Gene Expression from 1764 27-/4 pRIQIacZ is Maintained Over Time.

Following the observation that a high titre stock of vector 1764 27-/4- pR19CMV/acZ could efficiently reach peripheral ganglia following sciatic nerve inoculation (see section 4.1.2.2.), the virus was tested for long-term gene delivery to DRG. Here 2-5pl of a 1x10®pfu/ml stock of vector was administered unilaterally into mouse sciatic nerve. DRG were removed from 3 mice at 2days, Iweek and 1 month post inoculation and examined for lacZ activity following X- Gal staining. Results can be seen in figure 4.6 and show that transgene expression is maintained for at least 1 month and at levels equivalent to those seen at 2days p.i. This result highlights, for the first time in this thesis, a vector that is able to successfully reach peripheral ganglia and maintain high levels of transgene expression during latent infection, without an apparent drop-off in expression levels.

Results in figure 4.6 also show that X-Gal staining produced a punctate speckled pattern in some neurons. This phenomenon was not observed in results presented elsewhere in this thesis, but has been reported previously (Dobson at al. 1990) (Ho and Mocarski 1989). Ho at al suggested that this was because the p-galactosidase protein used contained no nuclear localisation signal and Dobson at al suggested that p-Gal, the product of the histochemical reaction, was limited to membrane bound organelles in these neurons. However, the actual reason for such a speckled appearance remains to be determined.

Chapter 4 Replication Incompetent Vectors

1764 27-/4- pR19CMV/acZ

A.

B.

c.

Figure 4.6. Gene delivery to peripheral ganglia following sciatic nerve inoculation with a replication incompetent vector deleted for IC27 and ICP4. 2-5^1 of 1x10Vu/ml of vector 1764 27-/4- pR19CMV/acZ was administered unilaterally to the rear sciatic nerve of mice. DRG were extracted at; A. 2days, B. Iweek and C. 1 month post-inoculation and examined for lacZ activity following X-Gal staining.

4.2. DISCUSSION.

Previously there has been only one documented report of a vector deleted for an essential gene that has been used for gene delivery to peripheral ganglia (Dobson et ai. 1990). Dobson et al used a vector deleted for ICP4 and showed that lacZ expression was maintained for at least 24weeks following sciatic nerve inoculation of virus. However here the efficiency of gene delivery was very low (an average of 60 cells per animal). In another report Marshall et ai (Marshall et al. 2000) used a conditionally replication competent vector that was inactivated for VP16 and ICPO and had a ts ICP4 mutation. This gave transgene delivery following peripheral administration of the vector that increased during latent infection. Marshall et al concluded that replication was therefore not necessary for HSV to reach DRG, since they expected their vector to be replication incompetent under the conditions used. However, since the mouse footpad is likely to be at a temperature somewhat below the fully non-permissive temperature for the ICP4 mutation (38°C), it is possible that the vector retained a degree of replication competence, which allowed gene delivery to occur. This possibility was however discounted by the authors since no replicating vector could be recovered from DRG of inoculated animals and in addition homogenised footpads from inoculated animals showed a drop in viral titre, consistent with a non-replicating vector. Based on the results presented in this thesis, using a number of replication incompetent vectors in the footpad, it would seem likely that replication was occurring in the vector described by Marshall et al.

Results presented here show that a fully replication competent vector deficient in ICP4, ICP27, VP16 and ICP34.5 when used at high titre and following sciatic nerve inoculation can efficiently reach DRG and express a delivered transgene for at least 1 month. The vectors 1764 27-/vhs-pR20.9 and 1764 27-/vhs-pR20.5 gave no gene delivery to DRG following sciatic nerve inoculation. However with hindsight improved results may be obtained if these vectors were also used at higher titres. Footpad inoculation of vectors deleted for the essential genes ICP27 alone or ICP27 and ICP4 gave very few lacZ positive neurons when used at a titre sufficient for delivery of replication competent vectors (1x10®pfu/ml). An increase of titre did not result in efficient gene delivery. These

C hapter 4________________________________________________Replication Inœ m petent Vectors

results collectively suggest that replication is required for vectors to reach peripheral ganglia following footpad inoculation. However, this problem can be overcome if sufficient pfu are used and vectors are administered directly into a nerve.

The results presented in figure 4.6 show that after injection of 1764 27-/4- pR19CMV/acZ, the numbers of lacZ positive neurons, determined by X-Gal staining, were consistent between 2days and 1 month p.i. Previously a similar vector had been constructed, 1764 pR19CMV/acZ (see section 3.2.2.1.), without ICP27 and ICP4 deleted, and assessed following sciatic nerve and footpad inoculation (see figure 3.2.). The replication competent vector gave high levels of gene delivery following sciatic nerve and footpad inoculation but results tended to be inconsistent between animals. The replication incompetent vector reported here, 1764 27-/4- pR19CMV/acZ gave highly consistent results between animals. This observation was possibly attributable to replication of the 1764 pR19CMV/acZ vector in the periphery (cells of the footpad or axonal bundle). This could function to increase vector toxicity and/or titre, and result in different amounts of vector potentially able to reach the DRG in different animals.

Previous work has reported that replication deficient vectors, whilst non-toxic, show a repression of transgene expression in cultured cells immediately after infection (Samaniego et al. 1998). This was suggested to be a result of the lack of expression of ICPO. The replication incompetent vector described here, 1764 27-/4- pR19CMV/acZ, expresses a transgene in the PNS for at least 1 month (Palmer et ai. 2000), in the CNS for at least 1 month and in primary DRG cultures for at least 1 week (Lilley et ai. 2001). A similar virus 1764 27-/4-/vhs- pR20.5, containing the pR20.5 cassettes in the vhs locus, was shown to express GFP and lacZ up to 3 weeks in organotypic hippocampal slice cultures and for 28days in cultured Vero cells (Lilley et ai. 2001). These findings, when compared to other vectors deficient in IE gene expression, suggest that elements of the LATP2 region included in the promoters used, are responsible for maintenance of transgene expression. Alternatively as the ICPO gene has

not been deleted, residual levels of ICPO expression may aid in the continued gene expression observed.

In conclusion v\/ork performed in this section has successfully identified a HSV-1 vector that is capable of both lytic and latent transgene expression in the PNS that is persistent and consistent over time. Previously (see chapters), successful transgene expression was achieved during acute infection but which declined rapidly during latent infection. The result seen collectively in chapters 3 and 4 suggest that long term expression can be achieved by a lack of IE gene expression in the vectors. These genes have previously been shown to be cytotoxic. It is likely that the increase expression observed with these replication incompetent vectors is due to a reduced cyctotoxicity and/or the lack of immune response to the vector. However the exact reasons remain to be seen