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The use of herpes simplex virus-based vectors for gene delivery to the nervous system
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The use of herpes simplex virusbased vectors for gene delivery to the nervous system Robin H. Lachmann and Stacey Efstathiou
The ability of herpes simplex virus (HSV) to establish a lifelong, latent infection within neurons has led to much interest in the development of HSV-based vectors for neuronal gene delivery. This review discusses the progress made towards the construction of safe, replication-disabled HSV vectors that are capable of directing long-term transgene expression in latently infected neurons. Such vectors are now being investigated in a variety of animal model systems, with a view to developing gene therapy approaches to a number of metabolic and degenerative neurological diseases. GENE therapy ha potential therapeutic applications in the treatment of a wide variety of di ea es, including inborn errors of metabolism, infectious disease. and cancer. Each of these potential applications involve a different trategy, be it stable, long-term gene expression to replace a missing enzyme, or transient production of an immunomodulatory mol ecule or a toxin within a malignant cell. In all ca. es, however, a vector of some description i required to deliver the therapeutic gene to the nucleus of the target cell.
Box 1. The use of HSV as a neuronal gene delivery vector Advantages of HSV vectors • Ability to establish lifelong latent infection in neurons. • High titre stocks (10 111 pfu ml - I) can be readily produced in ti ue culture. • 152 kb D A gerome containing multiple, non-essential genes that can be deleted to accommodate large in ert . Disadvantage of H V vectors • Wild-type virus is highly cytotoxic. • Wild-type virus can reactivate from latency. • High prevalence (-80%) of seropositivity in the general population. Categorie of vector • Replication-defective HSV vectors - at lea t one essential viral gene is deleted 0 that the viru can only be propagated in a complementing cell line. • Amplicon vectors - plasm ids containing both bacterial and HSV origins of replication and an HSV-packaging igna!. When amplicontransfected cells are superinfected with an HSV helper virus, the amplicon is packaged into HSV particles.
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Interest in herpes simplex virus (I-J V) as a gene deliver} vector (see Box]) ari es from its natural ability to establi h a lifelong latent infection in neurons!, making it a particularly attractive candidate as a vector for gene delivery to the nervou y. tern.
consists of a long and a short unique region, each flanked by inverted repeats (see Figs 1 and 2). Upon uncoating of the viral D A in the nucleus of the infected cell , immediate early (IE) gene expression is activated by the action of a structural component of the viral particle - Vmw65 (also known as VP16 or o:TIF). Vmw65 interacts with a specific sequence element in the promoters of the five IE genes4, in conjunction with cellular proteins Oct-1 and complex-forming factor (CFE also known as HCF, VCAF-l and C1). A number of IE protein fu nction to activate early viral gene expression which, in tum, leads to replication of viral 0 A followed by the expression of the late, mostly structural, genes and the formation of progeny virions.
Biology of herpes simplex virus Herpes simplex virus type] (HSV-l ) is a natural pathogen of humans, ca using recurrent oropharyngeal cold ores. The virus is tran mitted by close personal contact with an infected individual; initially, the vi ru establi hes a productive infection in epithelial cells. gain access to the sensory nerve end ing upplying the infected area of skin and travels by retrograde axonal flow to neuronal cell bodies within the respective dor al root ganglia~. Lytic viral replication in the nervous sy tern i generally limited, and the viru can e tablish a latent infection within sensory neurons. During latency, which la ts for the lifespan of the individual. the viral genome persists within the neuronal nucleu in the ab ence of any detectable viral protein expression 2• The lytic pathway of HSV infection in tis ue culture cells has been extensively studied) and is summarized in Fig. 1. The viral genome
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HSV latency The biological function of latency is to allow the virus to persist in the individual in the presence of an effective immune response. Reactivation then gives the virus an opportunity to infect other susceptible individuals, conferring a significant survival advantage to the virus within the human population. o vi ral gene express ion is required for either the establishment or the maintenance of viral latency. The latent genome exists in an
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Figure 1. The HSV-1 lytic cycle of Infection. (a) After binding and entry, the unenveloped VIrion IS (b) transported to the nuclear pore, where (c) uncoating of viral DNA takes place followed by CIrcularizalion of the genome. (d) Inside the nucleus, immediate-early (IE) gene expression is activated by the binding of a complex of the virion tegument protein Vmw65 (also known as VP16 or a-TIF), the cellular transcription factor Oct-1, and complex forming factor (CFF) to a common sequence element in the promoters of the five IE genes. The position of the IE transcription units within the HSV genome is shown. (e) The early genes, which are only expressed in the presence of IE gene products, are responsible for (~ replication of the viral DNA, which is thought to occur by (g) a rolling circle mechanism. (h) This is followed by expressIon of the late, (i) mostly structural genes, and (j) the assembly of progeny virions, which subsequently (k) gain an envelope and eXIt the cell. This lytic cascade of gene expression eventually leads to cytolysis and cell death.
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although the virus can establish latency even if the LAT region is deleted, such deletion mutants might not reactivate as effici ently as wi ld-type vi rus in some animal models7•M•
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[n developing HSV as a potential gene delivery vector, two basic issues mu t be addre sed. First, the virus vector must be 'safe', both to the patient and to the environment; and second, strategies for obtaining prolonged, stable transgene expression from the latent viral genome must be developed.
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Figure 2. (a) HSV-1 latent gene expression. The latent genome is represented as a circular episome. One of the repeat regions (F\-Rsl is expanded and the relative positions of a number of lytic transcripts and the latency-associated transcripts (LATs) are shown. During latency. LATs are transcribed from the latency-associated promoter (LAP) . It is believed that the high-abundance major LAT is a stable intron that has been spliced from the low-abundance minor LAT (mLAT) . All the LATs are located in the nucleus in the latently infected neuron. (b) A section of dorsal root ganglion from a mouse latently infected with HSV-1 . In situ hybridization has been performed with a major -LATspecific nboprobe. The arrows show the characteristic nuclear localization of major LAT. Scale bar. 10 fLm.
'endless' form , which is not integrated into cellular DNA, and eems most likely to be either a circular episome or a concatameric species5 . o viral protein production has been detected in latently infected neurons, but once latency has been established, a family of nuclear RNA species [the latency-associated transcripts (LATs») can be detected. These tran cripts are transcribed from a diploid region (the LAT region) located within the viral terminal repeats Oankjng UL (Fig. 2)6. The precise functions of the e transcripts are unknown and , 406
Generating safe HSV vectors Disabled viral veclors Although latent HSV infection of neurons does not cause any detectable disturbance to the physiology of the cell, the initiation of the lytic cycle of infection is highly cytotoxic. Any virus that is to be used for neuronal gene delivery will have to be fully attenuated, such that it can safely be given by direct intracranial injection, in a high dose. Strategies for producing attenuated viruse are described in Box 2. Because the virus can establish a latent infection without expression of any viral genes, such attenuation is unlikely to dec rea e the efficiency of latency establishment or gene delivery. Indeed, there i evidence that attenuated viruses are actually more efficient at e tablishing neuronal latency than wild-type viru es9, presumably because a higher proportion of infected cells becomes latently infected, rather than undergoing cytolysis. Viruses with mutations in the Vmw65 (Ref. ] 0) or lE3 (Ref. 1]) gene have been developed and used 111 vivo. They can establish neuronal latency and can be administered by direct intracranial injection. They would, therefore, seem to be promising candidates for gene delivery vectors. However, when such vectors are injected intracranially, although they do establish latency in a proportion of infected neurons, considerable acute cytotoxicity can till be demonstratedl ~ . To characterize this further, viruses deleted for various combinations of [E genes have been studied in tis ue culture. At high multiplicitie of infection, such viruses still display some cytotoxicityl3, which appears to be due to a low level of expression of the remaining, intact IE genes. If Vmw65 is mutated in addition to lEI and IE3, then it is possible to produce a virus that shows no cytotoxicity when cells are infected at high multiplicitie of infection l4. Alternatively, because a complementing cell line that expresse IE3 and IE2 (the only essential IE gene) has now been produced l 3, it should be possible to generate and propagate HSV mutants in which all potentially cytotoxic IE genes have been deleted. The is ues of cytotoxicity discussed above must be differentiated from those of environmental safety. Steps must be taken to prevent the generation of replication-competent virus carrying biologically active genes, either during vector propagation or following in vivo vector delivery. To prevent homologous recombination during vector propagation between the vector and the viral sequences contained within the complementing cell, it is necessary to ensure that all sequences that have been inserted into the cell line have been deleted from the virus. An example of such a system, where there is only a 92-base-pair overlap present between viral and cellular sequences l5, is based on the deletion of the essential HSV-l glycoprotein H (gH) gene l6 (Fig. 3). gH is essential for viral infectivity. Viru propagated in the complementing cell line has gH derived from the cell in its envelope, and is fully infectious. If, however, such a virus is used to infect cells not capable of
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Box 2. The generation of non-cytotoxic H5Vvectors Preventing entry into the lytic cycle If immediate-early (IE) gene expression can be prevented, then the virus cannot initiate the ca cade of lytic gene expression. Three groups of potential genes could be targeted: Essential IE genes IE3 (ICP4), a major transactivator of early gene expression. IE2 (ICP27), involved in the po t-transcriptional regulation of viral gene expression. Viruses deleted for these genes need to be propagated in a complementing cell line. Non-essential IE genes lEI (ICPO), a promiscuous transactivator of viral and cellular promoters IE 4 (/CP22) , modifies cellular RNA polymerase II Viru es deleted for these genes can be propagated in tissue culture, but often with very poor efficiency. The virion transacti vator protein Vmw65 (vp16, ex -TIF), an essential component of the viral coat, required for the efficient activation of IE gene expression. Viruses containing a mutant Vmw65 that is deficient in transactivating function can be propagated in ti ue culture using hexamethylene bisacetam ide44 . Attenuation of neurovirulence A num ber of genes required for virus neurovirulence are non-essential in tissue culture, and can be deleted. These include: thymidine kinase, -y34.5, ribonucleotide reductase Deletion of genes encoding essential structural components If the gene for an es ential tructural componenl of the virion is deleted fro m the viral genome and supplied ill trans by a complementing cell line, then the virus will not be able to generate infectious progeny when non-complementing cells are infected. These genes include: glycoprotei n H (gH), glycoprotein B (gB) and Vmw65.
