Gene transfer of wild-type apoA-I and apoA-I Milano reduce atherosclerosis to a similar extent

<p>Abstract</p> <p>Background</p> <p>The atheroprotective effects of systemic delivery of either apolipoprotein A-I (wtApoA-I) or the naturally occurring mutant ApoA-I Milano (ApoA-I_M) have been established in animal and human trials, but direct comparison studies evaluating the phenotype of ApoA-I or ApoAI-Milano knock-in mice or bone marrow transplantated animals with selectively ApoA-I or ApoAI-Milano transduced macrophages give conflicting results regarding the superior performance of either one. We therefore sought to compare the two forms of apoA-I using liver-directed somatic gene transfer in hypercholesterinemic mice – a model which is most adequately mimicking the clinical setting.</p> <p>Methods and results</p> <p>Vectors based on AAV serotype 8 (AAV2.8) encoding wtApoA-I, ApoA-I_Mor green fluorescent protein (GFP) as control were constructed. LDL receptor deficient mice were fed a Western Diet. After 8 weeks the AAV vectors were injected, and 6 weeks later atherosclerotic lesion size was determined by aortic <it>en face </it>analysis. Expression of wtApoA-I reduced progression of atherosclerosis by 32% compared with control (p = 0.02) and of ApoA-I_Mby 24% (p = 0.04). There was no significant difference between the two forms of ApoA-I in inhibiting atherosclerosis progression.</p> <p>Conclusion</p> <p>Liver-directed AAV2.8-mediated gene transfer of wtApoA-I and ApoA-I_Meach significantly reduced atherosclerosis progression to a similar extent.</p

Increased plasma zonulin in patients with sepsis

Introduction: Zonulin is a eukaryotic protein structurally similar to Vibrio cholerae’s zonula occludens toxin. It plays an important role in the opening of small intestine tight junctions. The loss of gut wall integrity during sepsis might be pivotal and has been described in various experimental as well as human studies. Increased levels of zonulin could be demonstrated in diseases associated with increased intestinal inflammation, such as celiac disease and type 1 diabetes. We therefore investigated the role of plasma levels of zonulin in patients with sepsis as a non-invasive marker of gut wall integrity. Materials and methods: Plasma level of zonulin was measured in 25 patients with sepsis, severe sepsis or septic shock according to ACCP/SCCM criteria at the first day of diagnosed sepsis. 18 non-septic post-surgical ICU-patients and 20 healthy volunteers served as control. Plasma levels were determined by using commercially available ELISA kit. Data are given as median and interquartile range (IQR). Results: Significantly higher plasma concentration of zonulin were found in the sepsis group: 6.61 ng/mL (IQR 3.51-9.46), as compared to the to the post-surgical control group: 3.40 ng/mL (IQR 2.14-5.70) (P = 0.025), as well as to the healthy group: 3.55 ng/mL (IQR 3.14-4.14) (P = 0.008). Conclusion: We were able demonstrate elevated levels of plasma zonulin, a potential marker of intestinal permeability in septic patients. Increased zonulin may serve as an additional mechanism for the observed increased intestinal permeability during sepsis and SIRS

AAV-mediated photoreceptor transduction of the pig cone-enriched retina

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases

Sequence Analysis of the Cloned mRNA Coding for Glyceraldehyde-3-Phosphate Dehydrogenase from Chicken Heart Muscle

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66279/1/j.1432-1033.1983.tb07239.x.pd

Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy

Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders

Placental Growth Factor Contributes to Micro-Vascular Abnormalization and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease

Efficacious and Safe Tissue-Selective Controlled Gene Therapy Approaches for the Cornea

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients

Low Adiponectin Levels Are an Independent Predictor of Mixed and Non-Calcified Coronary Atherosclerotic Plaques

Atherosclerosis is the primary cause of coronary artery disease (CAD). There is increasing recognition that lesion composition rather than size determines the acute complications of atherosclerotic disease. Low serum adiponectin levels were reported to be associated with coronary artery disease and future incidence of acute coronary syndrome (ACS). The impact of adiponectin on lesion composition still remains to be determined. We measured serum adiponectin levels in 303 patients with stable typical or atypical chest pain, who underwent dual-source multi-slice CT-angiography to exclude coronary artery stenosis. Atherosclerotic plaques were classified as calcified, mixed or non-calcified. In bivariate analysis adiponectin levels were inversely correlated with total coronary plaque burden (r = -0.21, p = 0.0004), mixed (r = -0.20, p = 0.0007) and non-calcified plaques (r = -0.18, p = 0.003). No correlation was seen with calcified plaques (r = -0.05, p = 0.39). In a fully adjusted multivariate model adiponectin levels remained predictive of total plaque burden (estimate: -0.036, 95%CI: -0.052 to -0.020, p<0.0001), mixed (estimate: -0.087, 95%CI: -0.132 to -0.042, p = 0.0001) and non-calcified plaques (estimate: -0.076, 95%CI: -0.115 to -0.038, p = 0.0001). Adiponectin levels were not associated with calcified plaques (estimate: -0.021, 95% CI: -0.043 to -0.001, p = 0.06). Since the majority of coronary plaques was calcified, adiponectin levels account for only 3% of the variability in total plaque number. In contrast, adiponectin accounts for approximately 20% of the variability in mixed and non-calcified plaque burden. Adiponectin levels predict mixed and non-calcified coronary atherosclerotic plaque burden. Low adiponectin levels may contribute to coronary plaque vulnerability and may thus play a role in the pathophysiology of ACS

Gene Therapy in a Humanized Mouse Model of Familial Hypercholesterolemia Leads to Marked Regression of Atherosclerosis

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr(-/-)Apobec1(-/-)) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr(-/-)Apobec1(-/-) mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10(9) genome copies/mouse. Whereas Ldlr(-/-)Apobec1(-/-) mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr(-/-)Apobec1(-/-) mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH