ENSAYOS DE VUELCO EN CARROCERÍAS DE BUSES

The virion host shutoff protein (vhs) is a tegument protein and is responsible for the shutdown of host protein synthesis by the non-selective degradation of mRNA (Kwong at al. 1988). Vhs mutants have also been shown to prolong the half-life of viral mRNAs (Kwong at al. 1988;Oroskar and Read 1989). Vhs mutants have been shown to have a reduced pathogenicity in vivo but replicate normally in vitro (Strelow and Leib 1995). It was thus speculated that vectors deleted for vhs, concomitantly with ICP34.5 and VP16, might prove successful vectors for gene delivery to the PNS and thus should be tested. The deletion of multiple viral genes also provides an extra safety margin for safe and effective vectors. Previously in our laboratory, a virus deleted for ICP34.5, VP16 and vhs had been tested for gene delivery to lumbar ganglia following footpad inoculation, this work is now published (Palmer at ai. 2000). Preliminary data had suggested that this virus might not be as efficient as one deleted for ICP34.5 and VP16 (1764), however further experiments were required.

3.2.4.1. Expression Cassettes and Vector Production.

Previously a 1764-based vector simultaneously deleted for vhs and UL43 had been created, termed 1764 UL43-MSVGFP vhs-pR15. Vhs was inactivated by the insertion of a reporter gene cassette, pR15 (Rob Coffin, UGL, London), at an Nru\ site (HSV nt 91854) and UL43 was inactivated by an MSV/GFP cassette at an A/s/l site (HSV nt 94911). See figure 3.7. pR15 contains LAP1 (HSV nt 118181 to 118768), a -700bp plasmid spacer sequence derived from pGem3Zf (Nda\-Xba\ [Promega]), and an MMLVLTR promoter driving iacZ. The pR15 cassette was known to give latent transgene expression when inserted either into vhs or UL43 (here called pR8c) as shown previously (Palmer at ai.

2000).

3.2.4.2. Deletion of Vhs Reduces the Efficiency of Reporter Gene Expression In Vivo Following Peripheral Administration.

The vector 1764 UL43-MSV/GFP vhs-pR15, was tested in vivo for gene delivery to peripheral ganglia following footpad and sciatic nerve inoculation (see figure 3.8). Results show that reporter gene expression is only efficient during the acute stage of viral infection, at 3days p.i and at later time points reporter gene expression drops. Following sciatic nerve inoculation, and during latent infection, no GFP or IacZ expression is observed and after footpad injection only low levels are observed, at 1 month p.i. which decline to undetectable levels at Smonths p.i. This result suggests that deletion of vhs does not enhance gene delivery to DRG following peripheral administration and is consistent with similar experiments performed earlier in the laboratory (Palmer at al. 2000).

Since the pRIS cassette previously supported latent transgene expression (Palmer at al. 2000), these results would suggest that the lack of GFP or IacZ

expression is here a result of failure of the virus to achieve a latent infection. This theory is substantiated by the fact that a vhs mutant was shown to have a reduced ability to establish latency in trigeminal ganglia compared to parental KOS virus (Strelow at al. 1997). Deletion of vhs together with ICP34.5 and VP16 may therefore further reduce the establishment of latency compared to a vhs deletion alone. To confirm this theory it will be necessary to quantify the amount of virus establishing a latent infection. This could be done by performing ISH for LAT RNA, or RT PCR or ISRTPCR for HSV-DNA (see section 1.4.3.8 ). In conclusion these results suggest that deletion of vhs together with deletion of ICP34.5 and VP 16 inactivation, is not advantageous in the vector described here. However this result may change if different expression cassettes, containing LATP2, were to be used in a similar vector backbone.

Chapter 3 Replication Competent Vectors

A.

nt 9 1 8 5 4 vhs IC P 34.5 V P 1 6 IC P 34.5

*

B.

LAP1 plasmid MMLVLTR LacZ pA vhs |—

/

C.

Figure 3.7. Expression cassettes and replication competent vector backbone. A. 1764 vector backbone deficient in ICP34.5 and VP16 and showing insertion sites in UL43 and vhs. B. The pR15 cassette C. An MSV/GFP expression cassette. In the same vector B. and C. were inserted into vhs and UL43 genes respectively and at the positions shown. The vector was tested for gene delivery to DRG following peripheral administration (see text for details). MSV, murine sarcoma virus promoter. MMLVLTR, Moloney murine leukemia virus promoter. GFP, Green fluorescent protein. LacZ, p-galactosidase. LAP1 is a HSV latency associated promoter. Arrows indicate orientation of the promoters.

Figure 3.8. Gene delivery to peripheral ganglia following footpad or sciatic nerve inoculation with a replication competent vector deleted for vhs.

2 0 |liI of 1x10®pfu/ml of virus was injected unilaterally

into the rear sciatic nerve of mice. DRG were removed at various times post inoculation, A. Sdays Bi. 2weeks Bii. Iweek C. 2months and D. Smonths and examined for GFP expression under a fluorescent microscope and for p-galactosidase activity by X-Gal staining.

Chapter 3 Replication Competent Vectors

A.

3davs

1764 UL43-MSVGFP vhs-pR15

Footpad Sciatic Nerve

Bi.

i

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■ i

Bii

c.

D.

3.2.5. Estimation of the Efficiency O f Vector Uptake by Ganglionic