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High Titre Figure 3.2. Replication competent vectors, 1764pR19MMLV/acZ and 1764pR19CMV/acZ tested for gene delivery to DRG during acute and latent infection. A. Footpad inoculation with 20pl of virus at 1x10®pfu/ml. B. Sciatic nerve inoculation with 2-5^1 of virus at 1x10^pfu/ml. C. Sciatic nerve inoculation with 2-5pl of high titre virus (1x10®pfu/ml). MMLVLTR, Moloney murine leukemia virus long terminal repeat promoter. CMV, cytomegalovirus promoter. lacZ, p-galactosidase.3.2.3. Multiple Transgene Expression from a Cassette in a Non-Essential Locus.
Many diseases might require the simultaneous delivery of two therapeutic genes as a means of treatment. This could be achieved by the use of two different vectors, each providing one transgene or by the insertion of two transgenes into the same vector. Previously in our laboratory promoter systems were constructed that allowed multiple gene delivery from a single expression cassette. These cassettes, pR20.5 and pR20.9, are shown in figure 3.3. It has been shown previously by others that elements found in the LAP2 region can confer long-term gene expression activity on the LAP1 promoter (Lokensgard at al. 1994;Lokensgard at al. 1997). In addition it was shown that the MMLVLTR promoter can give long term expression in combination with elements from LAP1 (Lokensgard at al. 1994). The LATP2 region provides the central element of both the pR20.9 and pR20.5 cassettes and is flanked on either side by two different promoters facing in opposite orientations (MMLV-LTR and LAP1 or CMV and RSV). These cassettes were inserted into either the UL43 or US5 loci of the HSV strain 1764.
3.2.3.1. Non-Essential Genes UL43 and US5.
US5 is a non-essential gene found in the unique short ( U s ) region of the HSV
genome and is one of 14 Us genes, 13 of which are non-essential (Rasty at al.
1997). As predicted by sequence analysis, it has recently been identified as encoding a surface glycoprotein (gJ) (Ghiasi at al. 1998). A virus deleted for the US5 gene was shown to be viable for growth in cell culture, as determined on Vero cells. (Weber at al. 1987). More recently, a /acZ-US5 insertion mutant has been shown to have a normal phenotype both in vivo and in vitro (Balan at al.
1994). Here the virus was shown to have normal particle to infectivity ratio in vitro and no phenotypic abnormalities were observed in BHK cells following high or low MO! infectivity. In vivo ear scarification of mice showed that US5 mutants had no effect on the ability of HSV to multiply at the inoculation site or on the ability to enter or multiply in the PNS or CNS.
UL43 is located in the unique long (UL) region of the HSV genome. The gene has no known function although it contains multiple hydrophobic stretches and
Chapter 3_________________________________________________ Replication Competent Vectors
is predicted to be a membrane channel protein (Carter et al. 1996). A UL43-/acZ insertion mutant and a UL43 deletion mutant were both shown to have no effect on the virus phenotype compared to the parental virus 17syn+, both in vitro, by comparing growth phenotypes, and in vivo (MacLean et al. 1991b). In vivo the viruses were compared in the mouse ear model, by their ability to replicate in the periphery or spread to and replicate within the nervous system. The UL43.5 ORF maps antisense to UL43, but is also known to be dispensable for growth in culture (Maclean et al. 1991b;Ward et al. 1996) and is of unknown function.
These suggested that UL43 and US5 were suitable sites for the insertion of expression cassettes.
3.2.3.2. Expression Cassettes and Vector Production.
Previously the two expression cassettes pR20.5 and pR20.9 had been constructed and have now been described (see figure 3.3) (Palmer at al. 2000) (Thomas at al. 1999b). Briefly, each cassette contains a central LATP2 region (HSV nt 118866 to 120219) flanked by two promoters. The 20.5 cassette contains CMV (Invitrogen) driving E-GFP (Clontech) and RSV (pRcRSV- Invitrogen) driving lacZ (pCHIIO- Pharmacia). The 20.9 cassette contains MMLVLTR (pJ4Q (Morgenstern and Land 1990)) driving E-GFP and the LAP1 promoter (HSV nt 118181 to 118878) driving lacZ. The cassettes were cloned into UL43 flanking regions at the unique A/s/1 site (HSV nt 94911 [in a BamHI- EcoRI fragment HSV nt 91619-96751 in pGem2]) or the US5 locus at the unique Sad site (HSV nt 137945 [in a BamH\- EcoNl fragment 136289 - 139328]). The viruses were produced by co-transfection of the plasmids with 1764 viral DNA (section 2.2.5) and purification of green and blue plaques from white plaques. Three viruses were made and purified, 1764pR20.9/UL43,
1764pR20.9/US5 and 1764pR20.5/UL43.
3.2.3.3. In Vitro Vector Analysis on Permissive Cells.
During the purification procedure it became apparent that the phenotypes of the pR20.9 viruses were different from that of the pR20.5 virus. During lytic replication on permissive BHK cells, both of the pR20.9 containing viruses showed only low levels of p-galactosidase activity upon X-Gal staining (driven by the LAP1 promoter). However, both pR20.9 containing viruses showed strong GFP expression (driven by MMLVLTR). The pR20.5 virus showed strong expression of both lacZ and GFP. See figure 3.4. This phenotype of the pR20.9 viruses appeared to be an active repression of LAP1 activity because when the virus is grown on BHK cells in the presence of acyclovir, which halts viral replication, p-galactosidase activity increases. However, here levels do not reach that observed with the pR20.5 virus (data not shown). In addition transient transfection of the pR20.9 plasmids results in strong X-Gal staining, i.e. in the absence of all viral proteins. Similar results to this phenomenon have been observed previously (Goins at al. 1994;Lachmann and Efstathiou 1997b). Lachmann at al describe vectors with either an 1RES p-Geo cassette or an 1RES lacZ cassette inserted 1.5kb downstream to the LAP1 TATA box. These
Chapter 3________________________________________________ Replication Competent Vectors
viruses did not express detectable lacZ upon lytic infection of Vero cells (with low levels only in a few cells), but lacZ was readily detectable in vivo. Goins et al investigated the phenomenon to a greater extent by creating two viruses with LAP2/p-gal insertions in the gC locus, one with the additional deletion of ICP4. The gC mutated virus showed no p-galactosidase expression in Vero cells but expressed lacZ in vivo, whereas the ICP4 deleted virus expressed lacZ both in vitro and in vivo. Goins at al suggested that in the gC-virus, the actions of the ICP4 or ICPO proteins on LAP2 during lytic infection were responsible for the loss of lacZ activity observed. It is already known that the ICP4 protein down- regulates LAP1 during productive infection, by binding to the ICP4 binding motif found in the cap site (Batchelor at al. 1994). It is thus possible that the action of either the ICP4 or ICPO proteins functions to silence LAP1 in the pR20.9 cassette. Since this repression is only evident in the pR20.9 cassette and no other cassettes studied in this thesis, it is likely that sequences in the LAP1 region must participate in this replication-dependent repression.