supplying gH in trans, it will only be able to undergo a single round of viral replication, because the progeny virions will lack gH and, therefo re, be non-infectious. It has been shown that vector tocks produced using the gH- supplying cell line do not contain detectable wild-type virus l6 . Although still cytotoxic to the initial cell it infects, such a disabled virus cannot spread, and poses no environmental hazard. If a reco mbinant HSV vector were to infect a human subject who already harboured a wild-type latent virus, then it is theoretically possible that recombination between the vector virus and a reactivated wild-type genome could produce a fully virulent virus that also carried the transgene. If the transgene is in erted in place of an essential gene, the possibility of generating a wild-type virus carrying the transgene by homologous recombination with a latent genome is abolished because the transgene could only be transferred to a reactivated wild-type genome in exchange for the essential gene.
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Figure 3. A system for propagating a glycoprotein H (gH) herpes simplex virus type 1 (HSV-1) vector in a complementing cell line. (a) The gH gene has been deleted from the genome of the virus (the deletion is represented by the broken line) . A complementing cell line (b) has been constructed that contains the gH gene. When the virus is propagated in the complementing cell line it can obtain the gH protein from the cell, and the viral progeny (e) are infectious. Virus that has been grown in the complementing cell line can be used to infect non-complementing cells (d). Progeny virus from this infection (e) will not contain gH in its envelope, and will be unable to infect further cells.
We must also consider the theoretical possibility that infection with an HSV vector virus might lead to reactivation of a wild-type latent genome, which could, in turn, lead to encephaliti . We know that expression of the lEI gene is closely involved in reactivation, and deletion of this gene would , therefore, improve vector safety, a well as reducing cytotoxicity. In summary, although we already have vectors that are suitable for the experimental investigation of gene therapy applications in animal models, if a safe, non-cytotoxic HSV vector suitable for clinical use is to be produced, it will be necessary to incorporate several mutations into the viral genome to prevent IE gene expres ion, and to construct a cell line capable of complementing a number of essential viral functions.
Amp/icon vectors An alternative approach to gene delivery using HSV has been the development of the amplicon system. An amplicon is a plasmid that can be propagated in both prokaryotic and eukaryotic cells. This is accomplished by incorporating the origin of replicat ion from HSV and an HSV-packaging signal into a plasmid. If such an amplicon i transfected into eukaryot ic cells that are then superinfected with an HSV helper virus, the amplicon will be replicated along with the viral genome, and packaged into viral capsids 17 • The progeny viruses will be a mixture of the helper virus and amplicon-containing virions, and can be used to infect more cells. The issues that must be addressed in developing the amplicon system for therapeutic gene delivery are similar to those encountered when using disabled viral vectors, namely the cytotoxicity and afety of the helper virus used, and the ability to obtain long-term gene expression from the amplicon-infected cell. A recent advance has been the development of an amplicon-packaging system that does not produce helper-virus particles 18 This system involves co-transfection of the amplicon plasmid with a et of cosmids that contain the whole HSV- l sequence, without the viral
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moters, except for the latency associated promoter (LAP), are tran criptiona ll y silenced I" . There have been two basic approaches to obtaining long-term gene expression in latently infected neurons. Th e first involves the insertion of strong heterologous promoter elements into the viral genome. The econd approach is to attempt to obtai n reporter gene expression from the endogenous vira l LAP.
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When otherwise trong R A polymerase II promoters, such as the cytomegalovirus (CMV) IE promoter or the Moloney murin e H H _ll _ _ _ leukaemia virus (MoMuLV) long terminal repeat (LTR). linked to the ~-galactosidase reporter gene. are inserted into the viral HSV-1 LBA genome at regions distal to the LAP, high IRES lacZ CMV poly A leve ls of reporter-gene express ion can be ob. erved during acute lytic infection, in tissue cu lture and in experimental anim als. If an an imal model of latency i used, however, gene expression is downregulated once the b c virus has established latency21J.21. Long-term gene express ion using neuron- pecific promote rs (e.g. neuron-specific enolase 22 , preproenkephalin promoter 23) has been reported, but thi is associated with a decrease in the number of transgene-expressing cells with time. Similar to these observations made with a number of heterologous pol ymerase II promoters. an RNA pol ymerase I promot er (which would be ex pected to be con litutively act ive in all cell types) could not drive tran gene express ion from the latent HSV- J Figure 4. (a) A schematic structure of the wild· type VIrUS HSV·l L~A. The Viral genome compnses two rege nome24. gions, unique long (UJ and unique short (Usl and IS shown With the internal repeat region expanded. The Therefore, exogenous promoters seem to POSition of minor and major latency·associated transcnpts (mLAT and LAT, respectively) is shown, along With the small Hpal (H) deletion used for insertion of the expression cassette. In this cassette, the lac Z rebe downregulated in a similar manner to the porter gene has been linked to an Internal nbosomal entry site (IRES). This IRES element functions to endogenous promoters of the virus, when allow nbosomes direct access to the mRNA. and to begin translation of the reporter gene without having to placed in the contex t of the latent genome. scan through the upstream leader sequence. The cytomegaloVIrUS polyadenylation Signal (CMV polyA) lies More recentl y, however, it has become clear downstream of the reporter gene. (b) Photomicrograph of a whole mount preparation of a murine cervical dorsal root ganglion. This was processed 190 days after the animal had been Infected In the ear with HSVth at this promoter sil encing might not apply 1 L~A. The ganglion has been histochemically stained for ~-galactosidase activity uSing the X-gal reagent, to all regions of the vira l genome equally. A which gives a blue preCIpitate In ~ ·galactos l dase-expresslng cells. Large numbers of latently Infected neunumber of groups have shown that if the rons are prodUCing ~ -galactosldase more than six months after Initial infection. Scale bar, 30 ~m . (c) A MoMuLV LTR is inserted within the LAT rephotomicrograph showing a section through the faCial nerve nucleus In the bralnstem of a mouse, 80 days gio n, it is possible to obtain long-term gene after penpheral ear Inoculation With HSV-l L ~A. It has been stained for ~ -galactosldase activity. A number of latently Infected neurons (stained blue) can be seen. Scale bar, 30 ~m . ex pression2) 21>. These re ult uggest that the LAT region must conta in elements that ensure it remains accessible to the transcrippackaging ignal. .· Lytic ' infection en\ue,>, but the only DNA that tional apparatu during neuronal latency. for transcription [rom the can be packaged into the progeny virion'> is the ampl icon pla~mid. core LAT promoter and from at lea. t some other promoter elements . Pure' amplicon \tock~ ~uch as the e shou ld be entirely free of any if they are inserted nearby . cytotoxic effect. that normally resu lt from viral gene express ion. mLAT
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Strategie to obtain prolonged tran gene expression from the latent HSV genome Although we know that the H V genome can per. ist 'indefinitely' in latently infected neuron. it i of note that latent transcription is highly re tricted . ln the context of the latent geno me. all vira l pro408
The LATs continue to be transcribed during latent infection and, therefore. the LAP would seem to be a good potential candidate as an elem ent capab le of driving long-term gene expre ion. Initi al attempt~ at harnessing this promoter to drive transgene expression in vo lved the insertion of reporter genes immediatel y downstream of
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Table 1. Examples of potential targets for HSV mediated gene delivery Disease
Therapeutic gene product(s)
Neurodegenerative diseases Parkinson 's disease
Tyrosine hydroxylase
Neurotropic factors
Cosm ids - Plasm ids that can be packaged by )., phage to allow easy purification.
