The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression.

BACKGROUND: In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. METHODS: We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. RESULTS: The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. CONCLUSION: This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.

Mutagenicity of herpes simplex virus type 1 in a shuttle vector

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced by herpes simplex virus type-1 (HSV-1). In cells infected by HSV-1 the frequency of mutation in supF gene, the mutagenesis marker, was increased over background by from two- to seven-fold, reaching 0.14-0.45%. No increase was induced by infection by vaccinia virus under the same conditions. Mutagenesis was an early event, showing a four-fold increase in mutation frequency at only two hours after infection, and peaking at a seven-fold increase at four hours after infection. DNA sequencing and gel electrophoresis analysis were performed on 105 HSV-1 induced mutants and 65 spontaneous mutants and provided the following information: (1) A change in plasmid size was seen in 54% of HSV-1 related mutants, compared with only 37% of spontaneous mutants. (2) Among point mutations, the predominant type was G:C to A:T transition, which accounted for 51% of point mutations in mutants isolated from cells infected with HSV-1, and 32% of point mutations in spontaneous mutants. (3) Deletions of DNA were seen in HSV-1 related mutants at a frequency of 40%, compared with 29% in spontaneous mutants. The HSV-1 related deletions were about half the length of spontaneous mutants and three contained short filler sequences. (4) Fifteen (15%) of HSV-1 induced mutants revealed the altered restriction patterns on agarose gel electrophoresis analysis and were due either to rearrangements of plasmid DNA, and/or to insertion of sequences derived from chromosomal DNA (seven plasmids). No insertions of DNA from HSV-1 were detected. Among spontaneous mutants, only 5 (7.7%) were rearrangements and none had inserted chromosomal DNA. (5) DNA sequence analysis of seven plasmids with inserted chromosomal DNA revealed that four cases had repetitive DNA sequences integrated and the other three were unidentified sequences from the GenBank database. Three repetitive DNA included $\alpha$ satellite, Alu and KpnI family sequences. The other sequence was identified as tRNA-like component. The observed mutations have implications for the mechanism of malignant transformation of cells by HSV-1. ^

Protease-activated receptor-1 signaling plays a major role in melanoma growth and metastasis

Overexpression of the thrombin receptor (Protease-Activated-Receptor-1), PAR-1, in cell lines and tissue specimens correlates with the metastatic potential of human melanoma. Utilizing lentiviral shRNA to stably silence PAR-1 in metastatic melanoma cell lines results in decreased tumor growth and lung metastasis in vivo. Since the use of viral technology is not ideal for clinical therapies, neutral liposomes (DOPC) were utilized as a delivery vehicle for PAR-1 siRNA. Our data suggest that PAR-1 siRNA-DOPC treatment by systemic delivery significantly decreases tumor growth and lung metastasis in nude mice. Concomitant decreases in angiogenic and invasive factors (IL-8, VEGF, MMP-2) were observed in PAR-1 siRNA-DOPC-treated mice. Utilizing a cDNA microarray platform, several novel PAR-1 downstream target genes were identified, including Connexin 43 (Cx-43) and Maspin. Cx-43, known to be involved in tumor cell diapedesis and attachment to endothelial cells, is decreased after PAR-1 silencing. Furthermore, the Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells suggesting transcriptional regulation of Cx-43 by PAR-1. ChIP analysis revealed a reduction in SP-1 and AP-1 binding to the Cx-43 promoter. Moreover, melanoma cell attachment to HUVEC was significantly decreased in PAR-1-silenced cells as well as in Cx-43 shRNA transduced cells. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Maspin, a serine protease inhibitor with tumor-suppressor function, was found to be upregulated after PAR-1 silencing. Our results indicate that PAR-1 transcriptionally regulates Maspin, as the promoter activity was significantly increased after PAR-1 silencing. ChIP analysis revealed that silencing PAR-1 increased binding of Ets and c-Jun to the Maspin promoter. As Maspin was recently found to be a tumor-suppressor in melanoma by reducing the invasive capacity of melanoma cells, invasion assays revealed a decrease in invasion after PAR-1 silencing and in cells transduced with a Maspin expression vector. We propose that PAR-1 is key to the progression and metastasis of melanoma in part by regulating the expression of Cx-43 and Maspin. Taken together, we propose that PAR-1 is an attractive target for the treatment of melanoma.^

