Gene Therapy (2015) 22, 8795; doi:10.1038/gt.2014.85; published online 18 September 2014

Autosomal dominant familial hypercholesterolemia (FH) is caused by mutations in the low-density lipoprotein receptor (LDLR).1 Homozygous FH patients present with massively elevated LDL cholesterol (LDL-C) and cardiovascular disease. They have severe atherosclerosis and die of ischemic heart disease usually in their third decade of life. The majority of homozygous and a substantial proportion of heterozygous patients are refractory to conventional pharmacological therapy. Therapeutic options for these resistant patients are limited to LDL apheresis, portacaval anastomosis or liver transplantation.2 Gene therapy has been explored as an alternative treatment. Liver is the main target organ for FH gene therapy because of its capacity to dispose excess cholesterol by diverting it into bile acids; it is also accessible to gene delivery via the intravenous (i.v.) route or the hepatic artery. A number of studies have shown that hepatic reconstitution of LDLR expression ex vivo can reverse hypercholesterolemia, including promising results in a rabbit model of FH. 3 In the only clinical gene therapy trial for FH to date, Grossman et al.4, 5 isolated hepatocytes from FH patients, transduced them ex vivo with retroviral vector expressing LDLR and reimplanted them into the liver of the patients. Only marginal therapeutic benefit was achieved in this study. It was difficult to determine whether the reduction in LDL-C level was the direct result of the gene transfer or other factors were involved. Plasma LDL level is determined by LDL production and removal. For example, the decline of LDL-C after portacaval anastomosis is caused by a decreased secretion of very-low-density lipoprotein, a precursor of LDL, not by an enhanced LDL removal.6 In this clinical trial, LDL turnover was not measured, which led to the comment a modest 17% fall in plasma cholesterol after 25% hepatectomy and re-infusion of hepatocytes infected with a retrovirus might have been due to either diminished lipoprotein production or to enhanced activity of the patients own receptor.7 The focus has shifted to in vivo gene therapy thereafter. Helper-dependent adenoviral vector (HDAd) is devoid of all viral protein genes and has shown considerable promise for liver-directed gene transfer with long-term transgene expression, which lasted a lifetime in mice.8 In a previous study in LDLR/ mice, we showed that a single injection of HDAd expressing monkey LDLR reduced plasma cholesterol over 2 years and attenuated atherosclerotic lesion progression. 9 We also demonstrated that LDLR gene therapy induces the regression of established atherosclerosis in LDLR/ mice.10 Despite promising results of gene therapy in small animal models, its efficacy in large animal models has not been tested; there are important differences in physiology and in immune responses between rodents and humans. This issue is particularly relevant in gene therapy for lipid disorders.11

A nonhuman primate model of FH has been described in rhesus monkeys,12, 13 which carried a heterozygous nonsense mutation involving codon Trp283 14 of the LDLR. Extensive cross-breeding of the affected monkeys failed to yield any homozygotes, indicating that the mutation may be linked to a lethal mutation. With the availability only of the heterozygous (LDLR+/) rhesus monkey, we will be modeling heterozygous FH in humans, a relatively common genetic disorder that affects about 1 in 500 people in most ethnic groups.15 Heterozygous LDLR-deficient monkeys displayed elevated plasma cholesterol (5.176.47mmoll1 or 200250mgdl1) compared with unaffected monkeys (2.593.36mmoll1 or 100130mgdl1); the plasma cholesterol level further increased to 12.9320.69mmoll1 (500800mgdl1) when the animals were fed a high-cholesterol diet.16 In this study, we tested the efficacy of HDAd-based monkey LDLR gene therapy in high-cholesterol diet-fed LDLR+/ rhesus monkeys. We compared the effect of i.v. injection of HDAd-LDLR with that of a balloon catheter-based procedure developed by Brunetti-Pierri et al. 17 We found that a single i.v. injection of HDAd-LDLR into LDLR+/ monkeys produced a >50% lowering of plasma cholesterol that lasted about a month. We next tested a modified percutaneous catheter-based gene delivery strategy also developed by Brunetti-Pierri et al. 18 In this refinement, the HDAd-LDLR was injected directly into the hepatic artery in the presence of increased intrahepatic pressure induced by transient blockage of hepatic venous drainage by a balloon catheter positioned in the inferior vena cava (IVC). The optimized gene delivery strategy was highly efficacious in reducing the vector dose while substantially prolonging the therapeutic hypocholesterolemic response to the treatment regimen.