Inborn errors of metabolism Mucopolysaccharidosis type VII
~·Glucuronidase
Tay-Sachs disease
Hexosaminidase A
Neoplasia Malignant gliomas
Complementing cell line - A cell line that has been stably trans· fected with the genes encoding one or more viral proteins. The cell line itself provides these proteins, and can, therefore, support the replication of a virus from which the relevant genes have been deleted. Concatamers - DNA molecules consisting of multiple linear copies of the VIral genome, joined up in a head·to-tail manner.
Neurotropic factors Huntington's disease
Glossary
p53 Direct cell killing by replication competent, non·neurovirulent virus
Ihe LAT transcriplional slart ite c-. This virus produced Iransient. high levels of reporter gene expression but. although there was . ome long-term expres ion, this wa at lower levels and in fewer cells than would be expected for LAT expre sion from wild-t) pe virus. Similarly, if the core LAP was linked to a reporter gene and in erted into an ectopic site in the viral genome. long-term gene expres ion was not observed c1 Interestingl), when the core LAP wa. linked to the MoMuLV LTR and inserted into the same site, long-term reporter-gene expression was observed in latently infected sensory neurons~l . Such hybrid promoters merit further investigation. The evidence from these and other tudies c' indicates that the LAT regulatory region is complex. and that elements both upstream and downstream of the transcription start point need to be left intact if we are to obtain reporter gene expres ion with kinetics that mimic wildtype LAT expression. A strategy has been devised in our laborator) that allows the insertion of a reporter gene. a full 1.5 kilobases downstream of the minor LAT transcriptional start site. maintaining all currently recognized elements of the LAP (Ref. 29) (Fig. 4). Using this strategy, we have constructed a \ iral vector th at can produce ~ galactosidase expression in the dorsal root ganglia and brainstems of latently infected mice. with kinetics closely resembling those that would be expected if it were under authentic LAP control. Although there is only low-level gene expre. sion during lytic infection, when the virus enter neuronal latenc). the reporter-gene expression is upregulated, and remains stable at high levels for at least six months~~. This ability to 'harne . the endogenou. LAP regi on of the virus to drive long-term reporter gene expression in neurons of both the peripheral and central nervou ystem (CNS) open. the way for the experimental evaluation of the use of disabled HSV vector to express therapeutic genes in the nervous . ystem.
Candidate diseases for gene delivery by an HSV vector Gene therapy has been suggested as a potential therapeutic approach to a vast range of neurological disorders. ranging from inherited metabolic disorders to degenerative disease and malignancy (sec Table I). The majority of these diseases affect large region. of the C S, and the delivery of a replication-defecti ve \irus to the whole of the brain
Immediate early genes - The first class of HSV genes to be expressed in the Iytically infected cell; they require no de novo protein synthesis for their expression. Internal ribosomal entry site - An RNA sequence that allows a ribosome to gain direct access to an mRNA molecule without having to bind to the cap structure at the 5' end and scan through the 5' leader sequence. Latent infection - In latently infected nervous tissue, viral DNA per· sists in the absence of any detectable infectious virus. Lysosoma l storage disorders - Disorders of lysosomal enzymes or transport that lead to the intraorganellar accumulation of un· degraded substrates derived from cellular macromolecules. Lytic infection - During lytic infection, the virus takes over the metabolic apparatus of the cell , in order to replicate itself. This re· suits in the production of progeny virions, and the eventual destruction of the host cell. Retrograde axonal flow - The process by which neurons transport material from the synapse to the cell body.
poses great technical problem . . By far the most widely used and well-characterized system for delivery is that of direct stereotactic injection into small. well-defined regions. Therefore, disease processes such as Parkinson 's disease 111 or malignancy, which cxhibit pathology at a di crete anatomical location within the nervous system. providc some of the most attractive models for genc therapy. An amplicon-based HSV vector has been used to deliver a tvrosine hydroxylase gene to striatal neurons in a rat model' of Parkinson's di, ease 1). with thc aim of increasing local levels of dopamine, the neurotransmitter that is deficicnt in this disease. It should be noted that this is an example of a di case model where careful regulation of the level of expression or the therapeutic gene will be required to avoid the potential side effects associated with o\'er-cxpression. The. e invc. tigator. reported some uccess in correcting the abnormal behaviour displayed by these animals, although they encountered considerable problems with vector toxicity beG1U~e of a reversion of the temperature-sensitive hclper virus (tsK) to the wild type. An approach that might ea ily be adapted to a range of neurodegenerative diseases has been the attempt to slow down or halt neuronal death_ by the local delivery of a vector canying a gcne for a ncurotrophin. Geschwind and colleagues.1~ have demonstrated that neurite outgrowth from auditory neurons can bc obtaincd when explant
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The outstanding questions • Will it be pos ible to con truct vectors that will be sufficiently non-cytotoxic for clinical use? • Will the currently defined promoter-constructs be able to mediate long-term gene expres ion in a wide variety of different neuronal types within the central nervou system (CNS)? If so, will they be able to mediate sufficient production of transgene to correct, or prevent, pathology? • For a succe sful gene therapy approach to most neurological disorder, large regions of the CNS will have to be transduced. Will it be pos ible to devise methods to deliver a vector throughout the C S? • What will be the con equence of u ing HSV vectors in patients who have already been exposed to natural HSV infection? Will the pre-existing immune response to HSV prevent efficient gene delivery? What ri ks will be associated with the pre ence of pre-existing latent genomes within the CNS? • Will fully disabled HSV-based vectors have therapeutic applications for gene delivery to non-neuronal tis ues?
cultures of murine spina l ga nglia are infected with an amplicon vector containing a cD A that encode brain-derived neurotrophic factor. The e results how that, in principle. this approach might be useful, although fu rther studies in animal models are required. Similar approaches might be used to delay a number of neurodegenerative diseases, even dementias with widespread pathology such as Al zheimer's disease; this is providing that a method could be devised to deliver the vector to a large enough area of tis ue. Other candidates for curative treatment by gene therapy are the inherited error of metabolism. Many of these produce devastating neurological disea e, and functional replacement of the affected gene might provide complete metabolic correction. Of the monogenic diseases that affect the brain, the Iy osomal storage diseases (examples of which are Gaucher' disease, Tay-Sachs disease and metachromatic leukodystrophy) represent attractive target for gene therapy" . Although these disorders affect the whole brain, the cellular defect can be corrected by the uptake of exogenously produced enzyme. Unfortunately, the blood-brain barrier prevents systemically administered enzyme (i.e. glucocerebrosidase u ed to treat patients with Gaucher's disease) from reachi ng the ceiL of the C S. but this can be circumvented if a gene therapy approach is used to produce the enzyme within the brain. The ecretion-recapture mechanism means that a significant effect could be achieved if only a . mall number of cell were tran duced, provided that they could be made to secrete sufficient enzyme. To date, the only model of a lysosomal storage disorder in which HSV vectors have been u ed is the mucopolysaccharidosis type VII (Sly disease) mouse, which i deficient in ~-glucuronidase34 . Wolfe and col leagues were. however, unable to demonstrate any correction of pathology. probabl y because on ly low levels of ~-glucuronidase were produced in a small number of neuron ~ following the peripheral administration of the replication-competent vi rus used in this study35 . one the Ie , this group did provide an importa nt proof of principle by demon trating that it was possible to obtain production of functional enzyme and, with the use of more recently developed replicat ion-defective HSV vectors, it might be possible to obtain more promising results. 410
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Genetically engineered HSV vectors are also being developed for use in the treatment of malignant gliomas. Here, the intention is not to produce long-term transgene expression in latently infected cells, but to selectively kill the malignant cells, while sparing the surrounding healthy neural tissue (reviewed in Ref. 36). A number of groups have achieved this in animal models by using HSV vectors, deleted for non-essential neurovirulence genes [i.e. "(34.5 (Ref. 37 and 38), thymidine kinase (tk) and ribonucleotide reductase (Ref. 39)]. These viru es will replicate in the dividing tumour cells, but not in terminally differentiated neurons. Using anim al models, it has been shown that when directly injected into gliomas, such viruses can produce tumou r regression with minimal bystander effects on surrou nding normal tissue.
