor development following gene therapy with HDAd expressing hFIX may be overestimated in animal models. Development of inhibitors at higher than expected human frequencies have also been reported in the rhesus macaque following administration of rAAV216 and scAAV817 expressing hFIX.

Lozier et al. also used the rhesus macaque to investigate AVC3FIX5, a first generation, E1-deleted Ad expressing hFIX from a cytomegalovirus promoter, for hemophilia B gene therapy.15,18 In that study, peripheral intravenous injection of AVC3FIX5 at 3.25 × 1012 vp/kg resulted in a peak of ~4,000 ng/ml hFIX at day 4 postinjection but declined to undetectable levels by day 22. At 1.625 × 1012 vp/kg, a peak of ~800 ng/ml hFIX was achieved 4 days postinjection but declined to undetectable levels by day 17; 3.25 × 1011 vp/kg yielded no detectable hFIX.15 The much lower levels of hFIX obtained by Lozier et al.15 compared to our results (on a per vp basis) is consistent with the enhancement in hepatocyte transduction afforded by our balloon occlusion catheter method of vector delivery. In contrast to our results, only transient hFIX expression was observed with ACV3FIX5. This difference in duration of expression is consistent with the difference between E1-deleted Ad and HDAd,19 although the different promoters used may have also contributed. Also in contrast to our study, Lozier et al. observed more severe and prolonged AST and ALT elevations that were dose-dependent.18 This difference is not surprising due to low level viral gene expression from the E1-deleted Ad which does not occur with HDAd.20 Indeed, we observed mostly negligible-to-transient mild elevations of AST and ALT in only some of the animals injected with HDAd, which did not appear to be dose-dependent and likely reflected a variable response to the balloon procedure.13 In addition, we carefully examined the livers from selected animals by ultrasound, gross inspection of thin slices, and histopathology at the end of the observation period (725–1,029 days post-vector) and the findings were all unremarkable. This was not surprising considering that HDAd do not cause chronic toxicity because they do not express any viral genes and because integration of Ad-based vectors into the host genome is negligible thus negating the risk of tumorigenesis.21

In accordance to general recommendations,22 IACUC (Institutional Animal Care and Use Committee) guidelines at the Texas Biomedical Research Institute, where these studies presented were performed, limited the amount of blood we could collect, and the numbers of sedations required for blood collection. Thus, we chose to focus our analyses on later time points (>7 days post-vector) to evaluate hFIX levels and duration, and adaptive immune response against the foreign hFIX xenoprotein and/or HDAd-transduced hepatocytes. This was important because long-term evaluation of hFIX expressed from an Ad-based vector in rhesus macaques had not been examined to date. Thus, we could not predict when such a putative adaptive immune response might develop, forcing us to evaluate frequent weekly blood samples starting 7 days post-vector (when ApoE genomic promoter first begins to actively drive hFIX expression) for the duration of the observation period. This frequent sampling precluded obtaining blood at early time points to assess the acute toxic response to HDAd which is important for assessing safety. In this regard, we have performed extensive analyses of early time points in a relatively large number of baboons given similar doses of HDAd by the identical route of administration in a previous study.13 That prior study revealed only mild to moderate elevation of AST and ALT peaking at 24–48 hours post-vector injection that were transient and appeared attributable to the balloon procedure, not the HDAd due to the low doses injected which were comparable with those used in the rhesus macaque in the current study.13 Mild to moderate transient elevation of serum IL-6, a marker of the innate inflammatory response was also observed with peak levels reached at 3.5 hours post-vector injection in those baboons which appeared attributable to the vector, not the balloon procedure.13 Thrombocytopenia was not observed in any baboons which we attributed to the low vector doses administered.13 In summary, while the acute toxic response in the rhesus macaques was not investigated in the current study, we expect that transient elevations of serum IL-6 (due to the vector), and AST and ALT (due to the balloon procedure) would have been observed at early time points. However, as was the case in baboons, we would also expect that these elevations in AST, ALT, and IL-6 be only modest considering the low vector doses administered and the minimally invasive nature of the balloon procedure.13 Further studies will be required to verify these assumptions and to fully evaluate safety of this modality.

We originally developed the balloon occlusion catheter method in baboons and no procedure-related complications occurred in over 14 baboons studied.13,23 However, the first two rhesus macaques suffered extravasation at the site of percutaneous access. A likely contributing factor is the rhesus macaques' higher level of physical activity following the procedure which would increase the likelihood of dislodging the blood clot at the site of percutaneous access. This was resolved for four subsequent rhesus macaques by prolonging manual compression of the inguinal area. This complication is unlikely to occur in humans as compliance to hazardous activities can be more easily assured. In the case of hemophilia patients, prophylactic factor coverage should mitigate this complication due to their underlying disease. Aside from the animals that suffered the avoidable post-procedural complication, all others recovered quickly from the minimally invasive procedure and returned to a healthy, active state with no signs of clinical toxicity for the duration of the observation period.

