B. De Geest

The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer

Fenestrae allow the passage of gene transfer vectors from the sinusoidal lumen to the surface of hepatocytes. We have previously shown that the diameter of fenestrae correlates with species and strain differences of transgene expression following intravenous adenoviral transfer. In the current study, we demonstrate that the diameter of fenestrae in humans without liver pathology is 107±1.5 nm. This is similar to the previously reported diameter in New Zealand White (NZW) rabbits (103±1.3 nm) and is significantly smaller than in C57BL/6 mice (141±5.4 nm) and Sprague–Dawley rats (161±2.7 nm). We show that the diameter of fenestrae in one male NZW rabbit and its offspring characterized by a more than 50-fold increase of transgene expression after adenoviral gene transfer is significantly (113±1.5 nm; P<0.001) larger than in control NZW rabbits. In vitro filtration experiments using polycarbonate filters with increasing pore sizes demonstrate that a relatively small increment of the diameter of pores potently enhances passage of adenoviral vectors, consistent with in vivo data. In conclusion, the small diameter of fenestrae in humans is likely to be a major obstacle for hepatocyte transduction by adenoviral vectors.

The size of sinusoidal fenestrae is a critical determinant of hepatocyte transduction after adenoviral gene transfer

The hepatotropism and intrahepatic distribution of adenoviral vectors may be species dependent. Hepatocyte transduction was evaluated in three rabbit strains after transfer with E1E3E4-deleted adenoviral vectors containing a hepatocyte specific α1-antitrypsin promoter-driven expression cassette (AdAT4). Intravenous administration of 4 × 1012 particles/kg of AdAT4 induced human apo A-I levels above 40 mg/dl in Dutch Belt, but below 1 mg/dl in New Zealand White and Fauve de Bourgogne rabbits. Diameters of sinusoidal fenestrae were significantly (P=0.0014) larger in Dutch Belt (124±3.4 nm) than in New Zealand White (108±1.3 nm) and Fauve de Bourgogne (105±2.6 nm) rabbits, suggesting that a smaller size constitutes a barrier for hepatocyte transduction. Indeed, intraportal transfer preceded by intraportal injection of sodium decanoate, which increases the diameter of sinusoidal fenestrae to 123±3.4 nm (P<0.01) in New Zealand White rabbits, increased human apo A-I levels 32- and 120-fold in New Zealand White and Fauve de Bourgogne rabbits, respectively, but did not affect expression in Dutch Belt rabbits. In conclusion, size of sinusoidal fenestrae appears to be a critical determinant of hepatocyte transduction after adenoviral transfer.

Species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the size of endothelial fenestrae

Sinusoidal fenestrae may restrict the transport of gene transfer vectors according to their size. Using Vitrobot technology and cryo-electron microscopy, we show that the diameter of human adenoviral serotype 5 vectors is 93 nm with protruding fibers of 30 nm. Thus, a diameter of fenestrae of 150 nm or more is likely to be sufficient for passage of vectors from the sinusoidal lumen to the space of Disse and subsequent uptake of vectors in hepatocytes. The average diameter of fenestrae in New Zealand White rabbits (103±1.3 nm) was 1.4-fold (P<0.0001) lower than in C57BL/6 mice (141±5.4 nm). The percentage of sinusoidal fenestrae with a diameter larger than 150 nm was 10-fold (P<0.01) lower in rabbits (3.2±0.24%) than in C57BL/6 mice (32±5%), and this resulted in 8.8-fold (P=0.01) lower transgene DNA levels in hepatocytes in rabbits after adenoviral transfer. Injection of N-acetylcysteine combined with transient liver ischemia preceding intraportal transfer in rabbits increased the percentage of sinusoidal fenestrae above 150 nm 2.0-fold (P<0.001) and increased transgene DNA levels in hepatocytes 6.6-fold (P<0.05). In conclusion, species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the diameter of fenestrae.

Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector

Gene transfer with ‘gutted’ vectors is associated with persistent transgene expression and absence of hepatotoxicity, but the requirement of helper viruses hampers efficient production and leads to contamination of viral batches with these helper-viruses. In the present study, gene transfer with a helper-virus independent E1/E3/E4-deleted adenoviral vector induced persistent expression of human apo A-I (200 ± 16 mg/dl at day 35, 190 ± 15 mg/dl at 4 months, 170 ± 16 mg/dl at 6 months) and stable transgene DNA levels (3.5 ± 0.60 at day 35, 3.3 ± 0.39 at 4 months, 3.1 ± 0.47 mg/dl at 6 months) in C57BL/6 mice in the absence of significant toxicity. The vector contained the 1.5 kb human α1-antitrypsin promoter in front of the genomic human apo A-I sequence and four copies of the human apo E enhancer (hAAT.gA-I.4xapoE) and was deleted in E1, E3 and E4. Reintroduction of E4 ORF 3 and E4 ORF 4 in the viral backbone caused a more than four-fold decline of transgene DNA between day 35 and 4 months after transfer both in wild-type and in C57BL/6 SCID and C57BL/6 Rag-1−/− mice, indicating that the effect of E4 ORF 3 and E4 ORF 4 is independent of a cellular immune response against viral epitopes. Co-injection of an E1-deleted vector containing no expression cassette and the E1/E3/E4-deleted vector containing the hAAT.gA-I.4xapoE expression cassette indicated that E4 gene products destabilize transgene DNA in trans. Gene transfer with an E1/E3/E4-deleted vector containing only E4 ORF 3 and the hAAT.gA-I.4xapoE expression cassette was associated with transgene DNA decline, but not with hepatotoxicity, indicating that transgene DNA persistence and hepatotoxicity are dissociated processes. After transfer with E1/E3/E4-deleted vectors containing expression cassettes with a different promoter or a different position of the apo E enhancers, transgene DNA levels were less stable than after transfer with the vector containing hAAT.gA-I.4xapoE, indicating that the expression cassette is an important determinant of episomal stability. In conclusion, gene transfer with an E1/E3/E4-deleted vector containing the hAAT.gA-I.4xapoE expression cassette induces persistent expression of human apo A-I in the absence of hepatotoxicity. Transgene DNA turnover is independent of an adaptive cellular immune response against viral epitopes and of hepatotoxicity. E1/E3/E4-deleted vectors containing transgenes under control of the hAAT promoter in combination with four copies of the human apo E enhancer may be suitable for hepatocyte-specific overexpression of transgenes after gene transfer.

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

Hepatocytes are a key target for treatment of inborn errors of metabolism, dyslipidemia and coagulation disorders. The development of potent expression cassettes is a critical target to improve the therapeutic index of gene transfer vectors. Here we evaluated 22 hepatocyte-specific expression cassettes containing a human apo A-I transgene following hydrodynamic transfer of plasmids or adenoviral transfer with E1E3E4-deleted vectors in C57BL/6 mice. The DC172 promoter consisting of a 890 bp human α1-antitrypsin promoter and two copies of the 160 bp α1-microglobulin enhancer results in superior expression levels compared to constructs containing the 1.5 kb human α1-antitrypsin promoter, the 790 bp synthetic liver-specific promoter or the DC190 promoter containing a 520 bp human albumin promoter and two copies of the 99 bp prothrombin enhancer. The most potent expression cassette consists of the DC172 promoter upstream of the transgene and two copies of the hepatic control region-1. Minicircles containing this expression cassette induce persistent physiological human apo A-I or human factor IX levels after hydrodynamic transfer. In conclusion, in this comparative study of 22 hepatocyte-specific expression cassettes, the DC172 promoter in combination with two copies of the hepatic control region-1 induces the highest expression levels following hydrodynamic and adenoviral transfer.

Sustained expression of human apo A-I following adenoviral gene transfer in mice

Elevation of HDL cholesterol, following adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. The choice of promoter may have significant impact on persistence of transgene expression. Human apo A-I expression was compared after adenoviral gene transfer with a cytomegalovirus promoter (CMV) driven construct (AdCMV/A-I.gA-I) and with a construct (AdA-I.gA-I.4xapoE) containing the endogenous 256 bp apo A-I promoter (A-I), the genomic human apo A-I DNA (gA-I) and 4 human apo E enhancers (4xapoE) in three different mouse strains: C57BL/6, Balb/c and Fvb. After gene transfer with 5 × 108 p.f.u. of AdCMV/A-I.gA-I, human apo A-I expression was observed for 35 days in C57BL/6 mice, but declined below 1 mg/dl within 14 days both in Balb/c and Fvb mice, due to a strong humoral immune response against human apo A-I. In contrast, after transfer with AdA-I.gA-I.4xapoE, human apo A-I expression persisted for 6 months in all three strains and no antibodies against human apo A-I occurred in Fvb or Balb/c mice. Human apo A-I transgene DNA level 35 days after transfer with AdA-I.gA-I.4xapoE was 4.6- to 5.5-fold higher than with AdCMV/A-I.gA-I. CMV promoter attenuation occurred in all three strains, but promoter attenuation was not observed in any strain after transfer with AdA-I.gA-I.4xapoE. In conclusion, gene transfer with AdA-I.gA-I.4xapoE is associated with absence of an immune response against human apo A-I, improved transgene DNA persistence and absence of promoter shut-off, resulting in human apo A-I expression for up to 6 months in three different mouse strains. Possibly, the absence of human apo A-I expression in antigen-presenting cells with the liver-specific apo A-I promoter containing construct abrogated the immune response against human apo A-I in Balb/c and Fvb mice.

