Joke Lievens

Human Apolipoprotein A-I Gene Transfer Reduces the Development of Experimental Diabetic Cardiomyopathy

Background— The hallmarks of diabetic cardiomyopathy are cardiac oxidative stress, intramyocardial inflammation, cardiac fibrosis, and cardiac apoptosis. Given the antioxidative, antiinflammatory, and antiapoptotic potential of high-density lipoprotein (HDL), we evaluated the hypothesis that increased HDL via gene transfer (GT) with human apolipoprotein (apo) A-I, the principal apolipoprotein of HDL, may reduce the development of diabetic cardiomyopathy. Methods and Results— Intravenous GT with 3×1012 particles/kg of the E1E3E4-deleted vector Ad.hapoA-I, expressing human apoA-I, or Ad.Null, containing no expression cassette, was performed 5 days after streptozotocin (STZ) injection. Six weeks after apoA-I GT, HDL cholesterol levels were increased by 1.6-fold (P<0.001) compared with diabetic controls injected with the Ad.Null vector (STZ-Ad.Null). ApoA-I GT and HDL improved LV contractility in vivo and cardiomyocyte contractility ex vivo, respectively. Moreover, apoA-I GT was associated with decreased cardiac oxidative stress and reduced intramyocardial inflammation. In addition, compared with STZ-Ad.Null rats, cardiac fibrosis and glycogen accumulation were reduced by 1.7-fold and 3.1-fold, respectively (P<0.05). Caspase 3/7 activity was decreased 1.2-fold (P<0.05), and the ratio of Bcl-2 to Bax was upregulated 1.9-fold (P<0.005), translating to 2.1-fold (P<0.05) reduced total number of cardiomyocytes with apoptotic characteristics and 3.0-fold (P<0.005) reduced damaged endothelial cells compared with STZ-Ad.Null rats. HDL supplementation ex vivo reduced hyperglycemia-induced cardiomyocyte apoptosis by 3.4-fold (P<0.005). The apoA-I GT-mediated protection was associated with a 1.6-, 1.6-, and 2.4-fold induction of diabetes-downregulated phospho to Akt, endothelial nitric oxide synthase, and glycogen synthase kinase ratio, respectively (P<0.005). Conclusion— ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

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.

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.

Wild-type apo A-I and apo A-I Milano gene transfer reduce native and transplant arteriosclerosis to a similar extent

Apolipoprotein (apo) A-IMilano is an apo A-I mutant characterized by a cysteine for arginine substitution at position 173. Apo A-IMilano carriers have much less atherosclerosis than expected from their low plasma high-density lipoprotein cholesterol levels, suggesting that this mutant may have superior atheroprotective properties. Here, we compare the effect of hepatocyte-directed gene transfer of wild-type human apo A-I and human apo A-IMilano on endothelial progenitor cell (EPC) biology and on the progression of native atherosclerosis and allograft vasculopathy in C57BL/6 apo E−/− mice. Human apo A-I and apo A-IMilano transfer resulted in an equivalent increase of EPC number and function as well as EPC incorporation and endothelial regeneration in allografts and inhibited the progression of native atherosclerosis and allograft vasculopathy to a similar extent. In conclusion, the current head-to-head comparison indicates that human apo A-IMilano transfer is not superior compared to wild-type human apo A-I transfer.

Hepatocyte-specific ABCA1 transfer increases HDL cholesterol but impairs HDL function and accelerates atherosclerosis

AIMS The ATP-binding cassette transporter A1 (ABCA1) lipidates apolipoprotein (apo) A-I. The hypothesis that hepatocyte-specific ABCA1 overexpression results in high-density lipoprotein (HDL) dysfunction was evaluated by comparing the effects of murine ABCA1 (AdABCA1) and human apo A-I (AdA-I) transfer on lipoprotein profile, HDL function, and progression of atherosclerosis. METHODS AND RESULTS Gene transfer in male and female C57BL/6 apo E(-/-) mice was performed at the age of 3 months with E1E3E4-deleted adenoviral vectors containing hepatocyte-specific expression cassettes. Atherosclerosis was quantified at baseline and 56 days later in AdABCA1, AdA-I, and control mice. HDL cholesterol after AdA-I transfer was 1.7-fold (P < 0.001) and 1.8-fold (P < 0.001) higher in male and female mice, respectively, and potently inhibited atherosclerosis progression compared with respective controls. Notwithstanding a 1.4-fold (P < 0.01) and a 1.7-fold (P < 0.01) increase of HDL cholesterol in male and female mice, respectively, after AdABCA1 transfer, the intima was 2.2-fold (P < 0.001) larger in male and 1.3-fold (P = NS) larger in female mice compared with respective controls. HDL isolated from control and AdA-I mice but not from AdABCA1 mice enhanced endothelial progenitor cell (EPC) migration in vitro and reduced endothelial cell death in vitro after serum and growth factor withdrawal. Scavenger receptor class B type I (SR-BI) protein level in the liver was significantly lower in AdABCA1 mice than in control and AdA-I mice. CONCLUSION Hepatocyte-specific ABCA1 transfer decreases SR-BI protein level in the liver and abrogates beneficial effects of HDL on EPCs and endothelial cells. Decreased HDL function may underlie accelerated atherosclerosis in AdABCA1 apo E(-/-)mice.