Future prospects The advent of fully replication-defective HSV vectors, capable of medi ating long-term gene expression in neurons, will open the door to a host of potential therapeutic uses. Each of the 'e will have to be carefully eva luated in a suitable animal model before clinical tri als can be considered. Perhaps the most difficult problem to overcome will be to devise methods to deliver a vector to large areas of the C S, and the possibility of an immune response to these vectors in patients, up to 80% of whom will have pre-existing immunity to HSV. A pre-existing immune response to HSV might hinder the ini tial delivery of the vector, and this might be overcome by transient immunosuppresion to cover the procedure. Once latency has been established, the immune re ponse to HSV should not pose a long-term problem, becau e no viral proteins are made in the latently infected cell. An immune response to the transgene product, however, might lead to problems in maintaining long-term transgene expression. The possibility of using HSV vectors to deliver therapeutic genes to non-neuronal tissue has al 0 been raised. If fully replicationdefective vectors that are no longer cytotoxic can establish latent infections in a wide range of non-dividing cell types, then they would have rea l potential as a universa l gene delivery system. However, at least in tissue culture studies, the LAP displays a degree of neuron specificity, so it may be necessary to use different promoter elements in non-neuronal tissues. [n the treatment of cancer there is no need to establish latency, and the ability of HSV to infect a wide range of human cells might make it an attractive candidate in the treatment of many different types of malignancies. A gH-deleted HSV vector that can transiently express immunomodulatory molecules in haemopoietic cells is currently being developed a a potential leukaemia therapy40. Encouraging results have been obtained using a number of vector ystems for gene delivery to neurons (reviewed in Ref. 41), including adenoviruses, adeno-associated virus and lentiviruses. In the deve lopment of all these vector systems, a with HSV, the issues of vector safet y and long-term transgene expression have to be addressed. With the advent of transgenic technology and the characterization of nat urall y occuring mutant trains, the number of small rodent models of genetic neurological diseases is ever increasing (i.e. mouse model of Tay-Sachs disease 42 and globoid cell leukodystrophy4J). The cha llenge we now face is to explore the therapeutic potential of neuronal gene therapy, using suitable animal models. References 1 Roizman , O. and Scars, A.E. (1987) An inquiry inlo Ihe mechanisms of herpes implex virus laleney. Ann. Rev. Microbiol. 41 ,543-571
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2 Whitle}, R.J. (1996) Herpes Simplex Viruses, in Fields l1rologl' (Fields, B. .. Knipe. D.M. and Howley. P.M.. cds). pp. 2297 - 2342. Lippwcotl-Ravcn 3 Roizman. B. and Scars. A E. (1996 ) Herpes sim plex viruses and their replication, in Fieldl Virologl (FIelds. B.N . Knipe. D.M and Howley, P.M.. cd~). pp. 2231 - 2295, Lippincotl-Ralcn 4 Bzik. D.J. and Preston. C.M. (19 6) Analy is of D A equences "hich regulate the transcription of herpe sim plex viru immediate early gene 3: D A sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide, Nile/. ACids Res. 14. 929-943 5 Ho. D.Y. (1992) Herpes simplex I'irus latency: molecular aspects. Prog. .\fet!. Virol. 39. 76-115 6 levens. J.G. el 01. (1987) RNA complementary to a herpes virus alpha gene mRNA i prominent in latentl) infected neurons, [lellC(, ~35. 1056-1059 7 lIill, J.M. el 01. (1990) Herpes simp lex lirus latent phase transcription facilitates in \,i\'o reactivation. I'irology 174.117-125 8 Maggioncalda. J el 01. (1996) herpes simplex virus t)pe I mutant "ith a deletion immediately upstream of tbe LAT locus establishes latenq and reactivates from latently infected mice with normal kinetic. 1. Neuromol. 2. 26 -27 9 Ecob-Prince, M. . el 01. (1993) Neuron containing latency-associated transcri pts are numerous and widespread in dorsal root ganglia following footpad inoculation of mice "ith herpe simplex virus t)pe I mutant inl814, 1. Gell. Virol. 74. 9 5-994 10 Ace. c.1. elili. (1989) Construction and characterization of a herpes sim plex viru type 1 mutant unable to tran induce immediate-early gene expression, 1. Virol. 63. 2260-2269 II DeLuca. .A .. McCarthy. AM. and Schaffer. P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type I in the gene encoding immediate-early regulatory protein ICN,J. \r,rol. 56. 55 -570 12 Ho, D.Y. el al. (1995) Herpe implex virus I'ector s}stem: analysi of its in vivo and in "ilro cytopathic effects,J. Neurosci. Melhods 57.205-215 13 Wu , N. el 01. (1996) Prolonged gene expre sion and cpll survhal after infection by a herpes sim plex yiru mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22,J. Virol. 70, 6358-6369 14 Preston. C.M .• Mabbs. R. and icholl. M.J. (1997) Construction and characterization of herpe simplex viru type 1 mutant with conditional defects in immediate early gene expression, Virology ~~9. 228-239 15 Boursnell, M.E. el al. (1997) genetically inactivated herpes implex virus type 2 (HSV.2 ) vaccine prol'ides effectil'e protection against primary and recurrent HSV-2 disea e,J. IlIfecI. DIS. 175, 16-25 16 Speck, P.G .. Minson, A.C. and Efstathiou. S. (1996) In \'i\'o complementation studies of a glycoprotein Ii-deleted herpes simplex I'irus-based lector,). Gen. Virol. 77. 2563-2568 17 Ho, D.Y. (1994) Amp licon-based herpes implex virus vectors, Melhods Cell Bioi. 43, 191-210 18 Fraefel, C. 1'1 al. (1996) Helper, iru -free tran fer of herpes implex virus type I plasmid vectors into neural cells,). I'irol. 70. 7190-7197 19 Fraser. .w. 1'1 al. (1991) A rel'ie" of the molecular mechanism of H V-I latency, Cllrr. E\'e Res 10, I- S 13 20 Ecob-Prince. M.. ell/I. (1995) Expre ion of beta-galactosida e in neuron of dorsal root ganglia which are latently infected with herpes simplex virus type 1,1. Gell. Virol. 76. 1527-1532 21 Lokensgard, J.R. 1'1 al. (1994) Long-term promoter activit" during herpes si mplex virus lat ency,.I. Virol. 6H, 714 -7158 22 Andersen, J.K. 1'1 al. ( 1 99~) Gene transfer into mammalian central nenous system usi ng herpes I iru lectors: extended expre ion of bacterial lacZ in neurons using the neuron - pecific enola e promoter. HI/m Gene Ther. 3. 4 7-499
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23 Kaplitt. M.G. 1'1 al. (1994) Preproenkephalin promoter yields region-specific and long-term expression in adult brain after direct in vi,'o gene transfer via a defecthe herpes si mplex viral vector, Proc. Natl. Mild SCI. U. S. A 91 . 8979-8983
24 Lachmann, R.II.. Brown. C. and Efstathiou, S (1996) A murine RNA polymerase I promoter inserted into the herpes simplex virus type I genome is functional during lytic, but not latent, infection,J. Gen. Vim/. 77. 25 75 2582 25 Dobson, AT el al. (1990) A latent, nonpathogenic Ii V-I-derived vector stably expresses beta-galactosidase in mouse neurons, Neuron 5, 353- 360 26 Carpenter. D.E. and Stevens, J.G. (1996) Long-term expression of a foreign gene from a unique position in the latent herpes simplex virus genome, Hum . Gent' Ther. 7, 1447- 1454 27 Dobson, AT el al. (1989) Identification of the latency-associated transcript promoter by expres ion of rabbit beta-globin mRNA in mouse sensory nene ganglia latently infected with a recombinant herpes simplex viru~, J. Virol. 63 , 3 44--3851 28 Margolis. TP. 1'1 al. (1993 ) Decreased reporter gene expre ion during latent infection with H V LAT promoter co nstructs, Virology 197, 585-592 29 Lachmann, R.II. and Efstathiou. S. (1997) Utilization of the herpes simple:\. viru type I latenc}-associated regulatory region to drive stahle reporter gene npre sion in the nen'ous sy tem,J. Virol. 71 . 3197-3~07 30 Bankiewicz, K., Mandel, R.J . and Sofroniew, M.V. (1993) Trophism, transplantation, and animal model of Parkinson's disease, Exp. Neurol. 124. 14(}-149 31 During. M.J. el al. (1994) Long-term behavioral recovery in parkinsonian rats by an HSV vector expressing tyrosine hydroxylase, SCiellce 266, 1399-1403 32 Geschwind. M.D. el al. (1996) Defective HSV-l vector expressing BD F in auditory ganglia elicit neurite outgrowth: model for treatment of neuron loss following cochlear degeneration, /fum. Gene Ther. 7. 173-1 2 33 Salvetti. A, Heard. J.M. and Uanos, O. (1995) Gene therapy of lysosomal storage disorders, Br. Met!. Bull. 51. 106-122 34 Wolfe , J.H.. Deshmane, .L. and Fraser. N.W. (1992) Herpesvirus ,ector gene transfer and expression of heta-glucuronidase in the central nervous system of MPS VII mice. Na l. Gellel. 1,379- 384 35 Deshmane, S.L. 1'1 al. (1995) An HSV-I containing the rat beta-glucuronidase cD A inserted within the LAT gene is less efficient than the parental strain at e tablishing a transcri ptionally active state during latency in neurons, Gelle Titer. 2,209-217 36 Kim. D.H. and McCorm ick. F. (1996) Replicating viruses a electi\'e cancer therapeutics, Mol. Met!. Today 2. 519-527 37 Chambers, R. el al. (1995) Comparison of genetically engineered herpes si mplex I'iruse for the treatment of brain tumors in a scid mouse model of human malignant glioma, Proc. atl. Acad. Sci. U. . A. 92, 1411 - 1415 38 Mincta. T el 01. (1995) Attenuated multi-mutated herpes implex viru -I for the treatment of malignant gliomas, al. Med. 1,93 943 39 Boviatsis, E.J. el 01. (1994) Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpe simplex virus vectors defective in thymidine kinase or ribonucleotide reductase, Gent' Ther. 1,323- 331 40 Dilloo, D. el III. ( 1997) A novel herpes vector for the high-efficiency lransduction of normal and malignant human hematopoietic cells, Blood H9. 11 9- 127 41 Blomer. . el al. (1996) Application of gene th rapy 10 the ,Hum. Mol Gellel. 5, 1397-1404 42 ango. K. el 01. (1995) Mouse models of Tay-Sachs and Sand hoff diseases differ in neurologic phenotYllc and ga nglioside metabolism, at. Gelle/. II , 170-176 43 Suzuki, K. and SUluki , K. (1995) The twitcher mouse: a model fur Krabbe disease and for experimental therapies, Braill Palhol. 5, 249- 25 44 McFarlane, M. Daksi~. J.l. and Preston. C.M. (1992) Hexamethylene bi 'acetamide stimulates herpe simplex virus immediate early gene expression in the absence of trans-induction by VmwIl5,.I. Gell. Viml. 73,285 292
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The use of herpes simplex virusbased vectors for gene delivery to the nervous system Robin H. Lachmann and Stacey Efstathiou
The ability of herpes simplex virus (HSV) to establish a lifelong, latent infection within neurons has led to much interest in the development of HSV-based vectors for neuronal gene delivery. This review discusses the progress made towards the construction of safe, replication-disabled HSV vectors that are capable of directing long-term transgene expression in latently infected neurons. Such vectors are now being investigated in a variety of animal model systems, with a view to developing gene therapy approaches to a number of metabolic and degenerative neurological diseases. GENE therapy ha potential therapeutic applications in the treatment of a wide variety of di ea es, including inborn errors of metabolism, infectious disease. and cancer. Each of these potential applications involve a different trategy, be it stable, long-term gene expression to replace a missing enzyme, or transient production of an immunomodulatory mol ecule or a toxin within a malignant cell. In all ca. es, however, a vector of some description i required to deliver the therapeutic gene to the nucleus of the target cell.