We treated four LDLR+/ monkeys as study subjects with a single i.v. injection of escalating doses of HDAd-LDLR. 9 We first treated monkey #8796 with 20ml of saline and found no significant changes in plasma cholesterol levels after treatment (Figure 1). As expected, we also failed to detect any change in plasma cholesterol when we treated another LDLR+/ monkey #9908 with an empty vector HDAd-0 (0.8 1012 viral particles (vp)kg). We next injected i.v. HDAd-LDLR into a third LDLR+/ monkey #7139 at a dose of 1.1 1012vpkg1, an HDAd dose that is 10-fold higher than the dose of HDAd--fetoprotein that stimulated significant elevation in -fetoprotein secretion in serum in baboons,17 and again failed to observe any change in plasma cholesterol level. We then treated a fourth monkey #13090 at an even higher i.v. dose of 5 1012vpkg1 of HDAd-LDLR. The treatment was well tolerated by the monkey and led to a 60% reduction in plasma cholesterol from a baseline of 14.95mmoll1 (578mgdl1) to 5.90mmoll1 (229mgdl1) on day 7. The plasma cholesterol lowering persisted until day 21, when it went up to 10.70mmoll1 (413mgdl1) on day 28, and toward pre-treatment levels on day 42. These results indicate that a dose higher than 1.1 1012vpkg1 was needed to reverse hypercholesterolemia in LDLR+/ monkeys, and a dose of 5 1012vpkg1 significantly restored normal plasma cholesterol in a heterozygous FH monkey, an effect that lasted for about a month. We next treated a fifth monkey #11226 with an even higher dose of 8.4 1012vpkg1, which was modestly below a dose that had previously proven to be lethal, 19 and observed severe acute toxicity and lethality within a day of treatment. The clinical picture and necropsy revealed hemorrhagic shock syndrome likely resulting from the high dose of HDAd vector used.

Efficacy of intravenous injection of HDAd expressing monkey LDLR in heterozygous LDLR-deficient rhesus monkeys. Four heterozygous LDLR-deficient monkeys were treated with a single intravenous injection of saline (#8796), empty vector at a dose of 0.8 1012vpkg1 (#9908) or HDAd-LDLR at a dose of 1.1 1012vpkg1 (#7139) or 5 1012vpkg1 (#13090). Baseline cholesterol levels were 18.0mmoll1 (696mgdl1) in monkey #8796, 9.5mmoll1 (368mgdl1) in monkey #9908, 8.0mmoll1 (308mgdl1) in monkey #7139 and 15.0mmoll1 (578mgdl1) in monkey #13090. The broken line shows pre-treatment cholesterol levels.

To improve on i.v. vector injection as a delivery method, Brunetti-Perri et al. developed a protocol 17, 18 to deliver the vector via an intrahepatic arterial catheter. Simultaneously, under fluoroscopic guidance, they inserted a balloon catheter into the IVC via the femoral vein and positioned it over the hepatic venous outflow (Figure 2a). Intrahepatic arterial HDAd injection when the balloon was inflated led to a 10-fold increase in efficiency in transgene expression ( Figures 2b and c). The IVC occlusion was also monitored by the venous pressure (Figure 2d). We performed the same procedure in rhesus monkeys and injected the HDAd vector (2ml) within a minute via a hepatic artery catheter immediately after the balloon was inflated.

Balloon catheter-based hepatic artery injection. (a) Schematic diagram of hepatic artery injection. Liver circulation is isolated by inserting a balloon catheter via the femoral vein and placing it in the IVC. A second intra-arterial catheter is inserted into the hepatic artery through the contralateral femoral artery. The placement of the catheter is visualized using fluoroscopy. Once occlusion of the hepatic circulation has been established via the balloon catheter in the IVC, the vector is injected via the arterial catheter. The occlusion is confirmed by monitoring hepatic venous pressure through the third catheter inserted into the femoral vein. BD, bile duct; HA, hepatic artery; HV, hepatic vein; PV, portal vein. (b) Fluoroscopy image to confirm the position of a balloon catheter. (c) Fluoroscopy after the balloon inflated. Contrast reagent was injected to confirm that the catheter was placed at the IVC. (d) Venous pressure. Occlusion was monitored by venous pressure.

