Analysis of factor VIII mediated suppression of lentiviral vector titres.

Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy.

Multiple control elements in the TRP1 promoter of Saccharomyces cerevisiae.

The TRP1 promoter generates two groups of mRNAs, transcript I and transcript II. The difference in size between the largest and smallest mRNAs is about 200 base pairs. A series of one-sided and internal deletions were constructed in vitro throughout the TRP1 promoter, and the effect of each deletion on transcription was assessed by Northern blotting. We showed that 395 base pairs of the TRP1 promoter were sufficient for the normal transcription of all RNAs and that the promoter contained two control domains. The control domain for transcript I consisted of one positive element and one negative element, while the control domain for transcript II contained two positive elements. The negative element, mapped between -293 and -318, expression of transcript I. Two regions of transcript I. Two regions (-280 to -236 and -235 to -209) were required for accurate initiation of transcript I. Each region contained sequences homologous to known consensus sequences of the TATA box.

Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1.

The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. The upstream activation sequence contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. We have demonstrated that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1. Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium. PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate. This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence. The promoters of many other yeast glycolytic genes also contain consensus RAP1-binding sites and copies of the CTTCC pentamer. This suggests that RAP1 may be involved in transcriptional control of many other glycolytic genes in addition to the PGK gene.

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences.

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

Complete nucleotide sequence of a mouse VL30 retro-element.

The complete nucleotide sequence of a mouse retro-element is presented. The cloned element is composed of 4,834 base pairs (bp) with long terminal repeats of 568 bp separated by an internal region of 3,698 bp. The element did not appear to have any open reading frames that would be capable of encoding the functional proteins that are normally produced by retro-elements. However, some regions of the genome showed some homology to retroviral gag and pol open reading frames. There was no region in VL30 corresponding to a retroviral env gene. This implies that VL30 is related to retrotransposons rather than to retroviruses. The sequence also contained regions that were homologous to known reverse transcriptase priming sites and viral packaging sites. These observations, combined with the known transcriptional capacity of the VL30 promoter, suggest that VL30 relies on protein functions of other retro-elements, such as murine leukemia virus, while maintaining highly conserved cis-active promoter, packaging, and priming sites necessary for its replication and cell-to-cell transmission.

A homologue of the human MSS1 gene, a positive modulator of HIV-1 gene expression, is massively expressed in Xenopus oocytes.

Here the nucleotide sequence of a Xenopus homologue of the human MSS1 gene, a positive modulator of the HIV-1 Tat mediated transactivation in mammalian cells, is presented. This gene is highly conserved and almost exclusively expressed in Xenopus oocytes. We speculate about a possible role of this gene in the HIV-1 Tat/TAR mediated transactivation in Xenopus oocytes.

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Purification and secondary structure determination of simian immunodeficiency virus p27.

We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.

Performance characteristics of a novel immunoassay based on hybrid Ty virus-like particles (Ty-VLPs): rapid differentiation between HIV-1 and HIV-2 infection.

Recombinant antigens containing all or parts of the HIV-1 proteins p24, Nef and gp41 and HIV-2 gp36 have been purified and used to develop a rapid immunoassay to detect and differentiate between HIV-1 and HIV-2 antibodies in a single test. The antigens were produced as particulate fusion proteins by exploiting the ability of a protein encoded by the yeast retrotransposon Ty to assemble into virus-like particles (Ty-VLPs). Hybrid HIV: Ty-VLPs carrying each of the antigens were applied to nitrocellulose strips at specified locations in a slot-blot format and then used to detect antibodies present in human serum and plasma samples of diverse geographical origin. Previously confirmed HIV-1- and HIV-2-positive samples were readily and reliably identified. The assay was used to identify a case of HIV-2 infection in an African woman who had been resident in the Oxford region for the last 3 years and to analyse the prevalence of anti-HIV antibodies in a longitudinal study of seroconverting patients. We also demonstrate that the assay works efficiently with whole blood.

A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs).

The self-assembly properties of a protein encoded by the yeast retrotransposon Ty can be exploited to produce large amounts of recombinant, particulate fusion proteins as hybrid Ty virus-like particles (Ty-VLPs). This system has now been adapted to allow the release of the additional protein by incorporation of a protease cleavage site between the yeast carrier protein and the protein of interest. The purification of the additional protein is facilitated by exploiting the ease with which Ty-VLPs can be purified from other yeast cell components due to their particulate nature. We have used this modified system to produce hybrid particles containing the HIV-1 p24 protein downstream of the recognition sequence for the blood coagulation factor Xa. The p24 was released from the particles by proteolytic cleavage and rapidly separated from the residual particulate material using centrifugation and standard chromatography techniques. This procedure has been used to purify milligram quantities of HIV-1 p24 protein that reacts with anti-p24 sera and elicits the production of p24-specific antibodies in experimental animals.