Box 1. The use of HSV as a neuronal gene delivery vector Advantages of HSV vectors • Ability to establish lifelong latent infection in neurons. • High titre stocks (10 111 pfu ml - I) can be readily produced in ti ue culture. • 152 kb D A gerome containing multiple, non-essential genes that can be deleted to accommodate large in ert . Disadvantage of H V vectors • Wild-type virus is highly cytotoxic. • Wild-type virus can reactivate from latency. • High prevalence (-80%) of seropositivity in the general population. Categorie of vector • Replication-defective HSV vectors - at lea t one essential viral gene is deleted 0 that the viru can only be propagated in a complementing cell line. • Amplicon vectors - plasm ids containing both bacterial and HSV origins of replication and an HSV-packaging igna!. When amplicontransfected cells are superinfected with an HSV helper virus, the amplicon is packaged into HSV particles.
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Interest in herpes simplex virus (I-J V) as a gene deliver} vector (see Box]) ari es from its natural ability to establi h a lifelong latent infection in neurons!, making it a particularly attractive candidate as a vector for gene delivery to the nervou y. tern.
consists of a long and a short unique region, each flanked by inverted repeats (see Figs 1 and 2). Upon uncoating of the viral D A in the nucleus of the infected cell , immediate early (IE) gene expression is activated by the action of a structural component of the viral particle - Vmw65 (also known as VP16 or o:TIF). Vmw65 interacts with a specific sequence element in the promoters of the five IE genes4, in conjunction with cellular proteins Oct-1 and complex-forming factor (CFE also known as HCF, VCAF-l and C1). A number of IE protein fu nction to activate early viral gene expression which, in tum, leads to replication of viral 0 A followed by the expression of the late, mostly structural, genes and the formation of progeny virions.
Biology of herpes simplex virus Herpes simplex virus type] (HSV-l ) is a natural pathogen of humans, ca using recurrent oropharyngeal cold ores. The virus is tran mitted by close personal contact with an infected individual; initially, the vi ru establi hes a productive infection in epithelial cells. gain access to the sensory nerve end ing upplying the infected area of skin and travels by retrograde axonal flow to neuronal cell bodies within the respective dor al root ganglia~. Lytic viral replication in the nervous sy tern i generally limited, and the viru can e tablish a latent infection within sensory neurons. During latency, which la ts for the lifespan of the individual. the viral genome persists within the neuronal nucleu in the ab ence of any detectable viral protein expression 2• The lytic pathway of HSV infection in tis ue culture cells has been extensively studied) and is summarized in Fig. 1. The viral genome
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although the virus can establish latency even if the LAT region is deleted, such deletion mutants might not reactivate as effici ently as wi ld-type vi rus in some animal models7•M•
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'endless' form , which is not integrated into cellular DNA, and eems most likely to be either a circular episome or a concatameric species5 . o viral protein production has been detected in latently infected neurons, but once latency has been established, a family of nuclear RNA species [the latency-associated transcripts (LATs») can be detected. These tran cripts are transcribed from a diploid region (the LAT region) located within the viral terminal repeats Oankjng UL (Fig. 2)6. The precise functions of the e transcripts are unknown and , 406
Generating safe HSV vectors Disabled viral veclors Although latent HSV infection of neurons does not cause any detectable disturbance to the physiology of the cell, the initiation of the lytic cycle of infection is highly cytotoxic. Any virus that is to be used for neuronal gene delivery will have to be fully attenuated, such that it can safely be given by direct intracranial injection, in a high dose. Strategies for producing attenuated viruse are described in Box 2. Because the virus can establish a latent infection without expression of any viral genes, such attenuation is unlikely to dec rea e the efficiency of latency establishment or gene delivery. Indeed, there i evidence that attenuated viruses are actually more efficient at e tablishing neuronal latency than wild-type viru es9, presumably because a higher proportion of infected cells becomes latently infected, rather than undergoing cytolysis. Viruses with mutations in the Vmw65 (Ref. ] 0) or lE3 (Ref. 1]) gene have been developed and used 111 vivo. They can establish neuronal latency and can be administered by direct intracranial injection. They would, therefore, seem to be promising candidates for gene delivery vectors. However, when such vectors are injected intracranially, although they do establish latency in a proportion of infected neurons, considerable acute cytotoxicity can till be demonstratedl ~ . To characterize this further, viruses deleted for various combinations of [E genes have been studied in tis ue culture. At high multiplicitie of infection, such viruses still display some cytotoxicityl3, which appears to be due to a low level of expression of the remaining, intact IE genes. If Vmw65 is mutated in addition to lEI and IE3, then it is possible to produce a virus that shows no cytotoxicity when cells are infected at high multiplicitie of infection l4. Alternatively, because a complementing cell line that expresse IE3 and IE2 (the only essential IE gene) has now been produced l 3, it should be possible to generate and propagate HSV mutants in which all potentially cytotoxic IE genes have been deleted. The is ues of cytotoxicity discussed above must be differentiated from those of environmental safety. Steps must be taken to prevent the generation of replication-competent virus carrying biologically active genes, either during vector propagation or following in vivo vector delivery. To prevent homologous recombination during vector propagation between the vector and the viral sequences contained within the complementing cell, it is necessary to ensure that all sequences that have been inserted into the cell line have been deleted from the virus. An example of such a system, where there is only a 92-base-pair overlap present between viral and cellular sequences l5, is based on the deletion of the essential HSV-l glycoprotein H (gH) gene l6 (Fig. 3). gH is essential for viral infectivity. Viru propagated in the complementing cell line has gH derived from the cell in its envelope, and is fully infectious. If, however, such a virus is used to infect cells not capable of
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Box 2. The generation of non-cytotoxic H5Vvectors Preventing entry into the lytic cycle If immediate-early (IE) gene expression can be prevented, then the virus cannot initiate the ca cade of lytic gene expression. Three groups of potential genes could be targeted: Essential IE genes IE3 (ICP4), a major transactivator of early gene expression. IE2 (ICP27), involved in the po t-transcriptional regulation of viral gene expression. Viruses deleted for these genes need to be propagated in a complementing cell line. Non-essential IE genes lEI (ICPO), a promiscuous transactivator of viral and cellular promoters IE 4 (/CP22) , modifies cellular RNA polymerase II Viru es deleted for these genes can be propagated in tissue culture, but often with very poor efficiency. The virion transacti vator protein Vmw65 (vp16, ex -TIF), an essential component of the viral coat, required for the efficient activation of IE gene expression. Viruses containing a mutant Vmw65 that is deficient in transactivating function can be propagated in ti ue culture using hexamethylene bisacetam ide44 . Attenuation of neurovirulence A num ber of genes required for virus neurovirulence are non-essential in tissue culture, and can be deleted. These include: thymidine kinase, -y34.5, ribonucleotide reductase Deletion of genes encoding essential structural components If the gene for an es ential tructural componenl of the virion is deleted fro m the viral genome and supplied ill trans by a complementing cell line, then the virus will not be able to generate infectious progeny when non-complementing cells are infected. These genes include: glycoprotei n H (gH), glycoprotein B (gB) and Vmw65.