Apolipoprotein A-I and lecithin:cholesterol acyltransferase transfer induce cholesterol unloading in complex atherosclerotic lesions

Plasma levels of high-density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo), apo A-I, are inversely correlated with the incidence of ischemic cardiovascular diseases. Reverse cholesterol transport is likely the main mechanism underlying the atheroprotective effects of HDL. Here, we investigated whether increased HDL cholesterol following hepatocyte-directed adenoviral rabbit apo A-I (AdrA-I) or rabbit lecithin-cholesterol acyltransferase (LCAT) (AdrLCAT) transfer may induce cholesterol unloading in complex atherosclerotic lesions in heterozygous low-density lipoprotein receptor-deficient rabbits fed a 0.15% cholesterol diet for 420 days before and for 120 days after transfer. HDL cholesterol levels increased 2.0-fold (P<0.001) and 1.9-fold (P<0.001) in the 120 days after transfer with AdrA-I and AdrLCAT, respectively, compared to levels just before transfer whereas non-HDL cholesterol remained unchanged. Increased HDL cholesterol following AdrA-I and AdrLCAT transfer resulted in a 31% (P<0.05) reduction of the intima/media ratio in comparison with the control progression group. Compared to the baseline group killed after 420 days of cholesterol diet, AdrA-I and AdrLCAT transfer reduced the percentage of Oil Red O area 1.6-fold (P<0.001) and 1.4-fold (P<0.001), respectively. In conclusion, increased HDL cholesterol after AdrA-I and AdrLCAT transfer inhibits progression of atherosclerosis and induces cholesterol unloading in complex lesions in rabbits.

The impact of antigen expression in antigen-presenting cells on humoral immune responses against the transgene product

Treatment of genetic diseases by gene therapy is hampered by immune responses against the transgene product. Promoter choice has been shown to be an important parameter of the presence or absence of antibodies against the transgene product after gene transfer. Here, the generality of some of these observations was tested by comparing different murine strains and different transgene products. We show immunological unresponsiveness for human apolipoprotein (apo) A-I in six murine strains after transfer with E1E3E4-deleted adenoviral vectors containing hepatocyte-specific expression cassettes. However, differences in the induction of a humoral immune response against human apo A-I after gene transfer with vectors driven by the major histocompatibility complex class II Eβ promoter and the ubiquitously active cytomegalovirus promoter were not consistent in these six murine strains. Furthermore, use of a potent hepatocyte-specific expression cassette did not prevent a humoral immune response against human plasminogen in C57BL/6 mice. In contrast, human microplasminogen transfer resulted in stable expression in the absence of an immune response against the transgene product. Taken together, the molecular design of strategies to abrogate or induce an immune response against the transgene product may be hampered by the multitude of parameters affecting the outcome, thus limiting the external validity of results.

Bilateral blurred vision and low HDL in a 69-year-old man

A 69-year-old man presented with progressive blurred vision and corneal clouding (Fig. 1A, B). No other ocular abnormalities were present. There was no history of cardiovascular problems. On slit-lamp examination, corneal clouding was seen (Fig. 1C: left eye). Lipid profile showed total cholesterol 186 mg/dl (normal e190), low-density lipoproteins 132 mg/dl (e115), high-density lipoproteins 9 mg/dl (Q40) and triglycerides 236 mg/dl (e180). Esterified cholesterol fraction in plasma of the patient (0.59) was decreased compared to controls (0.73), with the most marked decline observed in HDL particles (0.54 versus 0.81). The diagnosis of fish-eye disease (FED, OMIM #136120), caused by a mutation in the lecithin:cholesterol acyltransferase (LCAT) gene (Carlson and Holmquist 1985) was made, based on the presentation, the lipid profile and homozygosity for a known point mutation (440C>T, J Inherit Metab Dis DOI 10.1007/s10545-008-0853-6

4 REGRESSION AND STABILIZATION OF ADVANCED MURINE ATHEROSCLEROTIC LESIONS: A COMPARISON OF LDL LOWERING AND HDL RAISING GENE TRANSFER STRATEGIES

Both reductions in atherogenic lipoproteins and increases in high-density lipoprotein (HDL) levels may affect atherosclerosis regression. Here, the relative potential of low-density lipoprotein (LDL) lowering and HDL raising gene transfer strategies to induce regression of complex murine atherosclerotic lesions was directly compared. Male C57BL/6 LDL receptor (LDLr)−/− mice were fed an atherogenic diet (1.25% cholesterol and 10% coconut oil) to induce advanced atherosclerotic lesions. A baseline group was killed after 6 months and remaining mice were randomized into a control progression (Adnull or saline), an apolipoprotein (apo) A-I (AdA-I), an LDLr (AdLDLr), or a combined apo A-I/LDLr (AdA-I/AdLDLr) adenoviral gene transfer group and followed-up for another 12 weeks with continuation of the atherogenic diet. Gene transfer with AdLDLr decreased non-HDL cholesterol levels persistently by 95% (p < 0.001) compared with baseline. This drastic reduction of non-HDL cholesterol levels induced lesion regression by 28% (p < 0.001) in the aortic root and by 25% (p < 0.05) in the brachiocephalic artery at 12 weeks after transfer. Change in lesion size was accompanied by enhanced plaque stability, as evidenced by increased collagen content, reduced lesional macrophage content, a drastic reduction of necrotic core area, and decreased expression of inflammatory genes. Elevated HDL cholesterol following AdA-I transfer increased collagen content in lesions, but did not induce regression. Apo A-I gene transfer on top of AdLDLr transfer resulted in additive effects, particularly on inflammatory gene expression. In conclusion, drastic lipid lowering induced by a powerful gene transfer strategy leads to pronounced regression and stabilization of advanced murine atherosclerosis.