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Table 1. Recombinant HSV vectors

Virus name

Gene deletions

Transgenes, locus::promoter gene

Cell survival*, %

Source

KOS321

None

None

0

7

KHZ:tk

tk

tk::HCMV-lacZ

0

8

d120

ICP4

None

2

2

SHZ.1

ICP4, tk

tk::HCMV-lacZ

2

9

DHZ.1

ICP4, ICP22

ICP22::HCMV-lacZ

18–30

Unpublished data

5dL1.2

ICP27

None

N.D.

10

D0Z.1

ICP0, ICP27

ICP0::ICP0-lacZ

N.D.

Unpublished data

THZ.2

ICP4, ICP22, ICP27

ICP22::HCMV-lacZ

55–80

Unpublished data

T.2

ICP4, ICP22, ICP27

None

60–90

Unpublished data

S0Z.1

ICP4, UL41

UL41::ICP0-lacZ

N.D.

Unpublished data

T0Z.1

ICP4, ICP22, ICP27, UL41

UL41::ICP0-lacZ

N.D.

Unpublished data

THZ.3

ICP4, ICP22, ICP27, UL41

ICP22::HCMV-lacZ

60–90

Unpublished data

T.3

ICP4, ICP22, ICP27, UL41

None

N.D.

Unpublished data

*Cytotoxicity studies were performed in our lab and are expressed as the percentage of Vero cells that survive 48 hr after infection at a multiplicity of infection of 3.

HCMV, Human cytomegalovirus; N.D., not done.

neuron-specific enolase) promoters placed in various sites in these vectors all demonstrate robust transient transgene expression peaking 2–3 days after inoculation but disappearing by 1 week after inoculation. The loss of transgene expression is not due to elimination of latent virus from brain. Latent HSV genomes can be demonstrated by PCR analysis up to 1 year after inoculation (11), and the number of persisting genomes determined by quantitative competitive PCR does not change between 1 and 8 weeks after inoculation (12). The time course of transgene expression is similar to that of viral replication, despite the fact that these vectors are incapable of replicating in brain and early viral genes (e.g., gB) remain undetectable; by the time latency would normally be established, transgene expression is silenced.

Latent vector genomes like latent wt virus continue to produce HSV LATs detectable by in situ hybridization and by reverse transcription-PCR, long after transgene expression is no longer detectable. Therefore, one strategy we have pursued is the use of the LAP element to drive transgene expression. Two different LAP sequences have been identified. LAP1 is a TATA-box containing promoter but lies 700 bp upstream of the 5′ end of the LAT intron, whereas the weaker LAP2 element lies directly upstream of LAT and is homologous to mammalian housekeeping gene promoters. Vectors with the reporter gene inserted into the native LAT intron, and therefore lying downstream of both LAP1 and LAP2, continue to express lacZ detectable by reverse transcription-PCR at 4 weeks after inoculation. Others have demonstrated that the LAP1 element loses its activity when transported to an ectopic locus within the viral genome (13), but we have found that LAP2-driven lacZ expression is detectable by reverse transcription-PCR for at least 2 weeks after direct inoculation into the brain. Several strategies, including constitutive autoenhancing and drug-inducible enhancer elements, that we have engineered into transiently expressing HSV vectors might be applied to increase the low level of persistent transgene expression achieved with this promoter.

A surprising finding that has emerged recently in studies of the multiply deleted viruses is that there appears to be a “failure” of remaining IE gene shutoff in these vectors, with HCMV-driven transgene expression persisting in a similar fashion. The triply deleted (THZ.2) vector continues to express ICP0 RNA for 2 weeks after intracranial inoculation and in vitro in cultures of cortical neurons. With this vector, lacZ expression driven by the HCMV IE promoter can be detected at 4 weeks both in vivo and 2 weeks in vitro. This suggests that in addition to reduced cytotoxicity, the multiply deleted vectors may provide a platform for long-term gene expression by allowing a variety of promoters to escape the natural silencing mechanism characteristic of HSV latency.

The HSV vectors described above are substantially improved both in terms of cytotoxicity and transgene expression. Viral IE gene deletion mutants that express only the transgene and the IE ICP47 gene should be highly effective for gene transfer without toxicity for brain. Moreover, infected neurons should be greatly protected from immune recognition by the action of the ICP47 gene product.

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2. DeLuca, N.A., McCarthy, A.M. & Schaffer, P.A. (1985) J. Virol. 56, 558–570.

3. Johnson, P.A., Miyanohara, A., Levine, F., Cahill, T. & Friedmann, T. (1992) J. Virol. 66, 2952–2965.

4. Johnson, P. A, Wang, M.J. & Friedmann, T. (1994) J. Virol. 68, 6347–6362.

5. Oroskar, A. & Read, G. (1989) J. Virol. 63, 1897–1906.

6. York, I., Roo, C., Andrews, D., Riddell, S., Graham, F. & Johnson, D. (1994) Cell 77, 525–535.

7. Schaffer, P.A., Carter, V.C. & Timbury, M.C. (1978) Virology 27, 490–504.

8. Rasty, S., Goins, W.F. & Glorioso, J.C. (1995) Methods Mol. Genet. 7, 114–130.

9. Mester, J.C., Pitha, P. & Glorioso, J.C. (1995) Gene Ther. 3, 187–196.

10. McCarthy, A.M., McMahan, L. & Schaffer, P.A. (1989) J. Virol. 63, 18–27.

11. Fink, D.J., Sternberg, L.R., Weber, P.C., Mata, M., Goins, W.F. & Glorioso, J.C. (1992) Hum. Gene Ther. 11–19.

12. Ramakrishnan, R., Fink, D.J., Guihua, J., Desai, P., Glorioso, J.C. & Levine, M. (1994b) J. Virol. 68, 1864–1870.

13. Lokensgard, J.R., Bloom, D.C., Dobson, A.T. & Feldman, L.T. (1994) J. Virol. 68, 7148–7158.



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