In summary, we have shown that balloon occlusion catheter-based delivery of >3 × 1010 vp/kg HDΔ21.7E4gE-hFIX is well tolerated without evidence of long-term toxicity and can result in sustained therapeutic hFIX levels in rhesus macaques.

Materials and Methods

HDAd vector. The HDAd, HDΔ21.7E4gE-hFIX, contains a liver-restricted ApoE genomic promoter24 driving the expression of the hFIX. HDAd was produced in 11625 cells with the helper virus AdNG163,26 as described in detail elsewhere.25 Helper virus contamination levels were determined as described elsewhere25 and were <0.05%. DNA analyses of HDAd genomic structure was performed as described elsewhere.27 HDAds were tested using Multi-test Limulus Amebocyte Lysate (Pyrogent; Biowhittaker, Walkersville, MD) for endotoxin which were below the limit of detection (<0.5 EU/ml). Two preparations of HDΔ21.7E4gE-hFIX were used, designated lot no. 040908 (titer: 1.83 × 1013 vp/ml) and lot no. 102308 (titer: 1.16 × 1013 vp/ml).

Animals. Animal experiments were reviewed and approved by IACUC of Baylor College of Medicine or Texas Biomedical Research Institute. Six male rhesus macaques were used (Table 1); 6–8 weeks before vector delivery, animals 17633, 18407, 19152, and 19313 received weekly infusions of 100 units/kg recombinant hFIX (BeneFIX; Wyeth, Madison, NJ). Blood was collected for analyses from all animals just before vector injection and periodically after vector injection for the duration of the observation period. For animals pre-treated with BeneFIX, blood was also collected immediately before and 30 minutes after BeneFIX infusion. Before killing, animals underwent liver ultrasound using a standard echography machine; 9–12-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were injected by tail vein with vector diluted in 0.2 ml of saline.

Balloon catheter delivery. A 4 French sheath was placed in the left femoral vein, a 7 French sheath in the right femoral vein, and a 4 French sheath in the right femoral artery by standard sterile percutaneous technique. A 20-gauge arterial catheter was placed in the opposite femoral artery for continuous blood pressure monitoring. Animals were given 50 mg/kg of heparin as soon as all the percutaneous lines were placed in the vessels. Through the two small sheaths, four French catheters were placed selectively in a hepatic vein and in the hepatic artery. The 6 × 2 cm balloon occlusion catheter (NuMED, Hopkinton, NY), was introduced into the right femoral vein sheath and positioned in the inferior vena cava with its tip just within the inferior vena cava-right arterial junction. The balloon was inflated with saline diluted contrast. Occlusion of hepatic venous outflow by the inflated balloon was confirmed by injecting contrast solution into the hepatic vein catheter while monitored on fluoroscopy; 20 ml/kg of saline was infused systemically before balloon inflation to minimize hypotension. Immediately after balloon inflation, HDAd, diluted in 5 ml saline, was injected at a rate of 0.5 ml/15 seconds through the percutaneously placed hepatic artery catheter, and the balloon was deflated 7.5 minutes after inflation. Phenylephrine was titrated as needed during the occlusion to further minimize the hypotension. At the end of the procedure, after removal of catheters and sheaths, 2–4 mg of protamine sulfate was given to neutralize the heparin. Hemostasis was achieved by manual compression over the puncture sites (30–60 minutes) after catheter/balloon/sheath removal. This minimally invasive procedure was well tolerated by all animals. However, inferior vena cava occlusion resulted in transient systemic hypotension caused by obstruction of venous return to the heart from the lower extremities. Although systemic infusion of 20 ml/kg of saline before balloon inflation and phenylephrine titration during the occlusion period were implemented to minimize hypotension, a ~50% decrease in systemic systolic blood pressure was nevertheless observed during the 7.5 minutes of occlusion. Hypotension was immediately and completely resolved upon balloon deflation with no apparent ill effects. At necropsy, a wide variety of organs (aorta, bone marrow, brain, heart, kidney, liver, lung, pancreas, skeletal muscle, spleen, stomach, testis, thymus, and trachea) were collected for H&E histology.

hFIX antigen enzyme-linked immunosorbent assay. Ninety-six–well polystyrene plates were coated with anti-hFIX in 0.2 mol/l NaHCO3 overnight at 4 ºC and then blocked with 6% bovine serum albumin/phosphate-buffered saline for 2 hours at room temperature. Dilutions of rhesus plasma were incubated at 4 ºC overnight and washed. A 1:1,000 dilution of the anti-rhesus IgG:horseradish peroxidase was incubated at room temperature for 3 hours for detection. The plate was developed with 100 µl of 1 mg/ml o-phenylene diamine in 0.1 mol/l sodium citrate buffer (pH 4.5) supplemented with 2 µl of 30% hydrogen peroxide per 10 ml solution and stopped by adding 100 µl of 1 mol/l hydrochloric acid. Absorbance was read at 495 nm. The lower limit of detection was 0.78 ng/ml.