Species Differences in Hepatocyte-Directed Gene Transfer: Implications for Clinical Translation

The liver is a key organ in numerous metabolic pathways, in cholesterol metabolism, and in production of coagulation factors. Therefore, gene transfer to hepatocytes has been extensively pursued. There are numerous biological parameters that may affect the outcome of hepatocyte-directed gene transfer. Species or strain variation of any of these multiple determinants hinders the process of clinical translation. This review specifically focuses on functional aspects of liver histology that are pertinent for gene transfer to parenchymal liver cells. We discuss the reticulo-endothelial cells of the liver and the spleen, and their impact on innate immune responses after adenoviral transfer and on vector clearance. Liver sinusoidal endothelial cells contain pores, called fenestrae, and have no basal lamina. Fenestrae are clustered in sieve plates and may provide direct access for circulating gene transfer vectors to the space of Disse, in which microvilli of parenchymal liver cells protrude. We present multiple lines of evidence that the species differences in the diameter of sinusoidal fenestrae are a critical determinant of transgene expression after adenoviral transfer. The small diameter of fenestrae in humans should be considered in any rational design of gene transfer technologies for hepatocyte-directed transfer. Hydrodynamic gene transfer is highly successful in rodents. The significantly lower efficacy in higher species may also partially be due to species differences in liver architecture. Finally, we discuss species differences in adaptive immune responses against the transgene product that may constitute one of the most significant hurdles for clinical translation.

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.

The relative atherogenicity of VLDL and LDL is dependent on the topographic site

To evaluate whether the relative atherogenicity of VLDL and LDL is dependent on the topographic site, atherosclerosis was compared at four topographic sites in homozygous LDL receptor (LDLr)-deficient rabbits fed normal chow and in heterozygous LDLr-deficient rabbits with the same genetic background fed a 0.15% cholesterol diet to match cholesterol levels. VLDL cholesterol was significantly higher and LDL cholesterol significantly lower in LDLr+/− diet rabbits compared with LDLr−/− rabbits. Intimal area in the ascending thoracic aorta and in the abdominal aorta at the level of the renal arteries was 1.4-fold (P < 0.05) and 1.5-fold (P < 0.05) higher, respectively, in LDLr−/− rabbits than in LDLr+/− diet rabbits, whereas no significant difference occurred in the descending thoracic aorta and in the abdominal aorta just above the bifurcation. Differences remained statistically significant after adjustment for plasma cholesterol, triglycerides, and sex. Compared with LDLr+/− diet rabbits, higher intimal lipoprotein lipase (LPL) and apolipoprotein (apo) B levels were observed in LDLr−/− rabbits only at the level of the descending thoracic aorta. Intimal apo E levels in LDLr−/− rabbits were significantly lower in sites with a larger intima than in LDLr+/− diet rabbits. In conclusion, the relative atherogenicity of VLDL and LDL is dependent on the topographic site.

636. Long Term Elevation of HDL Cholesterol Induced by Rabbit apo A-I or Rabbit LCAT Gene Transfer Inhibits Media Thinning in a Rabbit Model of Advanced Atherosclerosis

Background: High density lipoprotein (HDL) cholesterol is the most powerful predictor of ischemic cardiovascular diseases in humans. Dyslipidemic rabbits are a more appropriate model of human dyslipidemia and atherosclerosis than mice. In contrast to mice and similar as humans, rabbits express cholesteryl ester transfer protein and rabbit liver does not edit apolipoprotein (apo) B mRNA. The purpose of this study was to evaluate the effect of rabbit apo A-I and rabbit lecithin:cholesterol acyltransferase (LCAT) transfer on the lipoprotein profile and on atherosclerosis in rabbits.