Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.     

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.     

Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector.

We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that all introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.     

A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli beta-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and beta-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.     

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.     

靶向WNT5A基因的shRNA逆转录病毒表达载体的构建与鉴定

目的:构建人WNT5A shRNA逆转录病毒表达载体.方法:根据人WNT5A基因mRNA序列设计并合成两条互补的DNA单链寡核苷酸,将退火后形成的双链连接于pSUPER Retro RNAi质粒,构建WNT5A shRNA逆转录病毒表达载体,经脂质体介导入GPG293细胞,包装成逆转录病毒.用该逆转录病毒感染人鼻咽癌细胞,Western blot法和RT-PCR检测细胞WNT5A的表达.结果:目的序列成功连接于载体并包装成逆转录病毒,免疫印迹检测和RT-PCR检测结果表明构建的WNT5A shRNA逆转录病毒表达载体能显著抑制鼻咽癌细胞WNT5A的表达.结论:成功构建人WNT5A shRNA逆转录病毒表达载体.     

Construction and hormone regulation of a novel retroviral vector

We report the analysis of a self-inactivating retroviral vector, constructed to allow inducible gene expression of inserted sequences from the mouse mammary tumour virus hormonal response element. The cloning strategy has been designed to allow for ease of insertion of the genes of interest. The vector contains the aph gene, allowing geneticin-resistance selection in mammalian cells. We have characterised dexamethasone (Dex)-induced increase in gene expression using the reporter gene encoding chloramphenicol acetyltransferase (CAT) inserted into the retroviral vector. We observe low basal levels of CAT activity in infected cells which is increased up to 50-fold by induction with Dex. The induction of pooled clones is 13.3-fold. Variation in Dex-induced CAT activity is observed in independently infected clones, which is not explained by proviral copy number.     

Development of a new bicistronic retroviral vector with strong IRES activity

Background  

Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies.     

Transduction of host cellular sequences by a retroviral shuttle vector.

We studied the frequencies and types of excision events which can occur with a retroviral shuttle vector containing the simian virus 40 origin of DNA replication. Analysis of the recloned vector plasmids by size and restriction enzyme mapping indicated that most contain one long terminal repeat. By hybridizing the plasmids to a mouse genomic repetitive DNA probe, we also determined that approximately 1 to 3% contain transduced cellular DNA sequences.     

Heart targeting of retroviral expression in avian embryos: a species-independent phenomenon

A replication-incompetent retroviral vector derived from spleen necrosis virus (SNV), in which the viral structural genes gag, pol, and env were replaced with the bacterial -galactosidase gene lacZ, was used to infect embryos from outbred and inbred chicken lines, japanese quail and duck between embryonic day 0 and 13. LacZ expression was restricted to a few organs or cell types, and this distribution was not influenced by the different routes of inoculation tested but was specified by the age of the embryo at the time of inoculation. Inoculations at E0-E1 beneath or onto the blastodisc resulted in lacZ expression in ectodermal derivatives, i.e. skin and neural structures. From E2 onwards, heart muscle and skin were the preferential targets in all the species or inbred lines tested. Heart muscle was positive in 100% of the embryos displaying lacZ+ clones. Skin exhibited on and off periods depending on the age at inoculation. No lacZ-positive clones were detected in chick embryos infected after Ell. Outbred chick embryos displayed the largest array of organs labelled (heart, skin, liver, gizzard) while quail and duck embryos exhibited a more restrictive pattern. These results are of import if the vector is to be used as a tool to map lineages or to transfer genes into the developing embryo.     

Generation of a replication-competent,propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome

Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E(+) packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Delta3abDeltaE) was successfully rescued in these cell lines. rTGEV-Delta3abDeltaE reached high titers (10(7) PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 x 10(5) PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Delta3abDeltaE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E(+) packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.     

Viral vector targeting.

The field of viral vector targeting is advancing rapidly. Recent advances include the successful use of bifunctional crosslinkers to target adenoviral and retroviral vectors, elucidation of the crystal structures of an adenoviral and a retroviral receptor-binding domain, and definition of strategies for inserting short targeting peptides and larger polypeptide-binding domains into the coat proteins of a number of different viral vectors. Novel targeting strategies based on host range restriction and protease activation have been developed, targeted replication-competent vectors have shown promise as anti-cancer agents and the possibility of selecting targeted vectors from vector display libraries has been established.