Retroelement particles as purification, presentation and targeting vehicles.

The manipulation of retrotransposon and retroviral particles to carry biologically active molecules is becoming feasible. In addition, recent experiments suggest that it may be possible to target these engineered particles to specific cell types. This has implications for gene therapy, biological drug delivery and vaccine design.

Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.

A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.

Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 alpha and HIF-2 alpha in a primary neuronal model of hypoxia.

The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1 alpha and HIF-2 alpha elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2 alpha overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia.

Sequence variation in the LEU2 region of the saccharomyces cerevisiae genome.

The LEU2 regions present on yeast plasmid vectors come from two sources, a series of strains derived from S288c and strain M127. The LEU2 region from the S288c series contains a Tyl-17 element with its associated delta sequences and a small repetitive RNA gene while the LEU2 region from M127 which is present on pJDB248, lacks the Tyl-17 element, but carries a delta sequence and a small RNA gene. The various LEU2 plasmids currently in use vary with respect to these sequences depending on which restriction fragment from the region is present on the recombinant molecule. In addition, strain M127 contains three LEU2 homologous sequences that are represented by different EcoRI fragments and which segregate independently at meiosis. Therefore, there are at least four forms of the centromere-distal EcoRI fragment of the LEU2 locus in the Saccharomyces cerevisiae gene pool; these are 7.1 kb, 1.9 kb, 1.48 kb and 1.15 kb long.

Factors affecting heterologous gene expression in Saccharomyces cerevisiae.

The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast. The yields of IFN alpha 2 are only 1-3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. IFN alpha 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN alpha 2 is that IFN-specific RNA levels are much lower. This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and 3' untranslated regions of the transcripts or DNA flanking regions. It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Expression vectors for the construction of hybrid Ty-VLPs.

Purification of expressed proteins can be facilitated by expressing the recombinant protein as a fusion with a carrier protein that assembles into particulate structures. This article describes the use of expression vectors in producing a hybrid of the yeast retrotransposon Ty, which self-assembles into virus-like particles (VLPs). Hybrid VLPs can be used in such laboratory applications as the production of polyclonal and monoclonal antibodies, structure/function analyses, the detection of important antigenic determinants, and epitope mapping of monoclonal antibodies.

Production and purification of hybrid Ty-VLPs.

This article describes how pure Ty-VLPs (virus-like particles) can be prepared from hybrid Ty-VLPs. Many different hybrid Ty-VLPs have been produced and may be easily purified. Since the sedimentation properties of different hybrid Ty-VLPs are similar, a simple purification process can be used for any VLP. This fast, versatile, and easy process allows for the production of a variety of recombinant proteins.

Production and Purification of Hybrid Ty-VLPs.

The self-assembly properties of a protein encoded by the TYA gene of the yeast Ty element can be exploited to produce hybrid Ty-VLPs (virus-like particles) (1,2). There has been developed a series of expression vectors that allow the construction of Ty fusion genes containing protein coding sequences of interest (see Chapter 24 ). Many different hybrid Ty-VLPs have now been produced that carry additional proteins that range in size from 3 to 42 kDa. These include regions from human immunodeficiency virus-1 (HIV-1) env, pol, tat, rev, nef, and vif genes; influenza virus hemagglutinin; human α-interferon, feline leukemia virus env, and bovine papillomavirus El and E2 (1-5 and unpublished data).

Expression Vectors for the Construction of Hybrid Ty-VLPs.

The synthesis of recombinant proteins or protein domains in microbial, insect, or mammalian systems is now commonplace in molecular biology laboratories. The gene or gene fragment encoding the protein of interest is inserted into a specialized expression vector, flanked by efficient transcription and translation control sequences. The expression vector is then inserted into recipient cells and expression of the protein induced. The expressed protein then has to be purified from other cellular or medium components. Purification can be facilitated by expressing the recombinant protein as a fusion with a carrier protein that assembles into particulate structures. This approach has been developed using a protein encoded by the yeast retrotransposon Ty, which self-assembles into virus-like particles (VLPs) (1,2). Additional protein coding sequences can be fused to the carrier protein gene and expressed in yeast to produce hybrid Ty-VLPs (3,4). The physical characteristics of the VLPs have been exploited to produce a rapid purification procedure that is essentially generic for any hybrid construction. Hybrid VLPs can be used in many laboratory applications (see elsewhere in this vol), including the production of polyclonal and monoclonal antibodies, structure/function analyses, the detection of important antigenic determinants, and epitope mapping of monoclonal antibodies.