Perspectives on the public health implications of global climate change and the epidemiology of vector-borne disease

Global climate change is becoming an increasing concern among the public health community. Some researchers believe the earth is rapidly undergoing changes in temperature, sea level, population movement, and extreme weather phenomenon. With these geographic, meteorological, and social changes come increased threats to human health. One of these threats is the spread of vector-borne infectious diseases. The changes mentioned above are believed to contribute to increased arthropod survival, transmission, and habitation. These changes, in turn, lead to increased incidence among neighboring human populations. It is also argued that human action may play more of a role than climate change. This systematic review served to determine whether or not climate change poses a significant risk to human exposure and increased incidence of vector-borne disease. ^

TRANSOVARIAL TRANSMISSION OF YELLOW FEVER VIRUS BY A SYLVATIC VECTOR, HAEMAGOGUS EQUINUS (THEOBALD)

The potential of a sylvatic vector to act as reservoir of yellow fever virus was assessed in Haemagogus equinus (Theobald). Mosquitoes from the Maje'75 laboratory colony and a Panamanian human isolated strain of yellow fever virus were used. Female mosquitoes infected with yellow fever virus had a minimum transovarial transmission rate of 1:5745. This is the first time transovarial transmission of yellow fever virus has been demonstrated in a New World sylvatic vector. Transovarial transmission of yellow fever virus in this sylvatic mosquito species may be an alternative mechanism for biological survival of the virus during adverse periods or in the absence of susceptible vertebrate hosts. ^

Cross -packing among retroviruses and characterization of the feline immunodeficiency virus (FIV) packaging signal: Implications for the development of FIV-based gene transfer systems

Feline immunodeficiency virus (FIV)-based gene transfer systems are being seriously considered for human gene therapy as an alternative to vectors based on primate lentiviruses, a genetically complex group of retroviruses capable of infecting non-dividing cells. The greater phylogenetic distance between the feline and primate lentiviruses is thought to reduce chances of the generation of recombinant viruses. However, safety of FIV-based vector systems has not been tested experimentally. Since primate lentiviruses such as human and simian immunodeficiency viruses (HIV/SIV) can cross-package each other's genomes, we tested this trait with respect to FIV. Unexpectedly, both feline and primate lentiviruses were reciprocally able to both cross-package and propagate each other's RNA genomes. This was largely due to the recognition of viral packaging signals by the heterologous proteins. However, a simple retrovirus such as Mason-Pfizer monkey virus (MPMV) was unable to package FIV RNA. Interestingly, FIV could package MPMV RNA, but not propagate it for further steps of replication. These findings suggest that upon co-infection of the same host, cross-packaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential. ^ In order to understand the packaging determinants in FIV, we conducted a detailed mutational analysis of the region thought to contain FIV packaging signal. We show that the first 90–120 nt of the 5′ untranslated region (UTR) and the first 90 nt of gag were simultaneously required for efficient FIV RNA packaging. These results suggest that the primary FIV packaging signal is multipartite and discontinuous, composed of two core elements separated by 150 nt of the 5 ′UTR. ^ The above studies are being used towards the development of safer FIV-based self-inactivating (SIN) vectors. These vectors are being designed to eliminate the ability of FIV transfer vector RNAs to be mobilized by primate lentiviral proteins that may be present in the target cells. Preliminary test of the first generation of these vectors has revealed that they are incapable of being propagated by feline proteins. The inability of FIV transfer vectors to express packageable vector RNA after integration should greatly increase the safety of FIV vectors for human gene therapy. ^