Anti-hFIX antibody assay. Ninety-six–well polystyrene plates were coated with 1 µg hFIX (BeneFIX) in 50 µl of 0.2 mol/l NaHCO3 (pH 9.2) per well overnight at 4 ºC and then blocked with 6% bovine serum albumin/phosphate-buffered saline Tween 0.05% for 2 hours at room temperature. Dilutions of rhesus plasma were incubated at 4 ºC overnight. For detection, a 1:1,000 dilution of goat anti-hFIX polyclonal antibody conjugated with horseradish peroxidase was incubated for 2 hours at 37 ºC and the plate was developed with 100 µl of 1 mg/ml o-phenylene diamine in 0.1 mol/l sodium citrate buffer (pH 4.5) supplemented with 2 µl of 30% hydrogen peroxide per 10 ml solution and stopped by adding 100 µl of 1 mol/l hydrochloric acid. Anti-hFIX antibodies were defined as the greatest dilution at which the absorbance at 495 nm exceeded the identical dilution of baseline serum by >0.05 absorbance units. The limit of detection was 1:10 dilution of test plasma.

Bethesda inhibitor assay. A modification of the Bethesda assay28 was performed to screen for the presence of inhibitory antibodies to FIX. Plasma was heat-inactivated by incubation at 56 ºC for 30 minutes,29 serially diluted, then incubated with an equal volume of normal pooled human plasma (George King Bio-Medical, Overland Park, KS) at 37 ºC for 2 hours. The residual human FIX activity was determined with a one-stage clotting assay with human FIX-deficient plasma (George King Bio-Medical), using an ACL 300 (Instrumentation Laboratories, Lexington, MA) with reagents from the manufacturer. One Bethesda unit was defined as the amount of inhibitor that reduced the residual FIX activity to 50%. BU of >0.5 were considered significant. Values below 0.5 BU were considered to be within the error range of the assay.

Vector biodistribution. Total DNA was extracted from the tissues using the QIAamp DNA extraction kit (Qiagen, Valencia, CA) and quantitated by absorbance at 260 nm. Quantitative real-time PCR was performed using the LightCycler FastStart DNA Master SYBR Green I (Roche, Indianapolis, IN) in a total volume of 20 µl with 100 ng of template DNA and 1 mmol/l of each HDAd specific primers (5′-TCTGAATAATTTTGTGTTACTCATAGCGCG-3′ and 5′-CCCATAAGCTCCTTTTAACTTGTTAAAGTC-3′). Cycling conditions were 95 ºC for 10 minutes followed by 45 cycles at 95 ºC for 10 seconds, 60 ºC for 7 seconds, and 72 ºC for 20 seconds. Serial dilutions of a plasmid bearing the PCR target sequence were used as a control to determine the amounts of HDAd and results were analyzed with LightCycler software version 3.5 (Roche).

SUPPLEMENTARY MATERIAL Figure S1. Hematoxylin and eosin histology of rhesus liver. Table S1. Raw data for plasma hFIX levels, serum AST, ALT, total anti-hFIX antibodies, and Bethesda assays.

Acknowledgments

This study was supported by the National Institutes of Health R01 DK067324 (P.N.), R00 DK077447 (N.B.-P.), and the Texas Medical Center Digestive Diseases Center grant DK56338 (M.F.). We thank Allen Tower and Doug Villnave of NuMED for supplying the balloon catheters. We thank Federico Mingozzi, PhD and Kathrine High, MD for providing protocols for hFIX detection in rhesus plasma. We are grateful to Keith Hoots, MD for advice regarding BeneFIX infusion protocol and Angela Major for hematoxylin and eosin histology. C.M. is a paid consultant of NuMED Inc, Hopkinton, Inc. The other authors declared no conflict to interest.

Supplementary Material

Figure S1.

Hematoxylin and eosin histology of rhesus liver.

Click here for additional data file. (6MB, tif)
Table S1.

Raw data for plasma hFIX levels, serum AST, ALT, total anti-hFIX antibodies, and Bethesda assays.

Click here for additional data file. (380KB, doc)

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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.

Hematoxylin and eosin histology of rhesus liver.

Click here for additional data file. (6MB, tif)
Table S1.

Raw data for plasma hFIX levels, serum AST, ALT, total anti-hFIX antibodies, and Bethesda assays.

Click here for additional data file. (380KB, doc)

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