supplying gH in trans, it will only be able to undergo a single round of viral replication, because the progeny virions will lack gH and, therefo re, be non-infectious. It has been shown that vector tocks produced using the gH- supplying cell line do not contain detectable wild-type virus l6 . Although still cytotoxic to the initial cell it infects, such a disabled virus cannot spread, and poses no environmental hazard. If a reco mbinant HSV vector were to infect a human subject who already harboured a wild-type latent virus, then it is theoretically possible that recombination between the vector virus and a reactivated wild-type genome could produce a fully virulent virus that also carried the transgene. If the transgene is in erted in place of an essential gene, the possibility of generating a wild-type virus carrying the transgene by homologous recombination with a latent genome is abolished because the transgene could only be transferred to a reactivated wild-type genome in exchange for the essential gene.
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We must also consider the theoretical possibility that infection with an HSV vector virus might lead to reactivation of a wild-type latent genome, which could, in turn, lead to encephaliti . We know that expression of the lEI gene is closely involved in reactivation, and deletion of this gene would , therefore, improve vector safety, a well as reducing cytotoxicity. In summary, although we already have vectors that are suitable for the experimental investigation of gene therapy applications in animal models, if a safe, non-cytotoxic HSV vector suitable for clinical use is to be produced, it will be necessary to incorporate several mutations into the viral genome to prevent IE gene expres ion, and to construct a cell line capable of complementing a number of essential viral functions.
Amp/icon vectors An alternative approach to gene delivery using HSV has been the development of the amplicon system. An amplicon is a plasmid that can be propagated in both prokaryotic and eukaryotic cells. This is accomplished by incorporating the origin of replicat ion from HSV and an HSV-packaging signal into a plasmid. If such an amplicon i transfected into eukaryot ic cells that are then superinfected with an HSV helper virus, the amplicon will be replicated along with the viral genome, and packaged into viral capsids 17 • The progeny viruses will be a mixture of the helper virus and amplicon-containing virions, and can be used to infect more cells. The issues that must be addressed in developing the amplicon system for therapeutic gene delivery are similar to those encountered when using disabled viral vectors, namely the cytotoxicity and afety of the helper virus used, and the ability to obtain long-term gene expression from the amplicon-infected cell. A recent advance has been the development of an amplicon-packaging system that does not produce helper-virus particles 18 This system involves co-transfection of the amplicon plasmid with a et of cosmids that contain the whole HSV- l sequence, without the viral
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When otherwise trong R A polymerase II promoters, such as the cytomegalovirus (CMV) IE promoter or the Moloney murin e H H _ll _ _ _ leukaemia virus (MoMuLV) long terminal repeat (LTR). linked to the ~-galactosidase reporter gene. are inserted into the viral HSV-1 LBA genome at regions distal to the LAP, high IRES lacZ CMV poly A leve ls of reporter-gene express ion can be ob. erved during acute lytic infection, in tissue cu lture and in experimental anim als. If an an imal model of latency i used, however, gene expression is downregulated once the b c virus has established latency21J.21. Long-term gene express ion using neuron- pecific promote rs (e.g. neuron-specific enolase 22 , preproenkephalin promoter 23) has been reported, but thi is associated with a decrease in the number of transgene-expressing cells with time. Similar to these observations made with a number of heterologous pol ymerase II promoters. an RNA pol ymerase I promot er (which would be ex pected to be con litutively act ive in all cell types) could not drive tran gene express ion from the latent HSV- J Figure 4. (a) A schematic structure of the wild· type VIrUS HSV·l L~A. The Viral genome compnses two rege nome24. gions, unique long (UJ and unique short (Usl and IS shown With the internal repeat region expanded. The Therefore, exogenous promoters seem to POSition of minor and major latency·associated transcnpts (mLAT and LAT, respectively) is shown, along With the small Hpal (H) deletion used for insertion of the expression cassette. In this cassette, the lac Z rebe downregulated in a similar manner to the porter gene has been linked to an Internal nbosomal entry site (IRES). This IRES element functions to endogenous promoters of the virus, when allow nbosomes direct access to the mRNA. and to begin translation of the reporter gene without having to placed in the contex t of the latent genome. scan through the upstream leader sequence. The cytomegaloVIrUS polyadenylation Signal (CMV polyA) lies More recentl y, however, it has become clear downstream of the reporter gene. (b) Photomicrograph of a whole mount preparation of a murine cervical dorsal root ganglion. This was processed 190 days after the animal had been Infected In the ear with HSVth at this promoter sil encing might not apply 1 L~A. The ganglion has been histochemically stained for ~-galactosidase activity uSing the X-gal reagent, to all regions of the vira l genome equally. A which gives a blue preCIpitate In ~ ·galactos l dase-expresslng cells. Large numbers of latently Infected neunumber of groups have shown that if the rons are prodUCing ~ -galactosldase more than six months after Initial infection. Scale bar, 30 ~m . (c) A MoMuLV LTR is inserted within the LAT rephotomicrograph showing a section through the faCial nerve nucleus In the bralnstem of a mouse, 80 days gio n, it is possible to obtain long-term gene after penpheral ear Inoculation With HSV-l L ~A. It has been stained for ~ -galactosldase activity. A number of latently Infected neurons (stained blue) can be seen. Scale bar, 30 ~m . ex pression2) 21>. These re ult uggest that the LAT region must conta in elements that ensure it remains accessible to the transcrippackaging ignal. .· Lytic ' infection en\ue,>, but the only DNA that tional apparatu during neuronal latency. for transcription [rom the can be packaged into the progeny virion'> is the ampl icon pla~mid. core LAT promoter and from at lea. t some other promoter elements . Pure' amplicon \tock~ ~uch as the e shou ld be entirely free of any if they are inserted nearby . cytotoxic effect. that normally resu lt from viral gene express ion. mLAT
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Th e LA T promoler
Strategie to obtain prolonged tran gene expression from the latent HSV genome Although we know that the H V genome can per. ist 'indefinitely' in latently infected neuron. it i of note that latent transcription is highly re tricted . ln the context of the latent geno me. all vira l pro408
The LATs continue to be transcribed during latent infection and, therefore. the LAP would seem to be a good potential candidate as an elem ent capab le of driving long-term gene expre ion. Initi al attempt~ at harnessing this promoter to drive transgene expression in vo lved the insertion of reporter genes immediatel y downstream of
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Table 1. Examples of potential targets for HSV mediated gene delivery Disease
Therapeutic gene product(s)
Neurodegenerative diseases Parkinson 's disease
Tyrosine hydroxylase
Neurotropic factors
Cosm ids - Plasm ids that can be packaged by )., phage to allow easy purification.
Inborn errors of metabolism Mucopolysaccharidosis type VII
~·Glucuronidase
Tay-Sachs disease
Hexosaminidase A
Neoplasia Malignant gliomas
Complementing cell line - A cell line that has been stably trans· fected with the genes encoding one or more viral proteins. The cell line itself provides these proteins, and can, therefore, support the replication of a virus from which the relevant genes have been deleted. Concatamers - DNA molecules consisting of multiple linear copies of the VIral genome, joined up in a head·to-tail manner.
Neurotropic factors Huntington's disease
Glossary
p53 Direct cell killing by replication competent, non·neurovirulent virus
Ihe LAT transcriplional slart ite c-. This virus produced Iransient. high levels of reporter gene expression but. although there was . ome long-term expres ion, this wa at lower levels and in fewer cells than would be expected for LAT expre sion from wild-t) pe virus. Similarly, if the core LAP was linked to a reporter gene and in erted into an ectopic site in the viral genome. long-term gene expres ion was not observed c1 Interestingl), when the core LAP wa. linked to the MoMuLV LTR and inserted into the same site, long-term reporter-gene expression was observed in latently infected sensory neurons~l . Such hybrid promoters merit further investigation. The evidence from these and other tudies c' indicates that the LAT regulatory region is complex. and that elements both upstream and downstream of the transcription start point need to be left intact if we are to obtain reporter gene expres ion with kinetics that mimic wildtype LAT expression. A strategy has been devised in our laborator) that allows the insertion of a reporter gene. a full 1.5 kilobases downstream of the minor LAT transcriptional start site. maintaining all currently recognized elements of the LAP (Ref. 29) (Fig. 4). Using this strategy, we have constructed a \ iral vector th at can produce ~ galactosidase expression in the dorsal root ganglia and brainstems of latently infected mice. with kinetics closely resembling those that would be expected if it were under authentic LAP control. Although there is only low-level gene expre. sion during lytic infection, when the virus enter neuronal latenc). the reporter-gene expression is upregulated, and remains stable at high levels for at least six months~~. This ability to 'harne . the endogenou. LAP regi on of the virus to drive long-term reporter gene expression in neurons of both the peripheral and central nervou ystem (CNS) open. the way for the experimental evaluation of the use of disabled HSV vector to express therapeutic genes in the nervous . ystem.
