Anti-tissue factor short hairpin RNA inhibits breast cancer growth in vivo.

In breast cancer, there is a correlation between tissue factor (TF) expression, angiogenesis and disease progression. TF stimulates tumour angiogenesis, in part, through up-regulation of vascular endothelial growth factor (VEGF). Therefore, this study aimed to establish whether TF stimulates angiogenesis and tumour progression directly and independent of VEGF up-regulation. Initially, the effects of TF and VEGF were assessed on endothelial cell migration (Boyden chamber) and differentiation (tubule formation on Matrigel). Subsequently, MDA-MB-436 breast cancer cells, which produce high levels of both TF and VEGF (western blot analysis), were established in vivo, following which tumours were treated three times per week for 3 weeks with intra-tumoural injections of either anti-VEGF siRNA, anti-TF shRNA, the two treatments combined, or relevant controls. Both VEGF and TF significantly stimulated endothelial cell migration and tubule formation (P < 0.02). Breast cancer xenografts (MDA-MB-436) treated with TF or VEGF-specific agents demonstrated significant inhibition in tumour growth (VEGFsiRNA 61%; final volume: 236.2 ± 23.2 mm(3) vs TFshRNA 89%; 161.9 ± 17.4 mm(3) vs combination 93%; 136.3 ± 9.2 mm(3) vs control 400.4 ± 32.7 mm(3); P < 0.005). Microvessel density (MVD), a measure of angiogenesis, was also significantly inhibited in all groups (MVD in control = 29 ± 2.9; TFshRNA = 18 ± 1.1; VEGFsiRNA = 16.7 ± 1.5; both = 12 ± 2.1; P < 0.004), whereas the proliferative index of the tumours was only reduced in the TFshRNA-treated groups (control = 0.51 ± 0.011; TFshRNA = 0.41 ± 0.014; VEGFsiRNA = 0.49 ± 0.013; both = 0.41 ± 0.004; P < 0.008). These data suggest that TF has a direct effect on primary breast cancer growth and angiogenesis, and that specific inhibition of the TF-signalling pathway has potential for the treatment of primary breast cancer.

Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs.

Many microRNAs (miRNAs) are encoded within the introns of RNA Pol II transcripts, often as polycistronic precursors. Here, we demonstrate the optimization of an intron encoding three endogenous miRNAs for the ectopic expression of heterologous anti-HIV-1 small interfering RNAs (siRNAs) processed from a single RNA polymerase II primary miRNA. Our expression system, designated as MCM7, is engineered from the intron-embedded, tri-cistronic miR-106b cluster that endogenously expresses miR-106b, miR-93 and miR-25. Manipulation of the miR-106b cluster demonstrated a strict requirement for maintenance of the native flanking primary miRNA (pri-miRNA) sequences and key structural features of the native miRNAs for efficient siRNA processing. As a model for testing the efficacy of this approach, we have replaced the three endogenous miRNAs with siRNAs targeting the tat and rev transcripts of human immunodeficiency virus type 1 (HIV-1). This study has enabled us to establish guidelines for optimal processing of the engineered miRNA mimics into functional siRNAs. In addition, we demonstrate that the incorporation of a small nucleolar RNA TAR chimeric decoy (snoRNA) inserted within the MCM7 intron resulted in a substantial enhancement of HIV suppression in long-term acute infectious HIV-1 challenges.

Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes.

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

Hammerhead ribozyme design and application.

The emerging knowledge about RNA-based enzymes has already had great impact on our concept of evolutionary history, making the 'RNA world' more likely. It may well have an equally important impact on the diagnostic and therapeutic practices of human and veterinary medicine in the next decade. We are not quite there yet. This review addresses the design and application of hammerhead ribozymes, two aspects of a conserved and most commonly studied and used enzymatically active entity among the RNA enzymes. The emerging picture is one of great diversity. There is at this stage no general cell model nor a clearly preferable ribozyme structure. Each and every cell line (and tissue) may be unique in that they vary with respect to structural requirements for optimal uptake, activity and stability of ribozymes. We may have seen only the tip of the iceberg when it comes to RNA-based enzymes and their roles in biology and medicine.

Extensive intimal apolipoprotein A1-derived amyloid deposits in a patient with an apolipoprotein A1 mutation.

In the aortic intima amyloid deposits are often associated with atherosclerotic plaques. In a recent study of one patient with aortic intimal amyloid the major fibril protein was an N-terminal fragment of apolipoprotein A1 (apoA1) consisting of 69 amino acid residues. In the present study, we have screened the apoA1 gene for mutations in autopsy cases with aortic intimal amyloid immunohistochemically positive for apoA1, using single stranded conformational polymorphism (SSCP) analysis and DNA sequencing. All cases except one had a normal apoA1 gene sequence. One case of exceptionally severe atherosclerosis combined with extensive intimal amyloid deposits showed an apoA1 deletion corresponding to Lys 107. Thus, wild type apoA1 is amyloidogenic but our findings suggest that the expression of a mutant apoA1-form may be associated with enhanced amyloidogenicity.