In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer.

Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer.

Incidence and risk factors of coronary artery disease in patients on chronic hemodialysis.

Aim of the study was to estimate the incidence of coronary heart disease (CAD) in patients (pts) with end stage renal disease (ESRD) maintained on chronic hemodialysis (HD) and its association with the presence of predisposing factors. The study included 171 dialysis pts (107 male (M) and 64 female (F)). Mean age of pts was 67+/-13 years, mean time on dialysis 52.7+/-44 months and Body Mass Index (BMI) 25.9+/-3.7 kg/m2. Fifty pts (29.2%) were clinically diagnosed with CAD. The diagnosis was established by coronary angiography in 24 (48%) and in 26 by combined dipyridamole-exercise thallium imaging (52%). Pts' data in association with the development of CAD that were recorded included age, sex, smoking habits, hypertension, obesity, the presence of diabetes mellitus (DM), hyperlipidemia, anemia, low albumin levels, secondary hyperparathyroidism (SHP), the presence of chronic inflammation, as evidenced by the presence of elevated levels of CRP and hyperhomocysteinemia. There was a statistically significant association of increasing age and CAD (p<0.0001). Relative risk (RR) was significantly increased i) in male pts compared to female pts (RR: 8.56, p<0.001), ii) in anemic pts compared to pts with hemoglobin levels< or =11 g/dL (RR: 8.26, p<0.0001), iii) in obese pts compared to pts with BMI < or =30 (RR: 5.09, p<0.005) and iv) in pts with increased levels of homocysteine compared to pts with levels of homocysteine <15 |IM (RR: 4.14, p<0.0001). Using linear regression analysis, CAD was associated with the inadequacy of HD (r = - 0.05, p<0.0001), time on HD (r =0.04, p =0.012) and increasing age (r =0.24, p<0.001). There was no statistically significant association between CAD and the presence of the other traditional risk factors. The incidence of CAD in dialysis pts is significantly increased with age, male sex, obesity, time on dialysis, the presence of anemia, hyperhomocysteinemia and inadequacy of HD.

Promoters and control elements: designing expression cassettes for gene therapy.

It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailor-made' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

Association of p53 codon 72 polymorphism with advanced lung cancer: the Arg allele is preferentially retained in tumours arising in Arg/Pro germline heterozygotes.

The association of p53 codon 72 polymorphism with cancer has been investigated by several scientific groups with controversial results. In the present study, we examined the genotypic frequency of this polymorphism in 54 patients with advanced lung cancer and 99 normal controls from the geographical region of Greece. Sputum and bronchial washing samples from each patient were assayed for the presence of human papillomavirus. Codon 72 heterozygous (Arg/Pro) patients were also analysed for loss of heterozygosity at the TP53 locus, in order to determine the lost p53 allele (Arg or Pro). p53 Arg/Arg genotype was significantly increased in lung cancer patients compared to normal controls (50% vs 24.2%, P<0.002). Human papillomavirus was detected only in two patients (3.7%). Loss of heterozygosity at the TP53 locus was found in 14 out of 27 Arg/Pro patients (51.85%). The Pro allele was lost in 11 cases (78.6%), while the Arg allele was lost in three (21.4%). Our results suggest that p53 codon 72 Arg homozygosity is associated with advanced lung cancer, and that the Arg allele is preferentially retained in patients heterozygous for this polymorphism. On the other hand, human papillomavirus infection does not seem to play an important role in lung carcinogenesis.