This paper was presented at a colloquium entitled “Genetic Engineering of Viruses and of Virus Vectors,” organized by Bernard Roizman and Peter Palese (Co-chairs), held June 9–11, 1996, at the National Academy of Sciences in Irvine, CA.
Replication-defective herpes simplex virus vectors for gene transfer in vivo
(gene therapy/neurons/latency)
PEGGY MARCONI*, DAVID KRISKY*, THOMAS OLIGINO*, PIETRO L. POLIANI†, RAMESH RAMAKRISHNAN*†, WILLIAM F. GOINS*, DAVID J. FINK*†, AND JOSEPH C. GLORIOSO*‡
*Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; and †Department of Neurology, University of Pittsburgh School of Medicine, and Veterans Affairs Medical Center, Pittsburgh, PA 15261
Herpes simplex virus 1 has a number of biological features which suggest that it could be engineered as a vector for direct transfer of therapeutic genes to neurons. These features include (i) its natural ability to establish life-long latency, a state in which the viral genome is not integrated, lytic genes are quiescent, and the metabolic functioning of the host cell is apparently undisturbed; (ii) the expression of latency-associated transcripts (LATs) driven by neuron-specific, latency-active promoter (LAP) elements, which may prove useful in expressing transgenes from latent viral genomes; and (iii) the observation that replication-defective mutants created by the deletion of essential genes retain the ability to establish a latent state in the nervous system (1). In addition, many of the 81 herpes simplex virus (HSV) genes are not required for viral replication in cell culture and may conveniently be deleted to provide space for incorporation of substantial foreign DNA, and almost all viral genes are contiguous units, making genetic manipulation feasible. The virus can also be grown to high titer, and viral infectivity is very efficient. The major impediments to the development of HSV-effective vectors relate to residual cytotoxicity of defective vectors and the limited duration of transgene expression. Even replication-incompetent mutant viruses are cytotoxic, readily killing neurons in vitro, and with the exception of the HSV LAP elements, viral and foreign promoters appear to come under control of the virus’ ability to rapidly induce mechanisms of promoter shutoff.
Two different types of HSV-based gene delivery systems have been developed. The first type consists of genetically engineered genomic vectors, which may be deleted in genes required for the virus to replicate in postmitotic cells such as neurons or may be completely replication-defective, requiring complementation for vector propagation. The second type of HSV-based vector system, referred to as amplicons, uses defective helper-virus mutants for packaging concatemeric plasmids containing an HSV origin of DNA synthesis and a packaging sequence. We have focused our efforts on the development of replication-defective genomic vectors.
The “first generation” defective genomic vectors were deleted in the single essential immediate early (IE) gene encoding ICP4 (e.g., d120) (2). These vectors can be propagated in ICP4-complementing cell lines, but on infection of neurons, viral gene expression is aborted at the level of IE gene expression. Although these vectors are of reduced pathogenicity and can be used to efficiently transfer and transiently express reporter genes in brain (see below), they are toxic to neurons in culture, producing cytopathic effects such as cytoplasmic blebbing, host cell DNA fragmentation, and chromosomal aberrations (3). It is presumed that residual cytotoxicity results from the expression of HSV gene products, because UV-irradiated viral particles are not toxic and interferon treatment to disrupt IE gene expression markedly reduces cytotoxicity. Although deletion of the gene coding for ICP4 aborts the expression of both early and late viral genes, the other four immediate early gene products and ICP6, the ribonucleotide reductase large subunit, are overexpressed in the absence of ICP4. ICP4, ICP0, ICP27, and ICP22 have all been shown to be toxic in stable transfection assays (4), so deletion of these genes in combination may be required to eliminate toxicity. UL41, although not an IE gene product, is present in the virion and is responsible for shutoff of host cell protein synthesis through destabilization of host cell mRNA (5); in addition, UL41 many reduce transgene expression from HSV vectors. ICP6 and ICP47 do not appear to contribute to viral toxicity, and indeed, ICP47 expression may prove to be an asset because it contributes to escape from host immune surveillance (6).
To date we have succeeded in constructing the required complementing cell line and producing vectors in which all five targeted genes have been deleted in various combinations, although we have not produced a single mutant lacking all the targeted genes (Table 1). The deleted genes include those encoding ICP0, ICP4, ICP27, ICP22(US1.5), and UL41; one mutant has been deleted for all IE genes except those coding for ICP0 and ICP47 and shows substantially reduced cytotoxicity on infection of Vero cells at multiplicities of infection up to 50. Infection with this vector results in both vector persistence without genome integration and sustained transgene expression, and it does not block cell division. Cortical neurons in culture infected with either the triple-deleted (THZ.2) and quadruple deleted (THZ.3) vectors show substantial neuronal survival up to 2 weeks, in contrast to the singly deleted ICP4− vector SHZ.1, which kills these cultured cells within 24 hr.
Experimental studies in rats have demonstrated relatively efficient and robust transient transgene expression in brain after direct intracranial inoculation with a number of different vectors. Although singly deleted viruses are toxic to neurons in culture, animals uniformly survive direct injection of these replication-defective or incompetent vectors into brain with only minor pathologic evidence of cell loss. Reporter genes under the control of herpes simplex viral (gC) or other viral human cytomegalovirus IE, mammalian neurofilament, or
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Abbreviations: HSV, herpes simplex virus; LAT, latency-associated transcript; LAP, latency-active promoter; IE, immediate early; HCMV, human cytomegalovirus.
‡ |
To whom reprint requests should be addressed at: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, Pittsburgh, PA 15261. e-mail: joe@server1.mgen.pitt.edu. |
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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