Candidate diseases for gene delivery by an HSV vector Gene therapy has been suggested as a potential therapeutic approach to a vast range of neurological disorders. ranging from inherited metabolic disorders to degenerative disease and malignancy (sec Table I). The majority of these diseases affect large region. of the C S, and the delivery of a replication-defecti ve \irus to the whole of the brain
Immediate early genes - The first class of HSV genes to be expressed in the Iytically infected cell; they require no de novo protein synthesis for their expression. Internal ribosomal entry site - An RNA sequence that allows a ribosome to gain direct access to an mRNA molecule without having to bind to the cap structure at the 5' end and scan through the 5' leader sequence. Latent infection - In latently infected nervous tissue, viral DNA per· sists in the absence of any detectable infectious virus. Lysosoma l storage disorders - Disorders of lysosomal enzymes or transport that lead to the intraorganellar accumulation of un· degraded substrates derived from cellular macromolecules. Lytic infection - During lytic infection, the virus takes over the metabolic apparatus of the cell , in order to replicate itself. This re· suits in the production of progeny virions, and the eventual destruction of the host cell. Retrograde axonal flow - The process by which neurons transport material from the synapse to the cell body.
poses great technical problem . . By far the most widely used and well-characterized system for delivery is that of direct stereotactic injection into small. well-defined regions. Therefore, disease processes such as Parkinson 's disease 111 or malignancy, which cxhibit pathology at a di crete anatomical location within the nervous system. providc some of the most attractive models for genc therapy. An amplicon-based HSV vector has been used to deliver a tvrosine hydroxylase gene to striatal neurons in a rat model' of Parkinson's di, ease 1). with thc aim of increasing local levels of dopamine, the neurotransmitter that is deficicnt in this disease. It should be noted that this is an example of a di case model where careful regulation of the level of expression or the therapeutic gene will be required to avoid the potential side effects associated with o\'er-cxpression. The. e invc. tigator. reported some uccess in correcting the abnormal behaviour displayed by these animals, although they encountered considerable problems with vector toxicity beG1U~e of a reversion of the temperature-sensitive hclper virus (tsK) to the wild type. An approach that might ea ily be adapted to a range of neurodegenerative diseases has been the attempt to slow down or halt neuronal death_ by the local delivery of a vector canying a gcne for a ncurotrophin. Geschwind and colleagues.1~ have demonstrated that neurite outgrowth from auditory neurons can bc obtaincd when explant
409
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The outstanding questions • Will it be pos ible to con truct vectors that will be sufficiently non-cytotoxic for clinical use? • Will the currently defined promoter-constructs be able to mediate long-term gene expres ion in a wide variety of different neuronal types within the central nervou system (CNS)? If so, will they be able to mediate sufficient production of transgene to correct, or prevent, pathology? • For a succe sful gene therapy approach to most neurological disorder, large regions of the CNS will have to be transduced. Will it be pos ible to devise methods to deliver a vector throughout the C S? • What will be the con equence of u ing HSV vectors in patients who have already been exposed to natural HSV infection? Will the pre-existing immune response to HSV prevent efficient gene delivery? What ri ks will be associated with the pre ence of pre-existing latent genomes within the CNS? • Will fully disabled HSV-based vectors have therapeutic applications for gene delivery to non-neuronal tis ues?
cultures of murine spina l ga nglia are infected with an amplicon vector containing a cD A that encode brain-derived neurotrophic factor. The e results how that, in principle. this approach might be useful, although fu rther studies in animal models are required. Similar approaches might be used to delay a number of neurodegenerative diseases, even dementias with widespread pathology such as Al zheimer's disease; this is providing that a method could be devised to deliver the vector to a large enough area of tis ue. Other candidates for curative treatment by gene therapy are the inherited error of metabolism. Many of these produce devastating neurological disea e, and functional replacement of the affected gene might provide complete metabolic correction. Of the monogenic diseases that affect the brain, the Iy osomal storage diseases (examples of which are Gaucher' disease, Tay-Sachs disease and metachromatic leukodystrophy) represent attractive target for gene therapy" . Although these disorders affect the whole brain, the cellular defect can be corrected by the uptake of exogenously produced enzyme. Unfortunately, the blood-brain barrier prevents systemically administered enzyme (i.e. glucocerebrosidase u ed to treat patients with Gaucher's disease) from reachi ng the ceiL of the C S. but this can be circumvented if a gene therapy approach is used to produce the enzyme within the brain. The ecretion-recapture mechanism means that a significant effect could be achieved if only a . mall number of cell were tran duced, provided that they could be made to secrete sufficient enzyme. To date, the only model of a lysosomal storage disorder in which HSV vectors have been u ed is the mucopolysaccharidosis type VII (Sly disease) mouse, which i deficient in ~-glucuronidase34 . Wolfe and col leagues were. however, unable to demonstrate any correction of pathology. probabl y because on ly low levels of ~-glucuronidase were produced in a small number of neuron ~ following the peripheral administration of the replication-competent vi rus used in this study35 . one the Ie , this group did provide an importa nt proof of principle by demon trating that it was possible to obtain production of functional enzyme and, with the use of more recently developed replicat ion-defective HSV vectors, it might be possible to obtain more promising results. 410
MOl rCI
I ~,\R
MI-:DICINIlTODAY. SEPTEMBFR 1997
Genetically engineered HSV vectors are also being developed for use in the treatment of malignant gliomas. Here, the intention is not to produce long-term transgene expression in latently infected cells, but to selectively kill the malignant cells, while sparing the surrounding healthy neural tissue (reviewed in Ref. 36). A number of groups have achieved this in animal models by using HSV vectors, deleted for non-essential neurovirulence genes [i.e. "(34.5 (Ref. 37 and 38), thymidine kinase (tk) and ribonucleotide reductase (Ref. 39)]. These viru es will replicate in the dividing tumour cells, but not in terminally differentiated neurons. Using anim al models, it has been shown that when directly injected into gliomas, such viruses can produce tumou r regression with minimal bystander effects on surrou nding normal tissue.
Future prospects The advent of fully replication-defective HSV vectors, capable of medi ating long-term gene expression in neurons, will open the door to a host of potential therapeutic uses. Each of the 'e will have to be carefully eva luated in a suitable animal model before clinical tri als can be considered. Perhaps the most difficult problem to overcome will be to devise methods to deliver a vector to large areas of the C S, and the possibility of an immune response to these vectors in patients, up to 80% of whom will have pre-existing immunity to HSV. A pre-existing immune response to HSV might hinder the ini tial delivery of the vector, and this might be overcome by transient immunosuppresion to cover the procedure. Once latency has been established, the immune re ponse to HSV should not pose a long-term problem, becau e no viral proteins are made in the latently infected cell. An immune response to the transgene product, however, might lead to problems in maintaining long-term transgene expression. The possibility of using HSV vectors to deliver therapeutic genes to non-neuronal tissue has al 0 been raised. If fully replicationdefective vectors that are no longer cytotoxic can establish latent infections in a wide range of non-dividing cell types, then they would have rea l potential as a universa l gene delivery system. However, at least in tissue culture studies, the LAP displays a degree of neuron specificity, so it may be necessary to use different promoter elements in non-neuronal tissues. [n the treatment of cancer there is no need to establish latency, and the ability of HSV to infect a wide range of human cells might make it an attractive candidate in the treatment of many different types of malignancies. A gH-deleted HSV vector that can transiently express immunomodulatory molecules in haemopoietic cells is currently being developed a a potential leukaemia therapy40. Encouraging results have been obtained using a number of vector ystems for gene delivery to neurons (reviewed in Ref. 41), including adenoviruses, adeno-associated virus and lentiviruses. In the deve lopment of all these vector systems, a with HSV, the issues of vector safet y and long-term transgene expression have to be addressed. With the advent of transgenic technology and the characterization of nat urall y occuring mutant trains, the number of small rodent models of genetic neurological diseases is ever increasing (i.e. mouse model of Tay-Sachs disease 42 and globoid cell leukodystrophy4J). The cha llenge we now face is to explore the therapeutic potential of neuronal gene therapy, using suitable animal models. References 1 Roizman , O. and Scars, A.E. (1987) An inquiry inlo Ihe mechanisms of herpes implex virus laleney. Ann. Rev. Microbiol. 41 ,543-571
MOIL ( t ' IA R MII)I (" I1'< 1 IOJ) ·'\) SI I'll M Il I ' J{ 1""7
2 Whitle}, R.J. (1996) Herpes Simplex Viruses, in Fields l1rologl' (Fields, B. .. Knipe. D.M. and Howley. P.M.. cds). pp. 2297 - 2342. Lippwcotl-Ravcn 3 Roizman. B. and Scars. A E. (1996 ) Herpes sim plex viruses and their replication, in Fieldl Virologl (FIelds. B.N . Knipe. D.M and Howley, P.M.. cd~). pp. 2231 - 2295, Lippincotl-Ralcn 4 Bzik. D.J. and Preston. C.M. (19 6) Analy is of D A equences "hich regulate the transcription of herpe sim plex viru immediate early gene 3: D A sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide, Nile/. ACids Res. 14. 929-943 5 Ho. D.Y. (1992) Herpes simplex I'irus latency: molecular aspects. Prog. .\fet!. Virol. 39. 76-115 6 levens. J.G. el 01. (1987) RNA complementary to a herpes virus alpha gene mRNA i prominent in latentl) infected neurons, [lellC(, ~35. 1056-1059 7 lIill, J.M. el 01. (1990) Herpes simp lex lirus latent phase transcription facilitates in \,i\'o reactivation. I'irology 174.117-125 8 Maggioncalda. J el 01. (1996) herpes simplex virus t)pe I mutant "ith a deletion immediately upstream of tbe LAT locus establishes latenq and reactivates from latently infected mice with normal kinetic. 1. Neuromol. 2. 26 -27 9 Ecob-Prince, M. . el 01. (1993) Neuron containing latency-associated transcri pts are numerous and widespread in dorsal root ganglia following footpad inoculation of mice "ith herpe simplex virus t)pe I mutant inl814, 1. Gell. Virol. 74. 9 5-994 10 Ace. c.1. elili. (1989) Construction and characterization of a herpes sim plex viru type 1 mutant unable to tran induce immediate-early gene expression, 1. Virol. 63. 2260-2269 II DeLuca. .A .. McCarthy. AM. and Schaffer. P.A. (1985) Isolation and characterization of deletion mutants of herpes simplex virus type I in the gene encoding immediate-early regulatory protein ICN,J. \r,rol. 56. 55 -570 12 Ho, D.Y. el al. (1995) Herpe implex virus I'ector s}stem: analysi of its in vivo and in "ilro cytopathic effects,J. Neurosci. Melhods 57.205-215 13 Wu , N. el 01. (1996) Prolonged gene expre sion and cpll survhal after infection by a herpes sim plex yiru mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22,J. Virol. 70, 6358-6369 14 Preston. C.M .• Mabbs. R. and icholl. M.J. (1997) Construction and characterization of herpe simplex viru type 1 mutant with conditional defects in immediate early gene expression, Virology ~~9. 228-239 15 Boursnell, M.E. el al. (1997) genetically inactivated herpes implex virus type 2 (HSV.2 ) vaccine prol'ides effectil'e protection against primary and recurrent HSV-2 disea e,J. IlIfecI. DIS. 175, 16-25 16 Speck, P.G .. Minson, A.C. and Efstathiou. S. (1996) In \'i\'o complementation studies of a glycoprotein Ii-deleted herpes simplex I'irus-based lector,). Gen. Virol. 77. 2563-2568 17 Ho, D.Y. (1994) Amp licon-based herpes implex virus vectors, Melhods Cell Bioi. 43, 191-210 18 Fraefel, C. 1'1 al. (1996) Helper, iru -free tran fer of herpes implex virus type I plasmid vectors into neural cells,). I'irol. 70. 7190-7197 19 Fraser. .w. 1'1 al. (1991) A rel'ie" of the molecular mechanism of H V-I latency, Cllrr. E\'e Res 10, I- S 13 20 Ecob-Prince. M.. ell/I. (1995) Expre ion of beta-galactosida e in neuron of dorsal root ganglia which are latently infected with herpes simplex virus type 1,1. Gell. Virol. 76. 1527-1532 21 Lokensgard, J.R. 1'1 al. (1994) Long-term promoter activit" during herpes si mplex virus lat ency,.I. Virol. 6H, 714 -7158 22 Andersen, J.K. 1'1 al. ( 1 99~) Gene transfer into mammalian central nenous system usi ng herpes I iru lectors: extended expre ion of bacterial lacZ in neurons using the neuron - pecific enola e promoter. HI/m Gene Ther. 3. 4 7-499
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23 Kaplitt. M.G. 1'1 al. (1994) Preproenkephalin promoter yields region-specific and long-term expression in adult brain after direct in vi,'o gene transfer via a defecthe herpes si mplex viral vector, Proc. Natl. Mild SCI. U. S. A 91 . 8979-8983
24 Lachmann, R.II.. Brown. C. and Efstathiou, S (1996) A murine RNA polymerase I promoter inserted into the herpes simplex virus type I genome is functional during lytic, but not latent, infection,J. Gen. Vim/. 77. 25 75 2582 25 Dobson, AT el al. (1990) A latent, nonpathogenic Ii V-I-derived vector stably expresses beta-galactosidase in mouse neurons, Neuron 5, 353- 360 26 Carpenter. D.E. and Stevens, J.G. (1996) Long-term expression of a foreign gene from a unique position in the latent herpes simplex virus genome, Hum . Gent' Ther. 7, 1447- 1454 27 Dobson, AT el al. (1989) Identification of the latency-associated transcript promoter by expres ion of rabbit beta-globin mRNA in mouse sensory nene ganglia latently infected with a recombinant herpes simplex viru~, J. Virol. 63 , 3 44--3851 28 Margolis. TP. 1'1 al. (1993 ) Decreased reporter gene expre ion during latent infection with H V LAT promoter co nstructs, Virology 197, 585-592 29 Lachmann, R.II. and Efstathiou. S. (1997) Utilization of the herpes simple:\. viru type I latenc}-associated regulatory region to drive stahle reporter gene npre sion in the nen'ous sy tem,J. Virol. 71 . 3197-3~07 30 Bankiewicz, K., Mandel, R.J . and Sofroniew, M.V. (1993) Trophism, transplantation, and animal model of Parkinson's disease, Exp. Neurol. 124. 14(}-149 31 During. M.J. el al. (1994) Long-term behavioral recovery in parkinsonian rats by an HSV vector expressing tyrosine hydroxylase, SCiellce 266, 1399-1403 32 Geschwind. M.D. el al. (1996) Defective HSV-l vector expressing BD F in auditory ganglia elicit neurite outgrowth: model for treatment of neuron loss following cochlear degeneration, /fum. Gene Ther. 7. 173-1 2 33 Salvetti. A, Heard. J.M. and Uanos, O. (1995) Gene therapy of lysosomal storage disorders, Br. Met!. Bull. 51. 106-122 34 Wolfe , J.H.. Deshmane, .L. and Fraser. N.W. (1992) Herpesvirus ,ector gene transfer and expression of heta-glucuronidase in the central nervous system of MPS VII mice. Na l. Gellel. 1,379- 384 35 Deshmane, S.L. 1'1 al. (1995) An HSV-I containing the rat beta-glucuronidase cD A inserted within the LAT gene is less efficient than the parental strain at e tablishing a transcri ptionally active state during latency in neurons, Gelle Titer. 2,209-217 36 Kim. D.H. and McCorm ick. F. (1996) Replicating viruses a electi\'e cancer therapeutics, Mol. Met!. Today 2. 519-527 37 Chambers, R. el al. (1995) Comparison of genetically engineered herpes si mplex I'iruse for the treatment of brain tumors in a scid mouse model of human malignant glioma, Proc. atl. Acad. Sci. U. . A. 92, 1411 - 1415 38 Mincta. T el 01. (1995) Attenuated multi-mutated herpes implex viru -I for the treatment of malignant gliomas, al. Med. 1,93 943 39 Boviatsis, E.J. el 01. (1994) Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpe simplex virus vectors defective in thymidine kinase or ribonucleotide reductase, Gent' Ther. 1,323- 331 40 Dilloo, D. el III. ( 1997) A novel herpes vector for the high-efficiency lransduction of normal and malignant human hematopoietic cells, Blood H9. 11 9- 127 41 Blomer. . el al. (1996) Application of gene th rapy 10 the ,Hum. Mol Gellel. 5, 1397-1404 42 ango. K. el 01. (1995) Mouse models of Tay-Sachs and Sand hoff diseases differ in neurologic phenotYllc and ga nglioside metabolism, at. Gelle/. II , 170-176 43 Suzuki, K. and SUluki , K. (1995) The twitcher mouse: a model fur Krabbe disease and for experimental therapies, Braill Palhol. 5, 249- 25 44 McFarlane, M. Daksi~. J.l. and Preston. C.M. (1992) Hexamethylene bi 'acetamide stimulates herpe simplex virus immediate early gene expression in the absence of trans-induction by VmwIl5,.I. Gell. Viml. 73,285 292
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