The monkeys used for this procedure are summarized in Table 1. We first performed the procedure in a chow-fed (Purina LabDiet5LEO, St Louis, MO, USA) normal LDLR+/+ (#19254) and a heterozygous LDLR+/ (#19499) monkey. The injection was done immediately after the balloon was deflated but while hepatic venous pressure remained high. As reported previously, 17,18 systemic blood pressure fell significantly when the balloon was inflated. We found that serum interleukin (IL)-6 level increased 30min after injection and peaked at 2h ( Figure 3a) but decreased to non-detectable levels by 72h. The procedure also led to transient and inconsistent changes in plasma liver enzymes ( Figures 3b and c). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels peaked at about 24h; the increase was mild and resolved by day 5. Plasma total cholesterol levels in the LDLR+/ (#19499) monkey decreased from a baseline of 5.70mmoll1 (219mgdl1) to 3.90mmoll1 (150mgdl1) within 24h. It gradually went back up over the next few days returning to baseline by day 5. The plasma cholesterol level did not change in the non-FH (LDLR+/+) (#19254) monkey ( Figure 3d).

Acute toxicity measurements associated with balloon catheter-based hepatic artery injection. One normal LDLR+/+ (#19254) and one heterozygous LDLR+/ (#19499) monkeys on normal chow were treated by an injection of saline and a complete blood test and IL-6 measurement were performed. (a) Plasma IL-6 levels. (b) Serum ALT levels. (c) Serum aspartate aminotransferase (AST) levels. (d) Plasma cholesterol levels.

We next fed monkeys with a rhesus Western diet (Texas Biomedical Research Institute, San Antonio, TX, USA) for 7 weeks before treatment and were kept on the diet afterward. We injected HDAd-LDLR (2 1012vpkg1) into four monkeys immediately after the balloon was deflated. The plasma cholesterol did not change in two wild-type LDLR+/+ monkeys (#19360 and #21588) suggesting that the gene delivery does not have an effect on the cholesterol dynamics in monkeys that express normal amounts of LDLR. Of the two heterozygous LDLR+/ monkeys, one (#19251) showed no change in plasma cholesterol ( Figure 4a, green line), whereas another LDLR+/ monkey (#19498) exhibited a 57% drop in plasma cholesterol level from 8.15mmoll1 (315mgdl1) to 3.25mmoll1 (126mgdl1) at day 7 ( Figure 4a, red line). So there was a heterogeneous response in heterozygous FH monkeys treated at this dose of HDAd-LDLR. The cholesterol-lowering effect of HDAd-LDLR in the LDLR+/ (#19498) monkey that responded to the treatment was sustained for about 100 days. The plasma-lowering effect reached its nadir 7 days, and stayed at or near the nadir for another 3 weeks. Afterward, it gradually rose to 5.09mmoll1 (197mgdl1) at day 78, and then to above the pre-treatment level (9.30mmoll1 or 361mgdl1) by day 105 ( Figure 4a, red line). The two wild-type LDLR+/+ monkeys maintained normal serum ALT throughout the observation period of 120 days. The LDLR+/ monkey (#19251) that did not show a hypocholesterolemic response also maintained normal ALT levels for 67 days, end of the observation period for this monkey. In contrast, the serum ALT of the LDLR+/ monkey (#19498) that showed a hypocholesterolemic response maintained a normal ALT level during the first 3 weeks of treatment when the plasma cholesterol showed an excellent response ( Figure 4a, red line). ALT began to edge above normal to 70Ul1 on day 36, and continued to go up to peak at 144Ul1 on day 72, before it started trending down, eventually returning to normal on day 120 ( Figure 4b, red line). It is noteworthy that this monkey that had responded to the treatment developed liver enzyme elevation late, and the delayed increase in serum ALT coincided with the onset of loss of the cholesterol-lowering effect of the treatment. Although the significance of the timing is unclear, we note that a similar pattern is evident in an experiment involving another LDLR+/ monkey (#19269, see below).

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